UBC Theses and Dissertations

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UBC Theses and Dissertations

Interaction of cellular and environmental factors in tumor histogenesis Auersperg, Nelly 1968

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THE INTERACTION OF CELLULAR AND ENVIRONMENTAL FACTORS IN TUMOR HISTOGENESIS  by  NELLY AUERSPERG  M.D.,  U n i v e r s i t y o f Washington,  1955  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE R'EQUIRMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n t h e Department of Zoology  We a c c e p t t h i s t h e s i s as conforming t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 1968  In  presenting  for  an.advanced  that  the  Study.  thesis  degree  I further  for  agree  partial  the  make  it  that  freely  representatives.  h.i)s  of  of  this  thesis  may  for  permission.  5 o ^ l o  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Columbia  be  for  granted  It  is  financial  of  British  available  permission  or  by  fulfilment  U n i v e r s i t y of  purposes  my w r i t t e n  Department  at  in  scholarly  publication  without  thesis  Library shall  Department  or  this  the  Columbia,  I  reference  and  for  extensive  by  the  requirements  copying  Head o f  understood  gain  shall  this  my  that  not  of  agree  be  copying  allowed  i i  The derived  histogenesis  ABSTRACT o f two squamous-carcinoma c e l l  lines,  originally  from t h e same human c e r v i c a l tumor b u t w i t h d i f f e r e n t morphol-  ogy lH v i t r o and in v i v o , was compared h i s t o c h e m i c a l l y and e l e c t r o n microscopically.  I n o c u l a t i o n i n t o . h a m s t e r cheek pouches was used  t o d i s t i n g u i s h in v i t r o from _in v i v o r e s p o n s e s i n t h e two c e l l  lines.  Normal c e r v i c a l e p i t h e l i u m was grown in v i t r o t o determine which h i s t o g e n e t i c c h a r a c t e r i s t i c s were due t o t h e m a l i g n a n t nature o f t h e cell  lines.  P o s s i b l e mechanisms u n d e r l y i n g h i s t o g e n e t i c v a r i a t i o n  were i n v e s t i g a t e d by c h e m i c a l and p h y s i c a l m o d i f i c a t i o n s o f t h e i n v i t r o environment. In v i t r o c e l l s of l i n e C-4c were more c o h e s i v e , and  l e s s deformable  l e s s a d h e s i v e t o s u b s t r a t a t h a n t h e c e l l s o f l i n e C-4s and t h e y  formed more compact and h i g h l y s t r a t i f i e d c o l o n i e s . t o crowding i n v i t r o by c e l l increased  stratification.  separation  L i n e C-4s responded  ( d i s p e r s a l ) w h i l e l i n e C-4c  Histochemically  t h e c e l l l i n e s were e s s e n t i a l l y  s i m i l a r but they d i f f e r e d i n u l t r a s t r u c t u r e , p a r t i c u l a r l y at the l e v e l of t i s s u e o r g a n i z a t i o n . nucleo-cytoolasmic cytoplasmic  The C-4s c e l l s were columnar, w i t h a h i g h e r  r a t i o , a p o l a r i t y of t h e o r g a n e l l e s ,  filaments, l i t t l e  between s u p e r f i c i a l c e l l s .  dispersed  s t r a t i f i c a t i o n and w i t h t e r m i n a l  In c o n t r a s t , . C-4c c e l l s were o v a l ,  f i e d and w i t h s u p e r f i c i a l c e l l s f l a t t e n e d , w i t h a lower mic r a t i o , w i t h o u t p o l a r i t y o f o r g a n e l l e s , and w i t h more  bars strati-  nucleo-cytoplascytoplasmic  f i l a m e n t s condensed i n t o bundles t h a t were a s s o c i a t e d w i t h desrnosom.es. In both l i n e s , but p a r t i c u l a r l y i n l i n e C-4c, i n t e r d i g i t a t i n g m i c r o villi  provided  modified  t h e main i n t e r c e l l u l a r c o n t a c t  and t h e c e l l s u r f a c e s were  i n a s s o c i a t i o n w i t h both s u b s t r a t a and t h e growth medium.  Most  iii i n v i t r o d i f f e r e n c e s between the  cell  l i n e s were r e t a i n e d  in vivo, and,  i n a d d i t i o n , incomplete basement membranes were formed i n both though more e x t e n s i v e l y  i n l i n e C-4.s.  I t appeared t h a t the  d i f f e r e n c e _in v i t r o between benign squamous c e l l s cells  l a y i n the  organization  ability  i n the  of the  l a t t e r t o m a i n t a i n an  complete absence of any  P o s s i b l e mechanisms u n d e r l y i n g genesis  were e x p e r i m e n t a l l y  i n d i c a t e d t h a t the  and  the  lines,  o n l y major  carcinoma  intercellular  supporting  tissue.  these observed d i f f e r e n c e s i n h i s t o -  investigated.  F e r r i t i n uptake _in v i t r o  d i f f e r e n c e i n s t r a t i f i c a t i o n was  not due  t o more  efficient  i n t e r c e l l u l a r c i r c u l a t i o n i n the more h i g h l y s t r a t i f i e d  line.  d i f f e r e n c e i n c e l l - s u r f a c e charges or d i s t r i b u t i o n of  No  surface  a c i d mucosubstances c o u l d be demonstrated, s u g g e s t i n g  ference  i n contact  It was  i n h i b i t i o n t o be  found t h a t C-4c  a s s o c i a t e d w i t h the  c e l l s were more c o h e s i v e  and  C-4c  cell-  no  dif-  stratification.  that t h i s  cohesion,  i n both l i n e s , depended p r e d o m i n a n t l y on the presence of d i v a l e n t In c o n t r a s t ,  adhesion to the  substrata required  cations.  extracellular proteins,  p r o b a b l y accompanied by the masking c f a c i d groups on the  cell  These and  i n deform-  ability  other  described  i n these c e l l  hesion  i n l i n e C-4c  tion.  The  and  observations  l i n e s w i t h the p r o b a b i l i t y t h a t the  was  due  of c e l l  the  separation  other  formation  and  hand, the cell  medium, to other of the  to the more e x t e n s i v e  in v i t r o maintenance of c e l l  shape, o f t i s s u e  adhesive f o r c e s and  specific, modifications,  stronger  co-  m i c r o v i l l u s popula-  ( d i s p e r s a l ) seemed t o i n v o l v e . a n  between t h e s e i n t e r c e l l u l a r On  suggested a d e f e c t  surface.  the  organization interaction  cytoplasmic  such as  filaments.  terminal-bar  f l a t t e n i n g , which appeared i n r e l a t i o n t o growth  c e l l s and  to substrata  c e l l s to the v i s c o s i t y of the  c o u l d be  shown to be  responses  immediate environment and  thus  iv experimentally m o d i f i a b l e . The r e s u l t s of t h i s i n v e s t i g a t i o n are discussed r e l a t i v e to the s p e c i f i c suggestion that much of the h i s t o g e n e t i c v a r i a t i o n could be explained by considering that the more cohesive c e l l l i n e (C-4c) had retained p r o p e r t i e s of normal epithelium stratum spinosum cells., while . the spreading c e l l l i n e (C-4s) e x h i b i t e d c h a r a c t e r i s t i c s of basal c e l l s , and r e l a t i v e to the i n t e r a c t i o n of d i f f e r e n t i a t i o n capacity and malignancy i n tumor development.  V  TABLE OF CONTENTS PAGE 1  INTRODUCTION • HISTORY OF THE CELL LINES  5  MATERIALS AND METHODS  13  RESULTS  24  Morphologic v a r i a t i o n i n c u l t u r e under maintenance conditions Histochemistry of i n s i t u  24 cultures  27  U l t r a s t r u c t u r e of in s i t u c u l t u r e s  29  Hamster  42  cheek-pouch i n o c u l a t i o n s  Cultures  o f normal c e r v i c a l e p i t h e l i u m  ~ 46  Summary of t h e h i s t o c h e m i s t r y and u l t r a s t r u c t u r e of t h e C-4 c e l l l i n e s and normal e p i t h e l i u m The d i f f e r e n c e i n growth p a t t e r n between the cell lines  56  The r e l a t i o n s h i p of c e l l p a t t e r n i n l i n e C-4s  69  separation  The r o l e o f s u b s t r a t u m - v i s c o s i t y of t h e C-4 l i n e s DISCUSSION SUMMARY BIBLIOGRAPHY  52  and growth  i n the histogenesis 73 •  83 '99 102  LIST OF TABLES  TABLE I II III  IV V VI  VII VIII  PAGE C h a r a c t e r i s t i c s of the C-4 Lines.  7  Proportion of C e l l s with Minute Marker.  9  Main U l t r a s t r u c t u r a l Differences between the C e l l Lines In v i t r o .  40  C e l l Electrophoresis.  58  Spreading of Single Subcultured C e l l s on Glass. The E f f e c t s of Trypsin Digestion and of Divalent-Cation Depletion on the Culture Morphology of the C-4 C e l l Lines.  61 63  V i a b i l i t y of C e l l s Shed into Growth Medium over 48-hour Periods.  68  Number of Colonies Formed by C e l l s Recovered from Growth Medium and Recultured.  70  vii  LIST OF ILLUSTRATIONS FIGURES 1-13  14-23  24-28  29-34 35-47 48-52  53-58  59-62  63-68  69-72  PAGE Morphologic v a r i a t i o n i n c u l t u r e maintenance c o n d i t i o n s  under 25-28  G e n e r a l o r i e n t a t i o n o f the c e l l s and o f i n t e r c e l l u l a r spaces - h i s t o l o g y  30  U l t r a s t r u c t u r e of i n s i t u - l i n e C-4c  cultures 32  U l t r a s t r u c t u r e of i n s i t u - l i n e C-4s  cultures 33  U l t r a s t r u c t u r e o f i_n s i t u c u l t u r e s - c e l l surface c h a r a c t e r i s t i c s  36-41  U l t r a s t r u c t u r e o f hamster cheek-pouch tumors  41  H i s t o l o g y and h i s t o c h e m i s t r y o f hamster cheek pouch tumors  44  C u l t u r e s of normal c e r v i c a l e p i t h e l i u m - Histology  48  U l t r a s t r u c t u r e o f normal e p i t h e l i a l growing from e x n l a n t s  48  cultures  U l t r a s t r u c t u r e o f normal c e r v i c a l e p i t h e l i a l c u l t u r e s growing w i t h o u t e x o l a n t s  50  73-74  F e r r i t i n uptake  50  75-00  U l t r a s t r u c t u r a l d i s t r i b u t i o n of a c i d mucosubstances D i s t r i b u t i o n of c e l l clumps superimposed upon c u l t u r e s of the same t y p e  81-82  83-90  50-51 51  The e f f e c t s o f d i v a l e n t - c a t i o n d e p l e t i o n and of t r y p s i n d i g e s t i o n on c u l t u r e morphology  65  91-98  The h i s t o l o g y of C-4 c o l o n i e s i n s u s p e n s i o n  76  99-107  The u l t r a s t r u c t u r e suspension  108-115  o f C-4 c o l o n i e s i n  The e f f e c t o f e n v i r o n m e n t a l v i s c o s i t y on the •morphology of C-4 c e l l s  77-78  81  viii Acknowledgements I wish t o express my  s i n c e r e a p p r e c i a t i o n t o Dr. C y r i l V. Finnegan  f o r h i s a d v i c e , guidance and encouragement throughout t h i s t i o n , and t o D r s . A.B. Acton, R.L. helpful  Noble and D.M.  investiga-  Whitelaw f o r t h e i r  s u g g e s t i o n s d u r i n g the i n v e s t i g a t i o n and c r i t i c a l  examination  of the m a n u s c r i p t . I a l s o wish t o thank Mrs. M. Douglas and Mr. L. Veto f o r t h e i r h e l p and  advice.  During the t e n u r e of t h i s  i n v e s t i g a t i o n the author was  of a f e l l o w s h i p from the N a t i o n a l Cancer r e s e a r c h was  the r e c i p i e n t  I n s t i t u t e o f Canada.  This  supported by g r a n t s from the N a t i o n a l Research C o u n c i l  of Canada t o Dr. C.V,  Finnegan.  Introduction One o f t h e c h a r a c t e r i s t i c s of. malignancy i s the c o n s i d e r a b l e s t r u c t u r a l v a r i a t i o n found among tumors.  T h i s r e s u l t s i n each l e s i o n  h a v i n g i t s own p e c u l i a r morphology and b e h a v i o u r , w i t h i n the broader framework o f p r o p e r t i e s  common t o a l l c a n c e r s .  This v a r i a t i o n i n  abnormal d i f f e r e n t i a t i o n , c a n c e r - c e l l a s s o c i a t i o n s and p a t t e r n s o f i n v a s i o n , has been known f o r a long time, as has t h e i n f l u e n c e o f . these c h a r a c t e r i s t i c s on the n a t u r a l h i s t o r y o f cancer However, n e i t h e r the mechanisms u n d e r l y i n g  (Willis,  1948).  changes i n d i f f e r e n t i a t i o n ,  nor  t h e causes which determine the growth p a t t e r n o f malignant tumors,  are  f u l l y understood.  Thus, p r o g r e s s i v e  anaplasia,  i . e . the gradual  l o s s o f t i s s u e - s p e c i f i c c h a r a c t e r i s t i c s , has been c o n s i d e r e d of i r r e v e r s i b l e , embryological  consecutive  development  as a s e r i  developmental changes, i n the sense o f  ( F c u l d s , 1954).  g e n e r a l l y accepted at p r e s e n t ,  there  A l t e r n a t i v e l y , and more  i s the concept t h a t the p r o g r e s -  s i v e l o s s o f s p e c i a l i z e d s t r u c t u r e s and f u n c t i o n s s e l e c t i o n o f i n c r e a s i n g l y autonomous c e l l s  i s due t o continuous  from t u m o r - c e l l  populations  c h a r a c t e r i z e d by a c t i v e p r o l i f e r a t i o n and a h i g h r a t e of g e n e t i c ability  (Sandberg, 1966).  In a d d i t i o n t o a n a p l a s i a ,  of cancer appears t o be i n f l u e n c e d by other have a s i m i l a r d i f f e r e n t i a t i o n c a p a c i t y vary t e c t u r e and h o s t  r e l a t i o n s h i p s (Leighton,  vari-  the h i s t o g e n e s i s  f a c t o r s , s i n c e tumors t h a t greatly i n their archi-  1967),  Among such  possible  f a c t o r s t h a t have been s t u d i e d e x t e n s i v e l y ,  are cancer c e l l m o t i l i t y ,  cell  as w e l l as host  and  surface  p r o p e r t i e s and l y t i c  activity,  immunological responses ( L e i g h t o n , To  d i s t i n g u i s h some c e l l u l a r  p l a y a r o l e i n the h i s t o g e n e s i s  stromal  1967; Weiss, 1967).  and environmental  f a c t o r s t h a t may  o f carcinomas, a comprehensive examin-  a t i o n o f carcinoma c e l l s i n v i t r o and i n v i v o was u n d e r t a k e n i n t h e present study.  However, i n c o n t r a s t t o t h e e x t e n s i v e amount o f i n f o r m a -  t i o n a v a i l a b l e i n r e g a r d t o t h e i n v i t r o growth o f f i b r o b l a s t - l i k e cells  ( A b e r c r o m b i e , 1966; H a y f l i c k , 1961) few e x p l a n a t i o n s e x i s t t o  date f o r t h e m o r p h o l o g i c v a r i a t i o n observed among e p i t h e l i a l Yet,  cultures.  i t would seem t h a t t h e u n d e r s t a n d i n g o f t h e b e h a v i o u r o f e p i t h e l i a l  c e l l s would be o f prime importance i n r e l a t i o n t o c a n c e r , s i n c e t h e g r e a t m a j o r i t y o f m a l i g n a n t tumors a r e o f e p i t h e l i a l  origin.  The r e t e n t i o n o f _in v i v o growth c h a r a c t e r i s t i c s by cancer c e l l s i n p r i m a r y c u l t u r e has been demonstrated f o r a v a r i e t y o f c a r c i n o m a s , i n c l u d i n g adenocarcinomas the  (Southam, 1954) and squamous carcinomas o f  c e r v i x (Gey e t a l . 1952; Gey, 1954-1955; M e l l g r e n e t a l . , 1962;  A u e r s p e r g and Worth, 1966) and l u n g (Sherwin _et a l . , 1967).  However,  these c u l t u r e s have a b r i e f l i f e span which l i m i t s t h e i r u s e f u l n e s s as e x p e r i m e n t a l m a t e r i a l , w h i l e those c u l t u r e s t h a t a r e propagated as l o n g - t e r m c e l l l i n e s u s u a l l y l o s e many o f t h e i r s p e c i f i c ( D a v i d s o n , 1964).  properties  A l s o , t h e v a l i d i t y and s p e c i f i c i t y o f c o r r e l a t i o n s  t h a t can be drawn from a comparison o f growth c h a r a c t e r i s t i c s i n a s e r i e s o f tumors i s l i m i t e d , by t h e m u l t i t u d e o f v a r i a b l e s t h a t a r e p r e s e n t due t o t h e g e n e t i c h e t e r o g e n e i t y o f t h e tumors.  I t appears  p a r t i c u l a r l y d e s i r a b l e t h e r e f o r e t o compare p r o p e r t i e s o f m a l i g n a n t c e l l s t h a t are c l o s e l y r e l a t e d g e n e t i c a l l y , a c o n d i t i o n f u l f i l l e d i n the  s t u d i e s o f t h e h i g h - and low c a n c e r - p r o d u c i n g l i n e s o f S a n f o r d  ( B a r s k i and W o l f f , 1965), which were d e r i v e d from one c l o n e o f c e l l s , and, t o a c e r t a i n e x t e n t , i n s t u d i e s o f v i r u s - t r a n s f o r m e d m a l i g n a n t c e l l s (Kirsten,  1966).  In t h e p r e s e n t i n v e s t i g a t i o n a comparison was made o f two carcinoma  3  cell  l i n e s with different  the u s u a l c h a r a c t e r i s t i c s  c u l t u r e morphology w h i c h had not y e t o f l o n g - t e r m c e l l T i n e s , but r a t h e r  t o have r e t a i n e d some o f t h e i r o r i g i n a l p r o p e r t i e s  seemed  (Auersperg and  H a w r y l u k , 1962; A u e r s p e r g , 1963; E a g l e et a l . ^ 1 9 6 6 ; G a r t l e r , These c e l l  assumed  l i n e s were i s o l a t e d from the same human c e r v i c a l  1967). carcinoma,  and l i k e l y o r i g i n a t e d from the same c l o n e o f c e l l s as i n d i c a t e d by a v e r y s i m i l a r , but abnormal k a r y o t y p e ( A u e r s p e r g , 1 9 6 4 ) .  In a d d i t i o n ,  t h e y showed c e r t a i n m e t a b o l i c , c y t o l o g i c a l and h i s t o l o g i c a l d i f f e r e n c e s commonly a s s o c i a t e d w i t h d i f f e r e n t below i n the these c e l l  degrees o f a n a p l a s i a ,  s e c t i o n on the H i s t o r y o f the C e l l  Lines.  as d e s c r i b e d Therefore,  l i n e s seemed t o r e p r e s e n t p o p u l a t i o n s from two s t a g e s i n  the development o f one tumor and t h u s a model system f o r the study o f c o n s e c u t i v e c e l l u l a r changes t h a t may t a k e p l a c e d u r i n g tumor sion.  I t a l s o seemed p o s s i b l e t o d i s t i n g u i s h c e l l - d e p e n d e n t  host-dependent h i s t o g e n e t i c between the c e l l  factors,  lines persisted  i n hamster  The comparison o f the two c e l l ultrastructural  since morphologic  differences  cheek pouch i m p l a n t a t i o n s .  t o observe the e f f e c t s  growth phases w i t h i n a on c u l t u r e morphology o f  changing growth r a t e s and c e l l c r o w d i n g , s i n c e no comparable existed.  To compare in v i t r o and i n v i v o c h a r a c t e r i s t i c s ,  s i s was performed on hamster malignant c h a r a c t e r i s t i c s  cheek-pouch tumors,  from r e s p o n s e s t o the c u l t u r e  tions revealed s i g n i f i c a n t differences  study  an a n a l y -  and t o d i s t i n g u i s h  normal c e r v i c a l e p i t h e l i u m was grown f o r c o m p a r i s o n .  t i o n of the c e l l  from  l i n e s i n c l u d e d a h i s t o c h e m i c a l and  a n a l y s i s of the d i f f e r e n t  passage _in v i t r o ,  progres-  environment,  These  observa-  i n the i n t e r c e l l u l a r o r g a n i z a -  l i n e s and some o f t h e s e d i f f e r e n c e s were  i n v i v o , w h i l e o t h e r s seemed dependent on the t i s s u e  retained  culture  environment.  A s t r i k i n g s i m i l a r i t y was observed i n some p r o p e r t i e s o f t h e carcinoma c e l l s and normal c e l l s i n c u l t u r e .  The b a s i s o f some o f t h e observed  d i f f e r e n c e s was f u r t h e r i n v e s t i g a t e d by examining hypotheses, e s t a b l i s h e d t o e x p l a i n t h e v a r i a t i o n i n c o h e s i v e n e s s between t h e c e l l  lines,  by examining t h e r e l a t i o n s h i p o f some u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s t o growth p a t t e r n s in v i v o and _in v i t r o , and by examining e f f e c t s o f the p h y s i c a l enviroament on t h e h i s t o g e n e s i s  o f the c e l l  lines.  •5 H i s t o r y of the C e l l  In 1960, carcinoma  a b i o p s y was  Lines  o b t a i n e d from an u n t r e a t e d , i n v a s i v e squamous  of the u t e r i n e c e r v i x .  The t i s s u e was washed i n Hanks' s a l i n e  and a r e p r e s e n t a t i v e p i e c e f i x e d i n f o r m o l - s a l i n e . H i s t o l o g i c examinat i o n of t h i s p i e c e showed compact clumps o f p o o r l y d i f f e r e n t i a t e d , s m a l l , u n i f o r m carcinoma  c e l l s , w i t h v e s i c u l a r n u c l e i , and.no evidence  t y p i c o r g a n i z a t i o n . The medium MB 752/1  remainder of the b i o p s y was  minced i n Waymouth's  u n t i l the f r i a b l e tumor t i s s u e formed a s u s p e n s i o n which  c o u l d be s e p a r a t e d from most of t h e c o n n e c t i v e t i s s u e stroma. s u s p e n s i o n was  of h i s t o -  This  c e n t r i f u g e d , and the sediment resuspended i n Waymouth's  medium MB 752/1  supplemented w i t h 10%  f e t a l c a l f serum, 10% u m b i l i c a l  cord  e x t r a c t , 100 ug/rnl of s t r e p t o m y c i n and 100 u n i t s / m l of p e n i c i l l i n . The  r e s u l t i n g c e l l s u s p e n s i o n was  d i s t r i b u t e d among f i v e L e i g h t o n t u b e s .  C u l t u r e s t h a t grew i n these tubes were kept s t r i c t l y  separate t h e r e a f t e r ,  and t h e r e f o r e represented, s u b l i n e s d e r i v e d from c e l l s t h a t had i n c o n t a c t i n the b i o p s y specimen. all  Epithelial-cell  sheets appeared, i n  f i v e c u l t u r e tubes w i t h i n two weeks a f t e r e x p l a n a t i o n .  c u l t u r e s were m a i n t a i n e d  l a s t been  Two  of t h e  as l o n g - t e r m c e l l l i n e s "C-4I" and " C - 4 I I " .  ( A u e r s p e r g and Hawryluk, 1962), and r e p r e s e n t t h e c u l t u r e s used i n t h e present  study.  I t was  found s u b s e q u e n t l y , t h a t the c u l t u r e morphology of the  c e l l l i n e s d i f f e r e d and, s i n c e the p r e s e n t study d e a l s w i t h the of t h i s morphologic for  d i f f e r e n c e , the names of the c e l l  easier identification.  f e r r e d t o as l i n e C-4c,  as l i n e C-4s,  nature  l i n e s were m o d i f i e d  Thus, i n t h i s t h e s i s , l i n e C-4I w i l l be r e -  because of the crowded growth p a t t e r n of the  and the f o r m a t i o n of compact c o l o n i e s , w h i l e l i n e C-4II w i l l be to  two  cells  referred  because t h e s e c e l l s were more s c a t t e r e d and the c o l o n i e s  6  were spread o u t . The c e l l s were s u b c u l t u r e d f o r the f i r s t time d u r i n g the  second  month i n v i t r o and t h e n were m a i n t a i n e d on Waymouth's medium MB w i t h 10?o f e t a l c a l f serum, 2-5% u m b i l i c a l c o r d e x t r a c t and as above.  antibiotics  They were s u b c u l t u r e d a t 1-2 weekly i n t e r v a l s w i t h  c r y s t a l l i n e trypsin/Moscona's s a l i n e .  752/1  0.12%  To keep the number o f s u b c u l t u r e s  low, s m a l l i n o c u l a were used, and heavy growth was a l l o w e d between passages.  Periodically,  c e l l samples were f r o z e n i n l i q u i d n i t r o g e n .  From 1960 t o 1964, c u l t u r e C-4c t u r e C-4s  grew through 132 passages, and  through 118 passages; i n 1964,  samples f r o z e n a t 73 and  cul74  passages r e s p e c t i v e l y , were r e e s t a b l i s h e d i n c u l t u r e and o b s e r v a t i o n s c o n t i n u e d on t h e s e .  The d o u b l i n g t i m e s of the c u l t u r e s , measured by  c e l l counts and by c e l l p r o t e i n d e t e r m i n a t i o n s , ranged from 1-2 d u r i n g the l o g a r i t h m i c growth phase t o 18-26 tures. cell  There was  lines.  The  days  days i n h e a v i l y grown c u l -  no s i g n i f i c a n t d i f f e r e n c e i n growth r a t e s between the cell  l i n e s seemed i d e n t i c a l d u r i n g the f i r s t y e a r i n  c u l t u r e , but t h e n , a number of d i f f e r e n c e s became a p p a r e n t , which have persisted since.  These i n v o l v e d chromosome p a t t e r n s , g l y c o l y s i s  and  m o r p h o l o g i c changes ( T a b l e I ) . Chromosome p a t t e r n s .  When f i r s t a n a l y s e d a t 3 and 7 months i n c u l t u r e ,  b o t h c e l l l i n e s had chromosome modes of 44-45. i n both l i n e s had s h i f t e d  t o 44.and remained  A t 13-15 months, modes  a t t h i s v a l u e up t o t h e time  of the l a s t chromosome a n a l y s i s a t 3 y e a r s i n c u l t u r e (Auersperg and Hawryluk,  1962} A u e r s p e r g , 1964).  lines differed  I n i t i a l l y , t h e k a r y o t y p e of b o t h  from normal as f o l l o w s :  one E18 chromosome were a b s e n t .  cell  one A2, one C o r D or b o t h , and  There was  an e x t r a A3 and an  abnormal  C h a r a c t e r i s t i c s o f t h e C-4 L i n e s Property  C-4c  C-4s  D e v i a t i o n s from normal f e male k a r y o t y p e  m i s s i n g : A2, 2x C and/or D, E18 e x t r a : A3, "A" marker, minute m e t a c e n t r i c marker  m i s s i n g : A2, C and/or D, E18 e x t r a : A3, "A" marker  Glycolytic  high  low  extensive s o l i d expanding mass  less extensive i n f i l t r a t i n g i n small cells  round v e s i c u l a r n u c l e i , clumped chromatin, i n d i s t i n c t c e l l bord e r s , no o r i e n t a t i o n  o v a l n u c l e i , more homogeneous chromatin, d i s t i n c t c e l l borders, some o r i e n t a t i o n i r r e g u l a r . spread c o l o n i e s , ly s t r a t i f i e d , cysts  rate  Growth i n hamster cheek pouches a) Quantitative b) Growth p a t t e r n  Tissue culture a) C y t o l o g y  b)  C o l o n y morphology  round compact c o l o n i e s , stratified  c)  Cell  marked  •moderate  d)  Adhesion t o glass  slow  more r a p i d  e)  Dependence on u m b i l i cal cord e x t r a c t f o r s p r e a d i n g on g l a s s  marked  little  f)  Growth on c o l l a g e n gel or u m b i l i c a l cord  no o r i e n t a t i o n  orientation  cohesion  highly  groups o f  slight-  chromosome grouped as A  because of i t s l e n g t h . This karyotype per-  s i s t e d e s s e n t i a l l y unchanged i n culture C-4s for the three years.  How-  ever, there was a change i n C-4c which was f i r s t observed at 15 months, and p e r s i s t e d t h e r e a f t e r .  I t involved loss of a further C and/or D  chromosome and the appearance of a minute metacentric marker chromosome. V a r i a t i o n around the predominant mode and karyotype was more pronounced i n l i n e C-4c throughout the chromosome s t u d i e s . The c e l l s with the new karyotype i n l i n e C-4c, characterized by the minute marker, could have o r i g i n a t e d through a mutation i_n v i t r o .  Alter-  n a t i v e l y , they may have been present at the time of i s o l a t i o n from the p a t i e n t , become l o s t i n c u l t u r e C-4s, but p e r s i s t e d i n small numbers and e v e n t u a l l y predominated  i n culture C-4c.  On reviewing chromosome  analyses with p a r t i c u l a r a t t e n t i o n t o the presence of the minute marker, i t was found that c e l l s with t h i s chromosome were present at d i f f e r e n t times as shown i n Table I I . I t seems probable t h e r e f o r e , that c e l l s with both karyotypes, i . e . with and without marker, were already present in vivo.  In view of the s i m i l a r i t y of the karyotypes, i t further appears  most l i k e l y that both c e l l l i n e s o r i g i n a t e d from the same clone. Morphology*  I n i t i a l l y , no d i f f e r e n c e was noted between the c e l l  lines.  The c u l t u r e s grew as cohesive e p i t h e l i a l sheets, s t r a t i f i e d and shed v i a b l e c e l l s into the medium.  A primary c u l t u r e , f i x e d and stained at  s i x weeks _in v i t r o , showed marked pleornorphism, v a c u o l i z a t i o n and necrosis of c e l l s , as w e l l as many m i t o t i c f i g u r e s , suggestion that c e l l degene r a t i o n took place concurrently with c e l l p r o l i f e r a t i o n i n these e a r l y stages.  During the second year i n c u l t u r e , i t was f i r s t noted that the  c e l l l i n e s d i f f e r e d from one another (Auersperg, 1963).  When grown on  glass and stained with hematoxylin and e o s i n , C-4c c e l l s appeared  larger,  Table I I P r o p o r t i o n o f C e l l s w i t h Minute Marker C-4c passages  months i n culture  2  3  3  C-4s c e l l s w i t h marker t o t a l // o f c e l l s  c e l l s w i t h marker t o t a l # of c e l l s  passages  months i n culture  2/14  11  7  0/8  4  5/23  17  11  3/9  21  15  17/20  19  13  0/22  46  25  14/14  48  25  0/12  57  29  14/14  62  30  0/16  80  36  14/16  87  38  0/10  U  vO  10 with round, v e s i c u l a r n u c l e i , i n d i s t i n c t i n t e r c e l l u l a r spaces and marked overlapping  of c e l l s and n u c l e i .  C-4s c e l l s seemed smaller, had  ovoid  n u c l e i , w e l l defined i n t e r c e l l u l a r ' s p a c e s , l e s s overlapping, and tended to be oriented along l i n e s of s t r e s s . The growth pattern on glass of C-4c c e l l s was more compact than that of C-4s:  C-4c c e l l s formed round  c o l o n i e s , while those of C-4s c e l l s had more i r r e g u l a r o u t l i n e s ; f u r t h e r , clumps of C-4c c e l l s d i d not adhere to glass as e a s i l y as did clumps of C-4s c e l l s , and C-4c c e l l s were more dependent on the presence of umbili c a l cord e x t r a c t i n the medium f o r spreading and attachment on g l a s s . C-4c colonies, s t r a t i f i e d e x t e n s i v e l y , even before becoming confluent, while C-4s c e l l s seemed to grow mainly as monolayers u n t i l they became crowded; then they formed c y s t - l i k e structures which r a i s e d c e l l s o f f the g l a s s , and also began to s t r a t i f y , but l e s s extensively than C-4c  cells.  When grown on collagen gel or on fragments of u m b i l i c a l cord, C-4s  cells  showed some o r i e n t a t i o n i n t o cuboidal or columnar epithelium, while C-4c c e l l s did not.  These morphologic d i f f e r e n c e s between the two c e l l l i n e s  have p e r s i s t e d with minor m o d i f i c a t i o n s . • The primary c u l t u r e , described above, showed some features of each c e l l l i n e .  I t resembled C-4c  cells  i n the degree of s t r a t i f i c a t i o n , but formed some c y s t - l i k e structures as with C-4s, and most but not a l l p e r i p h e r a l , a c t i v e l y growing areas resembled C-4s. During the fourth year i n c u l t u r e , c e l l s of both l i n e s were inoculated i n t o cheek pouches of Syrian hamsters.  C-4c c e l l s formed r e l a t i v e l y  large tumors, often several m i l l i m e t e r s i n diameter.  The c e l l s grew  as compact masses and c l o s e l y resembled the o r i g i n a l tumor biopsy from the p a t i e n t .  obtained  C-4s c e l l s formed smaller, often m u l t i p l e tumors,  with a tendency to invade as small nests and strands of c e l l s , rather  11 t h a n as compact clumps. Glucose metabolism.  D u r i n g t h e second y e a r i n c u l t u r e , i t a l s o became  apparent t h a t C-4c c e l l s lowered t h e pH o f t h e medium a t a much h i g h e r r a t e and t o a lower v a l u e t h a n d i d C-4s c e l l s , a l t h o u g h t h e c u l t u r e s had s i m i l a r growth r a t e s .  A number o f e x p e r i m e n t s were performed t o  s t u d y t h e r e l a t i o n s h i p s o f g l u c o s e uptake and o f l a c t i c a c i d p r o d u c t i o n to 1.  c e l l growth ( A u e r s p e r g , 1963).  R e s u l t s can be summarized as f o l l o w s :  G l u c o s e uptake and l a c t i c a c i d p r o d u c t i o n were h i g h e r by a f a c t o r  of 2-3 i n C-4c i n a l l phases o f growth, both p e r c u l t u r e and p e r mg o f cell protein.  2.  The p r o p o r t i o n o f l a c t i c a c i d produced t o g l u c o s e  used was h i g h e r i n C-4c.  3.  L a c t i c a c i d p r o d u c t i o n i n C-4c c e l l s  varied  o v e r a wide range depending o n t h e growth phase, and was a d i r e c t and a p p a r e n t l y f i x e d f u n c t i o n o f g l u c o s e u p t a k e ; i n C-4s c e l l s l a c t i c  acid  p r o d u c t i o n was always l i m i t e d t o a low range and seemed independent o f glucose uptake.  4.  Under c o n d i t i o n s o f low pH and h i g h l a c t i c  acid  c o n c e n t r a t i o n s , C-4s c e l l s c o u l d break down and a p p a r e n t l y u t i l i z e a c cumulated l a c t i c a c i d , w h i l e C-4c c e l l s c o u l d n o t .  5.  A nitrogen a t -  mosphere caused a much g r e a t e r r e l a t i v e i n c r e a s e i n g l u c o s e uptake and l a c t i c a c i d p r o d u c t i o n i n C-4s t h a n i n C-4c. These p r e l i m i n a r y o b s e r v a t i o n s suggested t h a t l i n e s C-4c and C-4s r e p r e s e n t e d two s t a g e s i n t h e development cancer c e l l s .  o f a tumor from one c l o n e o f  D u r i n g t h e f i r s t y e a r i n c u l t u r e , b o t h l i n e s resembled  C-4s, and l i n e C-4c o n l y assumed i t s p r e s e n t c h a r a c t e r i s t i c s d u r i n g t h e second y e a r i_n v i t r o .  However, t h e o r i g i n a l b i o p s y o b t a i n e d from t h e  p a t i e n t , h i s t o l o g i c a l l y resembled t h e h e t e r o t r a n s p l a n t e d tumors p r o duced by l i n e C-4c r a t h e r t h a n C-4s, and t h e p r i m a r y c u l t u r e had some c h a r a c t e r i s t i c s of both l i n e s .  These o b s e r v a t i o n s , as w e l l as t h e  12 changes i n chromosome p a t t e r n s , events: ant C-4s  suggested t h e f o l l o w i n g sequence o f  a t the time o f the b i o p s y ,  C-4c c e l l s  c o n s t i t u t e d t h e predomin-  c e l l type i n the tumor p o p u l a t i o n _in v i v o , but t h e r e were a l s o some cells.  selective  Explantation  cell  i n v i t r o was f o l l o w e d  by s e v e r a l weeks o f  p r o l i f e r a t i o n and d e g e n e r a t i o n w i t h i n the primary c u l t u r e s ,  d u r i n g which time most C-4c c e l l s d i e d and C-4s c e l l s  predominated,  p o s s i b l y because o f t h e i r b e t t e r a b i l i t y t o spread on, and adhere t o glass.  C-4c c e l l s were l o s t  i n the c u l t u r e that e v e n t u a l l y  became  l i n e C-4s, but some C-4c c e l l s p e r s i s t e d i n l i n e C-4c and became t h e predominant c e l l The  population  during  the second year i n c u l t u r e .  C-4c c e l l s are c y t o l o g i c a l l y and h i s t o l o g i c a l l y more m a l i g n a n t ,  s t r a t i f y more e x t e n s i v e l y ' i n c u l t u r e , grow more e x t e n s i v e l y  i n hamster  cheek pouches, do not o r i e n t on p h y s i o l o g i c a l s u b s t r a t a , have a h i g h e r rate of aerobic  and a n a e r o b i c g l y c o l y s i s and an impaired  a b i l i t y to  m e t a b o l i z e l a c t i c a c i d : they thus show s e v e r a l c h a r a c t e r i s t i c s which i n d i c a t e t h a t t h e y are more a n a p l a s t i c than C-4s c e l l s . a l s o appear- more c o h e s i v e  and l e s s deformable and would t h e r e f o r e be  expected t o be l e s s r e s p o n s i v e morphology.  C-4c c e l l s  t o environmental  i n f l u e n c e s on t h e i r  13  M a t e r i a l s and Methods Tissue  Culture  C e l l s f o r the p r e s e n t lishing  study were o b t a i n e d  by p e r i o d i c a l l y  re-estab-  i n c u l t u r e samples o f C-4c and C-4s c e l l s t h a t had been f r o z e n  and  preserved  i n liquid  and  glycolysis  n i t r o g e n a t t h e time o f the p r e v i o u s chromosome  s t u d i e s (see I n t r o d u c t i o n ) .  This p o l i c y provided  tively  uniform  mitted  c o r r e l a t i o n s o f the new r e s u l t s w i t h the known c y t o g e n e t i c and  metabolic The  c u l t u r e s throughout the d u r a t i o n o f the study,  rela-  and per-  c h a r a c t e r i s t i c s of the c e l l s .  c u l t u r e s were maintained  i n f l a t - b o t t o m c u l t u r e tubes  t u b e s ) on Waymouth medium'MB 752/1, supplemented by 10% f e t a l 3/o u m b i l i c a l c o r d e x t r a c t , 100 ug/ml o f s t r e p t o m y c i n of p e n i c i l l i n .  They were s u b c u l t u r e d  p e r s i o n i n O.125o c r y s t a l l i n e Initially, as f o l l o w s :  every  (Leighton calf  and 100 u n i t s / m l  1-2 weeks by p a r t i a l  trypsin/Moscona's  serum,  dis-  saline.  s e v e r a l m o d i f i c a t i o n s o f the growth medium were e v a l u a t e d  s e r i e s o f c u l t u r e tubes were d i v i d e d i n t o groups, and each  group o f tubes grown f o r up t o t h r e e months i n one o f t h e f o l l o w i n g media: 1.  Waymouth's medium 87%, f e t a l c o r d e x t r a c t 3?£  calf  serum 10%, u m b i l i c a l  2.  Waymouth !s medium 85%, f e t a l a c i d / s a l i n e 5%(0.1mg/ml)*  calf  serum 10%, h y a l u r o n i c  3.  Waymouth's medium • 85/b, f e t a l c a l f a c i d / s a l i n e 5, o(0.05mg/ml)"*  serum 10%, h y a l u r o n i c  q  4.  Waymouth's medium 85%, f e t a l c a l f a c i d / s a l i n e 5;b(0.025mg/ml ) *  serum 10}£, h y a l u r o n i c  5.  Waymouth's medium 90%, f e t a l  serum 10%,  6.  Eagle's  * The  final  calf  b a s a l medium 90?o, f e t a l  concentration  calf  serum 10%,,  i n medium  c u l t u r e s were observed f o r the i n i t i a l  pattern of c e l l  attachment,  14 f o r the growth rate and f o r the morphology of c e l l s and c o l o n i e s . I t was found that omission of u m b i l i c a l cord e x t r a c t had l i t t l e e f f e c t on the attachment of C-4s c e l l s , but d e f i n i t e l y retarded attachment and spreading of C-4c c e l l s .  This e f f e c t of cord e x t r a c t could be simulated  to a degree with h i g h l y polymerized hyaluronic a c i d , i n a l l the concentrations tested.  Once the c e l l s were attached, growth r a t e s and c u l t u r e  morphology were s i m i l a r with a l l s i x media.  For the sake of consistency  with previous experiments, medium number 1 was used throughout t h i s study. Culture conditions were modified as required by s p e c i a l problems under i n v e s t i g a t i o n : f o r the histochemical a n a l y s i s of i n s i t u c e l l s , c u l t u r e s were grown on glass c o v e r s l i p s , and f o r the u l t r a s t r u c t u r a l a n a l y s i s of i n s i t u c e l l s they were grown on a polyester p l a s t i c sheeting by the trade name of "Melinex 0" ( F i r k e t , 1966). were the same on glass and on p l a s t i c sheeting.  Growth c h a r a c t e r i s t i c s Suspension c u l t u r e s  were produced by maintaining c e l l s f o r several days i n polyethylene p e t r i dishes or i n s i l i c o n i z e d c u l t u r e tubes. of l i q u i d  To compare the e f f e c t s  and semisolid growth media oh c u l t u r e morphology, medium was  rendered semisolid by the a d d i t i o n of p u r i f i e d agar ('"Difco" c e r t i f i e d ) , as f o l l o w s :  a) Cultures grown on p l a s t i c sheeting to-the,.early l o g a r i t h -  mic growth phase were covered with a 2-3 mm layer of medium containing 0.33/0  agar, and then grown f o r approximately  another week,  b)  Trypsin-  ized c e l l s were suspended i n medium containing 0.33% agar, and plated on pre-established layers of medium containing 0.5% agar (Paul, 1965). The v i a b i l i t y of c e l l s shed i n t o the growth medium and t h e i r a b i l i t y to form colonies when r e c u l t u r e d were compared between the c e l l l i n e s . C e l l v i a b i l i t y was determined on groups of 12-15 c u l t u r e s f o r each growth phase of the two c e l l l i n e s .  Supernatant medium was removed from cultures  1 5  a t 48-hour i n t e r v a l s , and the t o t a l number of c e l l s , as w e l l as t h e proportions  of l i v e and dead c e l l s i n the medium, were determined  hemocytometer c o u n t s . a t room t e m p e r a t u r e was  E x c l u s i o n of 0.2%  Trypan blue  f o r 5-10  •  by  minutes  used as the i n d i c a t i o n o f c e l l v i a b i l i t y .  To  a s c e r t a i n whether c e l l s t h a t were v i a b l e were a l s o a b l e t o form c o l o n i e s , s u p e r n a t a n t c e l l s of a d d i t i o n a l c u l t u r e s were p o o l e d , a d j u s t e d  to  60,000-100,000 l i v e c e l l s / m l , suspended i n p a r t i a l l y c o n d i t i o n e d and  p l a t e d on a) g l a s s and b) c o l l a g e n g e l (Ehrmann and Gey,  The  number of c o l o n i e s per c u l t u r e was  counted t h r e e and  medium,  1956).  s i x days a f t e r  plating. The  a b i l i t y o f c e l l s t o a t t a c h when superimposed upon s o l i d  s h e e t s of the same c e l l type was  examined.  cell  C e l l s h e e t s were b r o k e n up  by p i p e t t i n g i n t o s m a l l clumps and t h e s e were suspended i n f r e s h medium o v e r c o n f l u e n t s h e e t s of the same c e l l l i n e . examined o v e r 3-day p e r i o d s  f o r attachment and  c e l l s into p r e - e x i s t i n g c e l l sheets. w i t h N i l e blue  The  c u l t u r e s were t h e n  i n c o r p o r a t i o n of the added  Attempts t o l a b e l the added c e l l s  s u l f a t e or carbon p a r t i c l e s were u n s u c c e s s f u l  t o x i c i t y of the s t a i n and the l a c k of uptake of carbon by the To  cells.  i n v e s t i g a t e the p o s s i b l e r o l e s t h a t e x t r a c e l l u l a r p r o t e i n s , b i -  v a l e n t c a t i o n s and of the c e l l s h e e t s ,  a c i d m u c o p r o t e i n s may  play i n maintaining  the  integrity  c u l t u r e s were t r e a t e d w i t h c r y s t a l l i n e t r y p s i n ,  b a l a n c e d s a l i n e w i t h and w i t h o u t C a was  because of  + +  , i<>g , and r u t h e n i u m r e d . ++  Trypsin  chosen because of i t s p r e d o m i n a n t l y s e l e c t i v e a c t i o n on e x t r a c e l l u l a r  proteins  ( P a u l , 1965), and  a l s o because of i t s a b i l i t y t o unmask neg-  a t i v e charges on c e l l s u r f a c e s  ( G a s i c et a l . , 1965).  Ruthenium r e d  was  used because of i t s c a p a c i t y t o b i n d t o m u c o p r o t e i n  and  t o be v i s i b l e w i t h both l i g h t m i c r o s c o p y and  a c i d groups  e l e c t r o n microscopy  16 (Gustavson and P i h l , were 1.  1967).  To t h i s end, the f o l l o w i n g experiments '/  performed:  .Growth medium i n c u l t u r e s was r e p l a c e d by: a.  Balanced s a l i n e  ++  (Hanks,  1949)  ++  b.  Ca  , Mg  free saline  c.  0.12% t r y p s i n / b a l a n c e d  d.  0.12% t r y p s i n / C a " , Mg " f r e e  The  c u l t u r e s were i n c u b a t e d and photographed  44  (Moscona, 1952) saline 44  saline. a t i n t e r v a l s over 2  days. 2.  C u l t u r e s grov/n on g l a s s c o v e r s l i p s were t r e a t e d w i t h the same f o u r s o l u t i o n s f o r 15 minutes  a t room temperature, w i t h and w i t h o u t sub-  sequent a d d i t i o n of ruthenium r e d t o a f i n a l  c o n c e n t r a t i o n of 0.2%.  A f t e r exposure t o ruthenium r e d f o r 3 minutes, a l l c o v e r s l i p s were placed  i n Carney's  fixative.  Those t r e a t e d w i t h ruthenium r e d were  s t a i n e d ' w i t h methyl green, and the o t h e r s with h e m a t o x y l i n and e o s i n . 3.  C u l t u r e s _in s_itu, as w e l l as p e l l e t s of t r y p s i n i z e d s t a i n e d with c o l l o i d a l Carnoy's  4.  iron  cells,  were  (Mowry, 1963), a f t e r b e i n g fixed, i n  f i x a t i v e or i n g l u t a r a l d e h y d e .  C u l t u r e s grown on g l a s s c o v e r s l i p s were t r e a t e d as f o l l o w s : a.  0.25% t r y p s i n / b a l a n c e d s a l i n e f o r 2-3 minutes, f o l l o w e d by f i x a t i o n i n Carnoy's and s t a i n i n g w i t h c o l l o i d a l i r o n .  b.  0,25% t r y p s i n / b a l a n c e d s a l i n e f o r 5-6 minutes, f o l l o w e d •by f i x a t i o n i n Carnoy's and s t a i n i n g w i t h c o l l o i d a l i r o n ,  c.  F i x a t i o n i n Carnoy's f o l l o w e d by t r y p s i n as above f o r 2, 4, and 6 minutes, then s t a i n e d with c o l l o i d a l i r o n , .  d.  F i x a t i o n i n Carnoy's, f o l l o w e d by balanced s a l i n e f o r 2, 4,'and 6 minutes, then s t a i n e d with c o l l o i d a l i r o n .  Primary c u l t u r e s of normal c e r v i c a l benign c o n t r o l ,  s i n c e no c e l l  e p i t h e l i u m were grov/n as the  l i n e s of normal  cervical epithelium  exist.  17  T i s s u e s were o b t a i n e d by punch b i o p s y from t h e area of the squamocolumnar j u n c t i o n of h e a l t h y c e r v i c e s .  The specimens were washed i n  c u l t u r e medium, g r o s s c o n n e c t i v e t i s s u e was removed, and the r e m a i n i n g t i s s u e minced w i t h f i n e s c i s s o r s . suspended for  The s u s p e n s i o n was  centrifuged, r e -  i n f r e s h medium and c u l t u r e d on p l a s t i c s h e e t i n g o r on g l a s s  p e r i o d s up t o t h r e e weeks.  The c u l t u r e s were f i x e d f o r e l e c t r o n  m i c r o s c o p y when e p i t h e l i a l s h e e t s up t o s e v e r a l m i l i m e t e r s i n d i a m e t e r had grown from t i s s u e f r a g m e n t s .  In a d d i t i o n , a number of c o l o n i e s were  a l s o f i x e d i n which t h e t i s s u e fragments had become d e t a c h e d .  These c u l -  t u r e s had assumed a more d i s o r g a n i z e d morphology, t y p i c a l of squamous c e l l s growing i n the absence of e x p l a n t s ( A u e r s p e r g and Worth, Cell  Electrophoresis E l e c t r o p h o r e s i s of b o t h c e l l  of  1966).  Dr. P. S. V a s s a r , Department  l i n e s was performed i n the l a b o r a t o r y o f P a t h o l o g y , t o determine whether a  d i f f e r e n c e i n c e l l - s u r f a c e charge between the c e l l  l i n e s might be i n - .  v o l v e d i n the d i f f e r e n t degrees of s t r a t i f i c a t i o n e x h i b i t e d .  C e l l sheets  i n t h e l a t e l o g a r i t h m i c growth phase were removed from the g l a s s by r u b b e r p o l i c e m a n , washed t h r e e t i m e s i n 0.15  molar NaCl and broken up  by s y r i n g i n g t h r o u g h 24-gauge n e e d l e s , a procedure which produced adequate numbers of s i n g l e c e l l s . c e l l s o f each c e l l t r o p h o r e s i s was Ag/AgCl  Two measurements per c e l l on  10-22  l i n e were performed on two s e p a r a t e o c c a s i o n s .  Elec-  c a r r i e d out i n a c y l i n d r i c a l apparatus equipped w i t h  e l e c t r o d e s (Seaman and Heard,  1961).  H e t e r o t r a n s p l a n t a t i o n experiments Tumors of both C-4 S y r i a n hamsters  l i n e s were grown i n cheek pouches  of u n c o n d i t i o n e d  ( F o l e y e t a l _ . , 1962), i n order t o determine which of t h e  c h a r a c t e r i s t i c s of the c e l l s i n v i t r o were a l t e r e d or absent and thus a response t o the c u l t u r e environment.  F u r t h e r , a comparison was made  18 of the h i s t o c h e m i c a l and u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s of the'two c e l l l i n e s under t h e s e in v i v o c o n d i t i o n s . Hamsters, w e i g h i n g 40-60 grams, were a n e s t h e t i z e d w i t h t o n e a l Nembutal (0.05  mg/gm h a m s t e r ) .  and p i n n e d t o a b o a r d .  The  intraperi-  cheek pouches were e v e r t e d ,  C e l l s i n the l o g a r i t h m i c growth phase were  t r y p s i n i z e d , washed t w i c e i n medium, suspended i n f r e s h medium and i n j e c t e d i n t r a d e r m a l l y i n t o the pouches. approximately  l - 2 x 10^ c e l l s i n 0.1  Each i n j e c t i o n c o n s i s t e d of  ml of medium.  A f t e r two weeks, the  a n i m a l s were a g a i n a n e s t h e t i z e d and most tumors f i x e d and as d e s c r i b e d under h i s t o c h e m i c a l  and e l e c t r o n m i c r o s c o p i c  processed techniques.  Three tumors, of each l i n e were e x c i s e d , minced i n Waymouth's medium, gross  connective  remaining  t i s s u e and  c e l l s u s p e n s i o n was  c u l a t e d i n t o f r e s h hamsters. had  pouch e p i t h e l i u m were removed, and concentrated  the  by c e n t r i f u g a t i o n and  A f t e r two weeks, these t r a n s p l a n t e d  reinoccells  a g a i n formed tumors and t h e s e were a l s o f i x e d and p r e p a r e d f o r e l e c -  t r o n microscopy. In a d d i t i o n , an attempt was  made t o grow C-4  tumors as p e r i t o n e a l ,  implants  i n c o n d i t i o n e d h a m s t e r s , f o r comparison w i t h , the cheek pouch  tumors.  T h i s was  w i t h and w i t h o u t  done as the r e c i p r o c a l experiment t o growing c e l l s agar i n c u l t u r e , i n an e f f o r t t o d e f i n e the e f f e c t s  of a c a v i t y , or l i q u i d , environment, on the h i s t o g e n e s i s of the tumors i n v i v o and 0.3  ijn v i t r o .  C e l l s and  ml of medium, c o n t a i n i n g s e v e r a l m i l l i o n c e l l s , was  the p e r i t o n e a l c a v i t i e s . ing,  animals were prepared as above,  injected into  As c o n t r o l s f o r any e f f e c t s of the c o n d i t i o n -  c e l l s were a l s o i n j e c t e d i n t o cheek pouches.  conditioned with cortisone acetate administered  and  simultaneously  The  hamsters were  i n subcutaneous doses of 2-3  mg,  w i t h the i m p l a n t a t i o n s , and t w i c e weekly  t h e r e a f t e r ( F o l e y et a l . . 1962).  The  a n i m a l s were a g a i n  anesthetized  19 a f t e r two weeks t o c o l l e c t tumors from cheek pouches and abdominal cavities. Histochemistry The  and H i s t o l o g y  f o l l o w i n g f i x a t i v e s were e v a l u a t e d  f o r use on c e l l  as t o t h e i r e f f e c t on subsequent demonstration and  extracellular  substances:  of c e l l  Carnoy's f i x a t i v e ,  s a l i n e , g l u t a r a l d e h y d e , paraformaldehyde v a p o r s ,  monolayers  surface materials  formol  a l c o h o l , formol  methyl a l c o h o l ,  c e t y l p y r i d i n i u m c h l o r i d e and c e t y l p y r i d i n i u r n bromide.  acetone,  Coverslips fixed  by t h e above m a t e r i a l s were s t a i n e d i n the f o l l o w i n g manner:  w i t h hema-  t o x y l i n and e o s i n or with t o l u i d i n e b l u e a t pH 4.5 f o r g e n e r a l morphology; with  a l c i a n blue/PAS, a l c i a n b l u e / n e u t r a l r e d , c o l l o i d a l  or t o l u i d i n e blue a t pH 4.5 f o r c e l l  iron/hematoxylin  s u r f a c e m a t e r i a l s ( C u l l i n g 1963,  Pearse 1961). Carnoy's, formol  a l c o h o l , methyl a l c o h o l and acetone gave  s i m i l a r r e s u l t s ; t h e r e was good c e l l u l a r d e t a i l but  some n o n s p e c i f i c s t a i n i n g with  fairly  and P A S - s p e c i f i c i t y , A l l three  alcian blue.  aldehyde  f i x a t i v e s augmented s t a i n i n g of e x t r a c e l l u l a r a c i d mucosubstences, but c e l l u l a r d e t a i l was d i m i n i s h e d . aldehyde-vapor f i x a t i o n . much o f which was p r o b a b l y  T h i s was most e v i d e n t a f t e r  O c c a s i o n a l l y t h e r e was widespread  paraformPAS-staining,  not s p e c i f i c , and t h e r e f o r e , aldehyde  fixatives  were not used t o study the d i s t r i b u t i o n o f P A S - p o s i t i v e m a t e r i a l . Cetylpyridinium  c h l o r i d e was u n s a t i s f a c t o r y because i t removed  from the g l a s s ; c e t y l p y r i d i n i u m bromide p r e s e r v e d i a l s w e l l , but i n t r o d u c e d to  cells  e x t r a c e l l u l a r mater-  i n t e n s e n o n s p e c i f i c s t a i n i n g with  a l c i a n blue,  the e x c l u s i o n of any PAS s t a i n i n g . F o l l o w i n g t h i s e v a l u a t i o n , Carnoy's was used when f i x a t i o n by pro-  t e i n d e n a t u r a t i o n was r e q u i r e d , and one o f the aldehydes when by c r o s s - l i n k i n g of p r o t e i n s was p r e f e r r a b l e .  fixation  T h i s d i s t i n c t i o n was  20 d e s i r a b l e i n the d e m o n s t r a t i o n of a c i d mucosubstances: t i v e s b i n d t o -NH2  aldehyde  fixa-  groups o f b a s i c p r o t e i n s and t h e r e b y unmask r e a c t i v e  groups on a c i d mucoproteins, w h i l e Carnoy's does not (Pearse,  1961).  Therefore,  material  s t a i n i n g f o r a c i d mucosubstances i n C a r n o y ' s - f i x e d  might be expected t o demonstrate most r e a d i l y those a c i d groups t h a t were not masked by p r o t e i n s , w h i l e i n a l d e h y d e - f i x e d staining in  should  material,  such  demonstrate a d d i t i o n a l a c i d groups t h a t were masked \  vivo. Some of the c u l t u r e s grown i n agar, as w e l l as some o f the tumors  grown i n hamsters, were f i x e d i n f o r m o l - s a l i n e ded  i n wax and the s e c t i o n s  addition, sections  or glutaraldehyde,  s t a i n e d with h e m a t o x y l i n and e o s i n .  of hamster tumors were s t a i n e d  embedIn  f o r collagen with  H e i d e n h a i n ' s Azan, Weigert-Van G i e s o n ( C u l l i n g , 1963) and Chromotrope 2R (Liisberg,  1962) and f o r mucoproteins o r mucopolysaccharides w i t h a l c i a n  blue/PAS and c o l l o i d a l  iron/hernatoxylin.  Salivary diastase  pretreat-  ment was used f o r the i d e n t i f i c a t i o n of g l y c o g e n . M a t e r i a l prepared f o r e l e c t r o n microscopy was a l s o examined h i s t o chemically.  Plastic  s e c t i o n s , 0.5-1.0 u t h i c k , of glutaraldehyde/csmium  f i x e d , Epon 812- embedded ultramicrotome.  t i s s u e s , were cut on an MT-2  The s e c t i o n s were t r a n s f e r r e d t o g l a s s  f i n e wire loop and h e a t - f i x e d the  Porter-Blum  t o the s l i d e s .  s l i d e s with a  For g e n e r a l  morphology,  s e c t i o n s were s t a i n e d with a drop o f 1% t o l u i d i n b l u e / 1 % borax a t  pH 11.0 (Pease, 1964). E l e c t r o n Microscopy 1.  General u l t r a s t r u c t u r e of the c e l l studied  i n d i f f e r e n t growth phases:  a.  Early logarithmic  b.  Late l o g a r i t h m i c  growth phase. growth phase.  lines.  Both c e l l  l i n e s were  21 c.  Stationary,  h e a v i l y grown c u l t u r e s .  d.  A p p r o x i m a t e l y one month o l d , d e g e n e r a t i n g  A l s o , the f o l l o w i n g c u l t u r e c o n d i t i o n s a.  Growth on p l a s t i c  sheeting,  cultures.  were compared:  f i x e d and embedded  i_n s i t u .  b. . Growth on g l a s s , detached by rubber policeman, t h e n f i x e d , l ) w i t h i n one minute, and 2) a f t e r one h o u r . c.  Suspension  cultures.  d.  Growth i n agar, as monolayers and i n s u s p e n s i o n .  In s i t u c u l t u r e s were washed g e n t l y c o l d 5?o g l u t a r a l d e h y d e / . M i l l o n i g ' s for  i n Waymouth's medium, f i x e d i n  buffer  (i-.'lillonig,  1-3 hours, washed i n b u f f e r 2-3 times and p o s t - f i x e d  OsO^/iv'.illonig's b u f f e r  f o r 15-30 minutes.  p r o p y l e n e oxide and embedded  After polymerization, in  situ  i n c o l d 1%  Then, the p l a s t i c  w i t h the c u l t u r e s were washed i n b u f f e r , dehydrated and  1962; Pease, 1964)  i n graded e t h a n o l s  i n 3-4 mm t h i c k l a y e r s of Epon 812.  the p l a s t i c was p u l l e d o f f , l e a v i n g the c e l l s  ( F i r k e t , 1966).  A p p r o p r i a t e areas of the embedded c u l t u r e s were,  s e l e c t e d by l i g h t microscopy, marked, photographed, e x c i s e d on b l o c k s  for sectioning.  centrifuged  sheets  Detached c e l l s  and c e l l  and mounted  suspensions were  i n t o p e l l e t s and then p r o c e s s e d as above.  Thin  sections  were cut w i t h g l a s s knives on an .MT-2 Porter-Blum microtome, mounted on  copper g r i d s and s t a i n e d with u r a n y l  citrate  (Venable and C o g g e s h a l l ,  1965).  acetate  (Watson, 1958) and lead  Thick sections  for light  micro-  scopy were u s u a l l y cut from a d j a c e n t areas i n the b l o c k s . 2.  A c i d mucoproteins.  C e l l s grown on p l a s t i c  s i o n , were s t a i n e d with c o l l o i d a l  sheeting,  i r o n (Wetzel e t a l . , 1966).  i r o n was prepared by the method o f R i n e h a r t and Abul-Haj t u r e s were f i x e d as d e s c r i b e d  above, s t a i n e d  a c e t i c a c i d at pH 2.0-2.2, r i n s e d and  embedded as f o r general  overnight  or i n suspenColloidal  (1951).  Cul-  in colloidal  iron/2  i n 2D% a c e t i c a c i d and then dehydrated  ultrastructure.  To determine whether l a c k  . of s t a i n i n g i n i n t e r c e l l u l a r s t a i n , or due  spaces was  due  2  2  t o poor p e n e t r a t i o n by  t o the absence of r e a c t i v e groups, some c e l l  fixed, carefully l i f t e d  o f f the growth s u r f a c e t o permit  sheets  then s t a i n e d w i t h  sections t r e a t e d with  i r o n were not p o s t s t a i n e d w i t h  acetate 3» Co.)  or l e a d  colloidal  iron.  Cadmium-free f e r r i t i n  solution, containing  added t o c u l t u r e s i n the incubated  ( N u t r i t i o n a l Biochemicals  at 37°C.  late  10%  Ferritin  l o g a r i t h m i c growth phase and  A f t e r 15 minutes, and  as f o r g e n e r a l  a f t e r 1, 3 and  the  was  cultures  7 hours, . embedded  u l t r a s t r u c t u r e . T h i n s e c t i o n s were examined w i t h o u t  f u r t h e r s t a i n i n g , or a f t e r l i g h t  s t a i n i n g with  Tumors orown i n hamsters.  Nembutal..  The  i n Waymouth's medium,  c u l t u r e s were r i n s e d r a p i d l y i n Waymouth's medium, f i x e d and  Everted  The  lead  citrate.  animals were a n e s t h e t i z e d  with  cheek pouches c o n t a i n i n g tumors, were immersed i n  c o l d 5/o g l u t a r a l d e h y d e / m ' i l l o n i g ' s tumors were e x c i s e d , buffer  uranyl  d i a l y s e d a g a i n s t Waymouth 's medium f o r 48 hours at 4°C.  resulting  4.  Thin  citrate.  F e r r i t i n uptake.  was  were  penetration  o f s t a i n from both s u r f a c e s and colloidal  the  fixed  b u f f e r f o r two  i n glutaraldehyde  i n which they were cut  i n t o small  minutes.  overnight,  Then the  rinsed in  pieces, postfixed i n cold  1%  O s O ^ / M i l I o n i a ' s b u f f e r f o r 20-30 minutes, r i n s e d , dehydrated, embedded, sectioned 5.  and  s t a i n e d as f o r g e n e r a l u l t r a s t r u c t u r e ,  Normal c e r v i c a l e p i t h e l i u m .  e p i t h e l i u m were grown on p l a s t i c ton tubes.  The  a l s o embedded as were C-4 with  sheeting  c u l t u r e s were f i x e d  regimen as c u l t u r e s of C-4  cells.  Primary c u l t u r e s of normal  nonpo.lymer.ized Epon 812,  and  or d i r e c t l y on g l a s s i n L e i g h -  in s i t u and Cultures  c u l t u r e s , but  cervical  the  dehydrated by the  on p l a s t i c Leighton  same  s h e e t i n g were  tubes were  filled  following polymerization, r a p i d l y  23 transferred  from 56°C. t o dry i c e or i c e water, to break the tubes and  release the blocks of Epon.  Sectioning and s t a i n i n g was the same as  for the general u l t r a s t r u c t u r e of the C-4 c e l l s .  24 Results  Morphologic v a r i a t i o n i n c u l t u r e under maintenance  Throughout the  course of the p r e s e n t  c h a r a c t e r i s t i c s of each c e l l  study, the  morphological  l i n e remained s u f f i c i e n t l y  maintenance c o n d i t i o n s t o permit easy i d e n t i f i c a t i o n . c o r r e l a t i o n s between experimental patterns  or c y t o l o g y  ly  all  a l t e r a t i o n s i n growth  from a 1 - 2  through the e a r l y and  day  gradual  lag-phase  immediate-  l o g a r i t h m i c growth phases,  l a s t i n g about a week, to a s t a t i o n a r y phase, which l a s t e d  other week or more. again  Thus, v a l i d  c u l t u r e s underwent a s e r i e s of  changes as they p r o g r e s s e d  following subculture,  together  v a r i a b l e s and  s t a b l e under  c o u l d be made.  During each passage, the consistent  conditions  subcultured.  During t h i s  i t was  g r e a t l y reduced.  another i n each growth phase as In l i n e C-4c,  the  time the m a j o r i t y  cells  were u s u a l l y  Some m i t o t i c a c t i v i t y p e r s i s t e d i n the  growth phases so t h a t  phase, but was  l a t t e r phase, the  not e l i m i n a t e d The  two  cell  cultures in  stationary  lines differed  from one .  follows:  lag-phase l a s t e d one  of c e l l  i n the  an-  to two  days, d u r i n g  clumps began t o a t t a c h and  spread  which  on the  glass.  D u r i n g the e a r l y l o g a r i t h m i c phase, small compact round c o l o n i e s  formed,  with  in  indistinct  t h e i r centers  intercellular  spaces and  ( F i g . l ) . In the  became c o n f l u e n t ,  and  possible s t r a t i f i c a t i o n  l a t e l o g a r i t h m i c phase, the  r i d g e s marked the  s i t e s of colony  suggesting  t h a t c e l l s of d i f f e r e n t  (Fig.  During the  s t a t i o n a r y phase, the  throughout  (Fig. 3).  2).  stratified  c o l o n i e s had  p i l e d up  colonies  fusion i n these  regions  c u l t u r e s became h e a v i l y  25  Plate I M o r p h o l o g i c v a r i a t i o n i n c u l t u r e under maintenance conditions (Figures 1-11) ( V i t a l microscopy, standard o p t i c s ) F i g u r e IS  C-4c  c u l t u r e , e a r l y l o g a r i t h m i c growth phase. x34  F i g u r e 21  C-4c  c u l t u r e , l a t e l o g a r i t h m i c growth phase. x34  F i g u r e 31  C-4c c u l t u r e , s t a t i o n a r y growth phase. x34  Figure 4t  C-4c c u l t u r e , one month o l d . R e t r a c t i n g edge of c o l o n y (arrow) and new o u t g r o w i n g c e l l s h e e t ( s ) . x34  F i g u r e 5'.  C-4s c u l t u r e , e a r l y l o g a r i t h m i c growth phase. x34  Figure 6 j  C-4s  c u l t u r e , l a t e l o g a r i t h m i c growth phase. x34  F i g u r e 7: . C-4s c u l t u r e , s t a t i o n a r y growth phase w i t h c y s t s . x l 4 0 F i g u r e 31  C-4s c u l t u r e , s t a t i o n a r y growth phase w i t h ridges. xl40  cell  F i g u r e 91  C-4s c u l t u r e , one month o l d . C y s t s f i l l e d d e b r i s . x34  with  26 In l i n e C-4s, the lag-phase was s h o r t e r .  Subcultured  cells at-  t a c h e d and spread on g l a s s f a s t e r , so t h a t small c o l o n i e s , c o n s i s t i n g of a few c e l l s , were o f t e n p r e s e n t the  early logarithmic  on the day o f s u b c u l t u r e .  During  phase, i r r e g u l a r l y shaped c o l o n i e s which appeared  mainly as monolayers, formed  (Fig. 5).  The d i r e c t i o n o f s p r e a d i n g  on  g l a s s o f s i n g l e c e l l s and o f c o l o n i e s seemed much more i n f l u e n c e d by contact  guidance  logarithmic  ('Weiss, 1958) t h a n i n C-4c c u l t u r e s .  phase, the C-4s c o l o n i e s had u s u a l l y fused  In t h e l a t e (Fig. 6).  c o n t r a s t t o C-4c c u l t u r e s , when two C-4s c o l o n i e s met, the c e l l s advancing edges tended t o o r i e n t p a r a l l e l than s t r a t i f y in  but the c e l l s  sharp  phases and  intercellular  U s u a l l y , but not always, c y s t s , i . e . groups o f adjacent  t h a t had l i f t e d stage  appeared as monolayers  seemed s m a l l e r than i n e a r l i e r  formed a r e g u l a r , pavement-like p a t t e r n with v e r y borders.  o f f t h e g l a s s w h i l e remaining c o h e s i v e ,  (Fig. 7).  at t h e  t o the l i n e o f f u s i o n r a t h e r  ( F i g s . 10, 1 1 ) . The c u l t u r e s s t i l l  most a r e a s ,  In  cells  appeared a t t h i s  The number o f . c y s t s i n these c u l t u r e s v a r i e d i n the course  of t h e study from v e r y  few or none t o more than one hundred per c u l t u r e .  However, c y s t s were always l i m i t e d t o l i n e C-4s and were never seen i n l i n e C-4c. did  C-4s d i d s t r a t i f y  C-4c c e l l s  increase  (Fig. 8).  somewhat, but t o much l e s s e r degree than  In the s t a t i o n a r y phase, t h e r e was o f t e n an  i n the number o f c y s t s , and small  peared t o extend towards the medium at  this  same stage,  layered  extensive  clumps and r i d g e s o f c e l l s ap-  (Fig. 8).  However, i n c o n t r a s t t o C-4c  areas within- t h e c u l t u r e s s t i l l  and i n such areas c e l l s  remained mono-  f r e q u e n t l y seemed t o separate from one  another. If c e l l s were not s u b c u l t u r e d tained  longer,  apparent.  i n the s t a t i o n a r y phase, but Were main-  a d d i t i o n a l d i f f e r e n c e s between t h e c e l l  l i n e s became  C-4c c u l t u r e s became i n c r e a s i n g l y more, s t r a t i f i e d and, about  27 a month a f t e r s u b c u l t u r e ,  the c e l l  sheets began t o p u l l  away from the  edges o f t h e c u l t u r e tubes, a p r o c e s s a s s o c i a t e d with the f o r m a t i o n concentric as  f o l d s which gave t h e impression  a c o n t i n u o u s t i s s u e under t e n s i o n .  gan  t h a t t h e c e l l s were behaving  Within  a day, new monolayers be-  t o grow from the r o l l e d - u p edges o f the o l d r e t r a c t i n g c e l l  onto the newly f r e e d spaces on the g l a s s  (Fig. 4 ) .  sheets-  I f C-4s c u l t u r e s  were m a i n t a i n e d beyond the s t a t i o n a r y phase, the c y s t s g r a d u a l l y f i l l e d w i t h d e b r i s , and l a t e r with c e l l s , stratified  became  as t h e c u l t u r e s became more  ( F i g . 9 ) . These c u l t u r e s tended t o degenerate i n s i t u  t h a n p u l l o f f t h e g l a s s and i f t h e c e l l  of  sheets were t o r n  rather  artificially,  t h e y r e t r a c t e d somewhat as i f under t e n s i o n , though much l e s s t h a n had C-4ccultures.  Histochemistry  Cultures  of i n s i t u  cultures  grown on c o v e r s l i p s and f i x e d i_n s i t u were s t a i n e d t o demon-  s t r a t e general  morphology, c y t o l o g i c d e t a i l , v i c i n a l  g l y c o l groups and  c e l l - s u r f a c e a c i d mucosubstances. Some o f the c h a r a c t e r i s t i c c y t o l o g i c d i f f e r e n c e s between t h e c e l l l i n e s are i l l u s t r a t e d late logarithmic  i n f i g u r e s 12 and 13, which show c u l t u r e s ' i n the  growth phase s t a i n e d with h e m a t o x y l i n and e o s i n .  C - 4 c c e l l s overlapped e x t e n s i v e l y , cell  showed no o r i e n t a t i o n , had i n d i s t i n c t  b o r d e r s and round, v e s i c u l a r n u c l e i with clumped c h r o m a t i n .  c e l l s o f t e n formed a p a v e m e n t - l i k e ' p a t t e r n with l i t t l e distinct  cell  borders:  s t r e s s , and i n o t h e r s ,  C-4s  c e l l - o v e r l a p and  i n some areas they were o r i e n t e d along  l i n e s of  they formed c y s t s .  smaller  The c e l l s appeared  than C-4c c e l l s , mainly because they appeared t o have l e s s c y t o p l a s m . N u c l e i were o f v a r i a b l e shapes, and chromatin was more homogeneous than  28  Plate I I F i g u r e 10?  C-4c c u l t u r e , l a t e l o g a r i t h m i c growth phase, j u n c t i o n between two c o l o n i e s . x l 4 0  F i g u r e 11:  .C-4s c u l t u r e , l a t e l o g a r i t h m i c growth phase, j u n c t i o n between two c o l o n i e s . x l 4 0  F i g u r e 12!  C u l t u r e C-4c, l a t e l o g a r i t h m i c growth phase. f i x a t i o n , . h e m a t o x y l i n a n d e o s i n . x660  Carnoy's  F i g u r e 13t  C u l t u r e C-4s, l a t e l o g a r i t h m i c growth phase. f i x a t i o n , h e m a t o x y l i n and e o s i n , x660  Carnoy's  i n C-4c  cells.  PAS-positive and  m a t e r i a l was  i n clumps of c e l l s .  cytoplasmic  granules.  present only i n h i g h l y s t r a t i f i e d  T h i s m a t e r i a l appeared t o c o n s i s t of The.PAS-positive material diminished  areas of t r a n s i t i o n t o t h i n n e r c e l l  l a y e r s , and  absent, from young, monolayered c o l o n i e s . was  observed and  blue p o s i t i v e  surface m a t e r i a l s .  and  in  diastase  staining labile.  l i n e s were observed i n the  There was  colloidal-iron positive  sharply  appeared c o m p l e t e l y  none of the P A S - r e a c t i v e m a t e r i a l was  staining for c e l l  intra-  Mo e x t r a c e l l u l a r PAS  No d e f i n i t e d i f f e r e n c e s between the c e l l  areas  a l a y e r of a l c i a n -  material  found  only  on p a r t s of c e l l s t h a t were not adherent t o g l a s s or t o o t h e r c e l l s . T h i s s t a i n i n g was  absent at s i t e s c f attachment t o the g l a s s , i n i n t e r -  c e l l u l a r , spaces and aldehyde-fixed  exposed t o the medium.  w i t h a l c i a n - b l u e and  i n a d d i t i o n , t h e r e was  f a i n t but d e f i n i t e s t a i n i n g  t o l u i d i n e b l u e of i n t e r c e l l u l a r spaces and  exposed t o the medium.  i n t e n s i v e i n C-4c  This  s t a i n i n g was  Areas r e p r e s e n t a t i v e  somewhat more  cultures  of s p e c i f i c growth phases, or o t h e r areas of  i n t e r e s t , were s e l e c t e d f o r e l e c t r o n m i c r o s c o p y by e x a m i n a t i o n of i n t a c t , Eoon-embedded c u l t u r e s .  was  of  cultures.  i n f r a s t r u c t u r e of i n s i t u  ing,  In  c u l t u r e s , the r e s u l t s were s i m i l a r t o t h o s e i n Carnoy's  f i x e d c u l t u r e s , but  the s u r f a c e s  on the s u r f a c e s  the e x a c t p o s i t i o n and  light-microscopic  Preceeding t h i n s e c t i o n -  o r i e n t a t i o n of the c e l l s w i t h i n these areas  f u r t h e r determined by l i g h t - m i c r o s c o p i c e x a m i n a t i o n of.0.5-1.0 u  30  "  Plate I I I  G e n e r a l o r i e n t a t i o n o f t h e c e l l s and o f i n t e r c e l l u l a r soaces ( F i o u r e s 14-23) ( H i s t o l o g y - Epon 812 embedding, a l k a l i n e t o l u i d i n e b l u e ) .  Figure  14'  C-4c c u l t u r e , e a r l y l o g a r i t h m i c growth phase. x470  Figure  lot  C-4c c u l t u r e , l a t e l o g a r i t h m i c growth phase. x470  Figure  16?  C-4c c u l t u r e , s t a t i o n a r y growth phase. x470  Figure  171  C-4c c u l t u r e , one month o l d c u l t u r e , r e t r a c t i n g edge of c o l o n y . x470  Figure  18:  C-4s c u l t u r e , e a r l y l o g a r i t h m i c growth phase. x470  Figures  19 and 20J  C-4s c u l t u r e s , l a t e l o g a r i t h m i c growth phase. x470  Figures  21 and 2 2 !  G-4s c u l t u r e s , s t a t i o n a r y growth phase w i t h f o r m a t i o n . x470  Figure  23!  C-4s c u l t u r e , one month o l d c u l t u r e . x470  cyst  31 plastic sections.  A l l s e c t i o n s were cut p e r p e n d i c u l a r  t o the  substra-  tum. •General o r i e n t a t i o n of the c e l l s and  i n t e r c e l l u l a r spaces.  During  the e a r l y l o g a r i t h m i c growth phase, the o r i e n t a t i o n i n the two l i n e s was  similar:  both C-4c  and C-4s  ed p a r a l l e l t o the growth s u r f a c e .  c e l l s were f l a t , t h i n and  of s e v e r a l c e l l and  made c o n t a c t  layers.  The  orient-  A l t h o u g h l i v e c u l t u r e s at t h i s  appeared as monolayers w i t h l i g h t m i c r o s c o p y , i t was i n b o t h l i n e s o v e r l a p p e d , and  cell  t h a t C-4c  stage  found t h a t the  c u l t u r e s almost always  cell  consisted  c e l l s were s e p a r a t e d by h o r i z o n t a l spaces,  l a t e r a l l y by o v e r l a p p i n g  microvilli  ( F i g s . 14,  18,  24,  As the c u l t u r e s matured, the d i f f e r e n c e s between the c e l l l i n e s i n creased.  In the l a t e l o g a r i t h m i c - s t a t i o n a r y phase the C-4c  cultures  became i n c r e a s i n g l y s t r a t i f i e d , forming up t o t e n of more c e l l  layers-  between the p l a s t i c s h e e t i n g  formation,  of more c e l l  and  the growth medium.  With the  l a y e r s , those c e l l s t h a t were i n d i r e c t c o n t a c t w i t h  medium became e x c e e d i n g l y  f l a t and  the  t h i n , r e s e m b l i n g squamous c e l l s ,  w h i l e most deeper c e l l s assumed i r r e g u l a r o v o i d or round o u t l i n e s , but r e t a i n e d a main a x i s p a r a l l e l t o the s u b s t r a t u m ( F i g s . 15, 16, 26, S t r a t i f i c a t i o n i n C-4s t h e r e was  c u l t u r e s was  much l e s s p r o m i n e n t .  27).  Rather,  a tendency f o r c e l l s of t h i s l i n e t o assume r h o m b o i c a l or  columnar shapes as t h e y became crowded, so t h a t even i n the phase, much of any C-4s  c u l t u r e remained monolayered.  exposed t o the medium were not perpendicular  t o the  f l a t t e n e d , and  substratum.  The  Those c e l l s  t h e i r main a x i s was  c y s t s , noted i n C-4s  u s u a l l y appeared w i t h i n columnar m o n o l a y e r s .  substratum and  often  cultures,  They were formed by groups  of c o h e r e n t , f l a t , and h o r i z o n t a l l y o r i e n t e d c e l l s t h a t had s e p a r a t e d from the  stationary  become  resembled c e l l s seen i n the  early  32  P l a t e IV U l t r a s t r u c t u r e of in s i t u c u l t u r e s - L i n e (Uranyl  acetate/Lead c i t r a t e .  f i g u r e s , the s u p e r f i c i a l  Note:  C-4c  In a l l  s i d e s of t h e c o l o n i e s are on  t h e t o p , and t h e s i d e s near t h e s u b s t r a t u m are orv t h e bottom.)  F i g u r e 24:  E a r l y l o g a r i t h m i c growth phase. x32500  F i g u r e 25:  E a r l y l o g a r i t h m i c growth phase. x l 8 0 0 0  F i g u r e 26a and 26b:  S t a t i o n a r y growth phase, s u p e r f i c i a l c e l l l a y e r s ( f i g u r e 26a) and deep c e l l l a y e r s ( f i g u r e 2 6 b ) . x6000  F i g u r e 27.:  Late l o g a r i t h m i c growth phase, s u p e r f i c i a l  c e l l s . xl2000  F i g u r e 28:  O l d c u l t u r e , r e t r a c t i n g c e l l edge, s u p e r f i c i a l X14000  cells.  33  Plate V U l t r a s t r u c t u r e of i n s i t u c u l t u r e s - Line (Uranyl a c e t a t e / l e a d the  superficial  and the  citrate.  Note:  C-4s  In a l l f i g u r e s ,  s i d e s of the c o l o n i e s are on the t o p ,  s i d e s near the substratum are on the  bottom.)  F i g u r e 29:  Early logarithmic  F i g u r e 30:  Late l o g a r i t h m i c  growth phase. x6500  F i g u r e 31:  Late l o g a r i t h m i c  - s t a t i o n a r y growth phase. xl6000  F i g u r e 32:  Stationary  growth phase, near c y s t . xl3000  F i g u r e 33:  Stationary  growth phase, edge of c y s t . x8000  F i g u r e 34:  Old  growth phase. x20000  culture, superficial  c e l l s . xl0500  34 l o g a r i t h m i c phase ( F i g s . 18-22, 2 9 - 3 3 ) .  D e g e n e r a t i n g o r dead c e l l s  were seen i n s t r a t i f i e d areas much more f r e q u e n t l y i n C-4s t h a n i n C-4c  cultures  c u l t u r e s ( F i g s . 23, 3 4 ) .  In both c e l l  lines, intercellular  i d e d by m i c r o v i l l i and desmosomes. i n a l l s t a g e s of C-4c  spaces were e x t e n s i v e and s u b d i v -  In young c u l t u r e s of both l i n e s  and  c u l t u r e s t h e s e spaces were s e p a r a t e d from t h e  growth  medium by o v e r l a p p i n g c e l l edges whose u l t r a s t r u c t u r e i n c r o s s - s e c t i o n resembled t y p i c a l tight  m i c r o v i l l i ( F i g s . 27, 2 9 ) .  intercellular  i n the l a t e As C-4c  But t h e y were s e p a r a t e d by  j u n c t i o n s w i t h the c h a r a c t e r i s t i c s of t e r m i n a l b a r s  l o g a r i t h m i c - s t a t i o n a r y phases o f C-4s  c u l t u r e s became o l d e r and more s t r a t i f i e d ,  spaces extended  intercellular  i n a l l d i r e c t i o n s around the c e l l s , r e m a i n i n g f a i r l y  form i n d i a m e t e r except f o r deeper c e l l  uni-  l a y e r s where spaces became more,  complex and w i d e r , r e a c h i n g d i a m e t e r s of 2-3 u. was  c u l t u r e s ( F i g s . 33, 3 4 ) .  The g r e a t e r c o m p l e x i t y  due t o a n . i n c r e a s e i n the number and l e n g t h of m i c r o v i l l i w i t h d i s -  t a n c e from t h e growth medium, as w e l l as t o an i n c r e a s e d i r r e g u l a r i t y of t h e c e l l s u r f a c e s ( F i g s . 2 4 - 2 7 ) . intercellular  On the o t h e r hand, i n C-4s  cultures,  spaces became v e r t i c a l l y o r i e n t e d d u r i n g t h e l o g a r i t h m i c  growth phases ( F i g s . 2 9 - 3 2 ) .  Although the i n t e r c e l l u l a r  spaces i n t h e s e  phases were a l s o s u b d i v i d e d by desmosomes and m i c r o v i l l i , t h e y were l e s s complex, and l e s s e x t e n s i v e t h a t i n C-4c the s t a t i o n a r y phase i n C-4s  c u l t u r e s ( F i g s . 35, 3 6 ) .  c u l t u r e s , the i n t e r c e l l u l a r  spaces seemed  t o become i n c r e a s i n g l y expanded, f u r t h e r s e p a r a t i n g a d j a c e n t c e l l s sometimes e x c e e d i n g t h e c e l l s i n w i d t h . served i n t h i s c e l l expanding  spaces  ( F i g s . 20-22, 31-33).  and  as i f t h e c y s t s  l i n e were formed by the c o a l e s c e n c e of s e v e r a l  intercellular  c a t i o n i n C-4s  I t appeared  During  I n a r e a s of  c u l t u r e s , the c o m p l e x i t y and d i a m e t e r s of  ob-  such stratifi-  intercellular  spaces and the number of m i c r o v i l l i a g a i n i n c r e a s e d w i t h d i s t a n c e  35  from t h e medium, as i n C-4c c u l t u r e s . cell  lines  The i n t e r c e l l u l a r  spaces o f both  c o n t a i n e d v a r i o u s forms o f amorphous and membraneous e x t r a -  c e l l u l a r m a t e r i a l ( F i g s . 24, 36, 4 2 ) , but t h i s m a t e r i a l was i n g r e a t e r amounts i n l i n e C-4s.  Finally,  i n both c e l l  present  lines, a layer  o f amorphous m a t e r i a l was d e p o s i t i e d i n v a r i a b l e amounts between t h e c e l l s and t h e p l a s t i c  sheeting  ( F i g s . 29, 31, 4 7 ) .  C u l t u r e s t h a t were m a i n t a i n e d  beyond t h e s t a t i o n a r y phase f o r a  t o t a l o f about one month, showed a d d i t i o n a l changes.  In C-4c c u l t u r e s  a t t h i s s t a g e , s t r a t i f i c a t i o n ranged from t h r e e t o over t e n c e l l l a y e r s , w i t h c e l l degeneration p a r t s of the c e l l  i n some o f t h e more s t r a t i f i e d a r e a s .  sheets were not i n c o n t a c t w i t h the' p l a s t i c  but had l i f t e d away from i t .  sheeting,  In p a r t s t h a t were not r e t r a c t i n g from  t h e s u b s t r a t u m , s u r f a c e c e l l s were a g a i n v e r y t h i n , nected  Large  and were o f t e n con-  l a t e r a l l y by desmosomes r a t h e r t h a n by o v e r l a p p i n g m i c r o v i l l i .  In p a r t s o f t h e c u l t u r e s t h a t were r e t r a c t i n g , t h e s u r f a c e c e l l s were t h i c k e r and i n t e r c e l l u l a r j u n c t i o n s c o n s i s t e d o f e x t e n s i v e r e g i o n s of interdigitating  m i c r o v i l l i as w e l l as desmosomes.  c u l t u r e s , c e l l degeneration  In comparable C-4s  was much more p r o m i n e n t .  There were r a r e -  l y groups o f more t h a n two or t h r e e a d j a c e n t h e a l t h y c e l l s .  C y s t s had  become f i l l e d w i t h d e b r i s , and t h e s u b s t r a t u m w i t h i n them had become covered  with c e l l s .  In c o n t r a s t t o t h e C-4c c u l t u r e s , c e l l s i n these  c u l t u r e s remained a t t a c h e d t o t h e s u b s t r a t u m ( F i g s . 17, 23, 28, 3 4 ) .  Cell  surface c h a r a c t e r i s t i c s .  M i c r o v i l l i were t h e most  striking  f e a t u r e on t h e s u r f a c e s o f b o t h c e l l t y p e s , b u t were more numerous and l o n g e r i n l i n e C-4c. and  T h e i r w i d t h u s u a l l y ranged from 600 t o 800 A,  they sometimes reached l e n g t h s o f s e v e r a l u.  They extended perpen-  36  Plate VI  .  i n f r a s t r u c t u r e of i n s i t u c u l t u r e s - C e l l s u r f a c e c h a r a c t e r i s t i c s ( F i g u r e s 35-47) (Uranyl acetate/lead c i t r a t e )  F i g u r e 35:  T y p i c a l i n t e r c e l l u l a r space i n a C-4c c u l t u r e , w i t h d i g i t a t i n g m i c r o v i l l i . xlSOOO  inter-  F i g u r e 36:  T y p i c a l i n t e r c e l l u l a r space i n a C-4s c u l t u r e , w i t h m i c r o v i l l i and areas- o f p a r a l l e l c e l l - s u r f a c e a p p o s i t i o n . x l 8 5 0 0  F i g u r e 37:  Phagocytosis. . M i c r o v i l l i of t h e p h a g o c y t i s i n g c e l l o r i e n t p a r a l l e l t o the s o l i d s u r f a c e . x26000  F i g u r e 38:  A d v a n c i n g c e l l edge on p l a s t i c  F i g u r e 39.  M i c r o v i l l i ; c r o s s - s e c t i o n s showing f i l a m e n t c o r e s . X590O0  Figure 40.  M i c r o v i l l i ; c r o s s - s e c t i o n s showing m i c r o t u b u l e s and f i l a m e n t s . x57000  Figure 41.  T a n g e n t i a l s e c t i o n .through c e l l near s u r f a c e . t i o n s and p r o t r u d i n g m i c r o v i l l i . x25000  Figure 42.  T y p i c a l desmosome i n l i n e C-4c. x43000  Figure 43.  T y p i c a l desmosome i n l i n e C-4s. X42000  sheeting. xl7000  (arrows) Invagina-  37 d i c u l a r t o t h e c e l l s u r f a c e s , and on c o n t a c t w i t h s o l i d  structures  changed d i r e c t i o n and a l i g n e d p a r a l l e l t o them, always r e m a i n i n g sepa r a t e d by spaces o f about 100 A ( F i g s . 35, 36, 37, 3 8 ) . They were uniformly  d i s t r i b u t e d on t h e s u r f a c e o f n e a r - s p h e r i c a l c e l l s , but i n  o t h e r s t h e i r number and s i z e i n c r e a s e d cells.  fairly  i n the p e r i p h e r a l parts of the  In e a r l y growth p h a s e s , m i c r o v i l l i were r e l a t i v e l y few and s h o r t ,  and t h e y became l o n g e r and more d e n s e l y d i s t r i b u t e d as t h e c u l t u r e s aged, p a r t i c u l a r l y i n l i n e C-4c ( F i g s . 24-27, 2 9 - 3 2 ) . i n both c e l l  At a l l stages  l i n e s , m i c r o v i l l i were absent from p e r i n u c l e a r  surfaces adjacent to p l a s t i c sheeting,  s h o r t e r on s u r f a c e s  cellf a c i n g the  medium, and l o n g e r and more numerous i n i n t e r c e l l u l a r s p a c e s .  The  a d v a n c i n g edges of c e l l s on. t h e p l a s t i c as w e l l as t h e b a r r i e r s between medium and i n t e r c e l l u l a r spaces i n e a r l y c u l t u r e s o f b o t h l i n e s and i n all  s t a g e s o f C-4c c u l t u r e s demonstrated t h e same c r o s s - s e c t i o n a l u l t r a -  s t r u c t u r e as m i c r o v i l l i  ( F i g s . 27, 31, 3 8 ) . The s u r f a c e s o f c e l l s ,  p a r t i c u l a r l y i n t h e deep l a y e r s , were f r e q u e n t l y f u r t h e r i n c r e a s e d i n area by i n v a g i n a t i o n s ' i n t o which m i c r o v i l l i p r o t r u d e d  (Fig. 41).  mosomes were s c a t t e r e d i n C-4c c u l t u r e s , and u s u a l l y a s s o c i a t e d dense b u n d l e s of c y t o p l a s m i c  filaments  ( F i g . 42), while  Des-  with  i n C-4s c u l t u r e s ".  t h e y were somewhat more common but u s u a l l y not a s s o c i a t e d w i t h prominent f i l a m e n t bundles ( F i g . 4 3 ) .  I n C-4s c u l t u r e s i n t h e l a t e  logarithmic  phase and t h e r e a f t e r , i n t e r c e l l u l a r spaces between columnar c e l l s were c o n s i s t a n t l y s e p a r a t e d from t h e growth medium by t e r m i n a l b a r s ; t h e t h r e e components of these i n t e r c e l l u l a r j u n c t i o n s c o u l d u s u a l l y be i d e n tified  ( F i g s . 34, 44, 4 5 ) .  A cell  c o a t , e x t e r i o r t o the plasma membrane, o f t e n seemed t o con-  s i s t of s h o r t f i l a m e n t s , s e v e r a l hundred A l o n g , t h a t were o r i e n t e d perpendicular  t o the c e l l  surface.  T h i s coat was absent i n areas o f  38 c l o s e c o n t a c t w i t h o t h e r c e l l s or s u b s t r a t a  ( F i g . 3 7 ) , appeared  wide i n t e r c e l l u l a r spaces ( F i g . 4 6 ) , and was  most prominent on  along surfaces  f a c i n g the medium, i . e . i t became more prominent w i t h i n c r e a s i n g d i s t a n c e from o t h e r were s h o r t and villi  solid structures.  evenly  In e a r l y growth phases, the  filaments  d i s t r i b u t e d , but as the c u l t u r e s aged, and  micro-  became d e n s e r , the f i l a m e n t s tended t o l o c a l i z e on the t i p s  m i c r o v i l l i and  appeared somewhat l o n g e r  ( F i g s . 24, 3 4 ) .  of  There were no  o b v i o u s d i f f e r e n c e s between the c e l l l i n e s i n amounts or d i s t r i b u t i o n of the c e l l  coat.  P i n o c y t o t i c v e s i c l e s were f r e q u e n t cell  surfaces  on  f a c i n g the p l a s t i c s h e e t i n g , but r a r e o n - s u r f a c e s exposed  t o t h e medium.  P i n o c y t o t i c a c t i v i t y was  showing e x t e n s i v e m i c r o v i l l i Organelles.  i n i n t e r c e l l u l a r spaces and  Organelles  a x i s of the c e l l s .  ( F i g s . 27,  o f t e n associated with areas 31). -  were g e n e r a l l y o r i e n t e d p a r a l l e l t o the main  Thus, i n f l a t  c e l l s d u r i n g e a r l y growth phases,  t h e y were o r i e n t e d h o r i z o n t a l l y , w h i l e i n columnar c e l l s t h e y were o r i e n t ed v e r t i c a l l y .  They tended t o be c o n c e n t r a t e d  i n the p e r i n u c l e a r  area  r a t h e r t h a t the p e r i p h e r a l c y t o p l a s m , and were commonly absent from t h o s e r e g i o n s w i t h i n the c y t o p l a s m t h a t were near the growth medium. l i n e s , n u c l e i were o v o i d matured, n u c l e a r  i n the e a r l y growth phases, and  as  In both  cultures  o u t l i n e s became more i r r e g u l a r and o f t e n developed  deep i n d e n t a t i o n s .  This i r r e g u l a r i t y  l y p r e s e n t i n columnar C-4s  cells.  was  most marked, and most c o n s i s t e n t -  Very l a r g e n u c l e o l i and  t i n were common, the l a t t e r p a r t i c u l a r l y i n o l d e r c u l t u r e s .  clumped chromaRibosomes  were p r e s e n t i n l a r g e numbers d u r i n g the l o g a r i t h m i c growth phases, mostl y as polysomes, w h i l e  i n the s t a t i o n a r y phase, t h e y were more o f t e n s i n g l e .  There were s c a t t e r e d s t r a n d s  of  rough endoplasmic r e t i c u l u m , and  a moderate  39 number o f w e l l formed m i t o c h o n d r i a i n both l i n e s ; however, m i t o c h o n d r i a i n C-4s  c e l l s were o f t e n l a r g e r , and had b e t t e r formed c r i s t a e .  Golgi  complexes were p r e s e n t i n both c e l l l i n e s , but were more prominent l i n e C-4s,  in  e s p e c i a l l y i n the s t a t i o n a r y phase when t h e y were most always .  found on t h e l a t e r a l or b a s a l s i d e s of columnar c e l l s .  Lysosomes appeared  i n o l d e r c u l t u r e s i n which p h a g o c y t o s i s , a s s o c i a t e d w i t h c e l l a l s o seemed t o t a k e p l a c e ( F i g s . 24-34).  C y t o p l a s m i c f i l a m e n t s , ap-  p r o x i m a t e l y 40 A i n d i a m e t e r , were p r e s e n t i n both c e l l more e v i d e n t i n C-4c  cultures.  degeneration,  l i n e s , though  In s u p e r f i c i a l c e l l s of both  lines,  t h e s e f i l a m e n t s i n p a r a l l e l a r r a y , u s u a l l y o c c u p i e d much of the c y t o plasm a d j a c e n t t o the growth medium.  In c e l l s l o c a t e d deeper w i t h i n ,  c u l t u r e s t h e i r d i s t r i b u t i o n was more v a r i a b l e as t h e y were found a d j a c e n t t o c e l l s u r f a c e s , a l o n g n u c l e i and i n t e r s p e r s e d among o t h e r organelles.  In g e n e r a l , f i l a m e n t s were arranged p r e d o m i n a n t l y  t h e main c e l l a x i s .  along  Thus, i n young c u l t u r e s o f both l i n e s , and i n C-4c  c u l t u r e s a t a l l s t a g e s , t h e y tended t o be o r i e n t e d p a r a l l e l t o the subs t r a t u m , w h i l e i n columnar C-4s  c e l l s t h e r e were prominent  of v e r t i c a l l y o r i e n t e d f i l a m e n t s between t h e n u c l e i and spaces.  In l i n e C-4c,  f i l a m e n t s tended t o aggregate  and t h e s e became more conspicuous  intercellular  into tight  as c u l t u r e s matured.  t h e f i l a m e n t s remained more d i f f u s e l y d i s t r i b u t e d .  groups  In C-4s  bundles, cultures  Juxtaposed t o plasma  membranes, which were a d j a c e n t t o the s u b s t r a t u m , a g g r e g a t i o n s of c y t o p l a s m i c f i l a m e n t s were o c c a s i o n a l l y seen i n a s s o c i a t i o n w i t h accumulations  of e x t r a c e l l u l a r m a t e r i a l .  These were r e m i n i s c e n t of  hemidesrnosomes but q u i t e i n d e f i n i t e and p o o r l y formed C y t o p l a s m i c f i l a m e n t s a l s o extended core which was  s e p a r a t e d from  ( F i g . 47).  i n t o m i c r o v i l l i t o form  the  the plasma membrane by a l e s s e l e c t r o n  Table I I I Main U l t r a s t r u c t u r a l D i f f e r e n c e s between t h e C e l l L i n e s I n V i t r o Cell Observation  Line  C-4c  C-4s  Predominant C e l l O r i e n t a t i o n a) E a r l y growth phases b) Late.growth phases  horizontal horizontal  horizontal vertical  Stratification  extensive  limited  Degeneration i n s t r a t i f i e d areas  limited  extensive  Cysts  absent  present  C e l l shape a d j a c e n t t o medium . ( l a t e phases)  very  columnar  Cell-contact  persistent  diminished  I n t e r c e l l u l a r j u n c t i o n s a t medium  overlapping m i c r o v i l l i •  terminal bars  Intercellular  complex  less  Microvilli  numerous  l e s s numerous  Desmosomes  associated with cytoplasmic f i l a m e n t bundles  not a s s o c i a t e d w i t h c y t o plasmic f i l a m e n t bundles  Mitochondria  smaller  larger  Golgi  less  more  (late  phases)  (late  phases)  spaces  complexes  Cytoplasmic  filaments  flat  frequent  more p r o m i n e n t , i n b u n d l e s  inclusions  many  frequent  l e s s prominent, d i s p e r s e d  a  Cytoplasmic  complex  some  41  Plate VII  F i g u r e 44;  C-4s c u l t u r e . T e r m i n a l b a r showing f u s i o n o f o u t e r l a y e r s of plasma membranes, o-zonula o c c l u d e n s , a-zonula adherens, d-desmosorne. x87000  Figure 45:  C-4s c u l t u r e . T e r m i n a l . b a r showing c e n t r a l f u s e d membrane, c, a, d as i n f i g u r e 44. x33000  F i g u r e 46:  F i l a m e n t o u s c e l l coat on m i c r o v i l l i i n an i n t e r c e l l u l a r s o a c e . x29000  F i g u r e 47:  E x t r a c e l l u l a r m a t e r i a l between plasma membranes and p l a s t i c s h e e t i n g , and. a s s o c i a t e d a c c u m u l a t i o n s o f c y t o p l a s m i c f i l a m e n t s ( a r r o w s ) . X26000  plasma  i n f r a s t r u c t u r e o f hamster cheek-pouch tumors ( F i g u r e s 48-52.) (Uranyl acetate/lead c i t r a t e ) Figure 48:  C-4c tumor. x6000  F i g u r e 49:  C-4s tumor. x6000  F i g u r e 50:  C-4c tumor. E p i t h e l i o - m e s e n c h y m a l j u n c t i o n , l a g e n f i b e r s . x9000  Figure 51:  C-4s tumor. E p i t h e l i o - m e s e n c h y m a l j u n c t i o n . t-tumor c e l l , f - f i b r o b l a s t , b-basement membrane. x l 9 0 0 0  Figure 52:  C-4s tumor. T e r m i n a l b a r f o r m a t i o n (arrows) around l a r g e i n t e r c e l l u l a r space. x23000  c-col-  42  dense r e g i o n .  W i t h i n t h i s c o r e , the f i l a m e n t s sometimes seemed con-  c e n t r i c a l l y arranged The. c y t o p l a s m contained  around a c e n t r a l m i c r o t u b u l e  i n both c e l l  ( F i g s . 39,  40).  l i n e s , though more f r e q u e n t l y i n C-4c  i n c l u s i o n s t h a t were m o d e r a t e l y e l e c t r o n dense i n the  but more e l e c t r o n dense at the p e r i p h e r y .  cells, center  They c o u l d r e p r e s e n t e i t h e r  l i p i d , d r o p l e t s or s e c r e t o r y p r o d u c t s , and were f r e q u e n t l y - a s s o c i a t e d w i t h rough endoplasmic r e t i c u l u m ( F i g . 2 6 a ) . glycogen The  i n any o f the observations  There was  no e v i d e n c e of  preparations.  of the u l t r a s t r u c t u r e of the two C-4  cell  lines  are summarized i n T a b l e I I I .  Hamster cheek-nouch i n o c u l a t i o n s The  h i s t o l o g y , h i s t o c h e m i s t r y and u l t r a s t r u c t u r e of hamster cheek-  pouch tumors were s t u d i e d t o d i s t i n g u i s h _in v i v o from i n v i t r o responses i n the two The  cell  l i n e s , and t o compare t h e i r h i s t o g e n e s i s In v i v o .  t y p i c a l growth p a t t e r n s of two-week o l d cheek-pouch tumors are  shown i n f i g u r e s 53 and 54. w h i l e the C-4s infiltration  The.C-4c tumors grew as compact masses,  tumors were b r o k e n up i n t o s m a l l e r clumps by abundant  of c o n n e c t i v e t i s s u e .  Near the c e n t e r o f each tumor t h e r e  appeared an area of host c o n n e c t i v e t i s s u e w i t h a f o r e i g n body r e a c t i o n c h a r a c t e r i z e d by i n f i l t r a t i o n multinucleated h i s t i o c y t e s .  o f l e u k o c y t e s , macrophages 3nd In a d d i t i o n , i n C-4c  giant  tumors t h e r e were  a r e a s o f f o c a l n e c r o s i s a t some d i s t a n c e from the host t i s s u e s . n e c r o t i c f o c i were p r e s e n t questionable  i n 14 of 14 C-4c  cases among 18 C-4s  tumors.  Such  tumors, but o n l y i n t h r e e  The maximum-width o f . t h e  rims  43  of h e a l t h y C-4c was  c e l l s s e p a r a t i n g c o n n e c t i v e t i s s u e from n e c r o t i c areas  i n the o r d e r of 100-200 u.  The C-4s  tumors were more e x t e n s i v e l y  i n f i l t r a t e d by c o n n e c t i v e t i s s u e and i t was  not p o s s i b l e to. determine  any c o n s i s t e n t v a l u e f o r maximal d i s t a n c e s of h e a l t h y C-4s  cells  from  host t i s s u e s . The  s t a i n i n g of t h e s e tumors f o r a c i d mucoproteins  demonstrated  a l c i a n - b l u e p o s i t i v e and c o l l o i d a l - i r o n p o s i t i v e m a t e r i a l s e p a r a t i n g cancer c e l l s from c o n n e c t i v e t i s s u e .  A c i d mucoprotein s t a i n i n g  a l s o i n t e n s e i n the n e c r o t i c areas i n C-4c  was  tumors and, i n a d d i t i o n ,  c o l l o i d a l i r o n s t a i n e d the i n t e r c e l l u l a r spaces between i n d i v i d u a l cancer c e l l s .  There were no obvious d i f f e r e n c e s i n s t a i n i n g of a c i d  m u c o p r o t e i n s between the c e l l  lines.  P A S - p o s i t i v e m a t e r i a l was  present  i n the form of e x t r a c e l l u l a r . , homogeneous d e p o s i t s between the tumors and c o n n e c t i v e t i s s u e s i n both c e l l l i n e s , but these d e p o s i t s were much more prominent  i n l i n e C-4s  where t h e y resembled basement membranes.  H e a l t h y cancer c e l l s of both c e l l  l i n e s were PAS-negative, but the  c e l l u l a r d e b r i s i n the n e c r o t i c areas i n C-4c  tumors was  intensely  P A S - p o s i t i v e , as were cancer c e l l s near such n e c r o t i c c e n t e r s . c e l l s seemed t o be d i s i n t e g r a t i n g , and t h e i r c y t o p l a s m was PAS-positive granules.  These  f i l l e d with  I t was a l s o observed t h a t the c o n n e c t i v e t i s s u e  a s s o c i a t e d with- f o r e i g n body r e a c t i o n s was u s u a l l y P A S - p o s i t i v e , poss i b l y due t o the presence o f p h a g o c y t i z e d degenerated ( F i g s . 55, 5 6 ) . diastase  cancer  cells  A l l P A S - p o s i t i v e m a t e r i a l i n t h e hamster tumors  was  insensitive.  The two c e l l lagen f i b e r s .  l i n e s d i f f e r e d i n the amount and d i s t r i b u t i o n of c o l -  In C-4c,  c o l l a g e n was  t h e tumors and much of i t was  c o n c e n t r a t e d a t the p e r i p h e r y of  arranged p a r a l l e l t o the advancing edge  44  Plate VIII H i s t o l o g y and H i s t o c h e m i s t r y o f hamster cheek-pouch tumors  Figure  53.  C--4c tumor.  H e m a t o x y l i n and E o s i n . x l 4 0  Figure  54.  C-.4s tumor.  H e m a t o x y l i n and E o s i n . x l 4 0  Figure  55.  c--4c tumor.  A l c i a n blue/PAS. x l 4 0  Figure  56.  c--4 s tumor.  A l c i a n blue/PAS. x l 4 0  Figure  57.  c--4c tumor.  H e i d e n h a i n ' s Azan. x l 4 0  Figure  58.  c- -4 s tumor.  Heidenhain's Azan. x l 4 0  45 o f t h e c e l l mass, w h i l e i n C-4s,  t h e c o l l a g e n network was more e x t e n -  s i v e and complex, a p p a r e n t l y b r e a k i n g up t h e tumors i n t o s m a l l e r  nodules  ( F i g s . 57, 5 8 ) . The  u l t r a s t r u c t u r e o f t h e tumors, i n areas l o c a t e d a t a d i s t a n c e  from h o s t t i s s u e s , as w e l l as r e g i o n s o f d i r e c t c o n t a c t w i t h  connective  t i s s u e , was examined. In areas removed from h o s t t i s s u e s , c e l l s o f both l i n e s were roughly spherical orientation.  o r p o l y g o n a l i n shape, w i t h v e r y i r r e g u l a r o u t l i n e s  and no  The i n t e r c e l l u l a r spaces were about as e x t e n s i v e as  i n v i t r o and t h e y were a g a i n more complex i n C-4c tumors, because m i c r o v i l l i were l o n g e r and more d e n s e l y spaced ( F i g s . 48, 4 9 ) . C-4s  tumors, p a r a l l e l , c l o s e l y opposed c e l l  than i n c u l t u r e  s u r f a c e s were more common  ( F i g . 5 2 ) , w h i l e i n C-4c tumors desmosomes a s s o c i a t e d  w i t h dense i n t r a c y t o p l a s m i c f i l a m e n t bundles in v i t r o .  In  exceeded t h e number seen  The appearance and d i s t r i b u t i o n o f o r g a n e l l e s i n both  l i n e s e s s e n t i a l l y resembled  cell  t h a t seen i n s t r a t i f i e d areas i n v i t r o ,  however, t h e amount o f d e g e n e r a t i o n observed  i n t h e C-4s tumors was  reduced  i n comparison w i t h c e l l masses o f comparable s i z e i n c u l t u r e .  The  j u n c t i o n s between cancer c e l l s , as w e l l as t h e i r s u r f a c e mod-  i f i c a t i o n s i n r e g i o n s o f c o n t a c t between C-4 tumors and h o s t t i s s u e s , resembled  connective  t h o s e seen i n v i t r o i n c e l l s a d j a c e n t t o t h e p l a s t i c  s h e e t i n g , i . e . t h e i n t e r c e l l u l a r j u n c t i o n s were open o r were l i m i t e d by m i c r o v i l l i o r i e n t e d predominantly enithelio-mesenchymal  junction.  p a r a l l e l t o each o t h e r and t o t h e The i n t e r c e l l u l a r spaces were g e n e r a l l y  o r i e n t e d p e r p e n d i c u l a r t o t h e c o n n e c t i v e t i s s u e , and t h e l a t t e r t i s s u e , consisting  o f f i b r o b l a s t s and abundant c o l l a g e n f i b e r s , p e n e t r a t e d  into  46  t h e s e spaces i n both c e l l l i n e s , b u t p a r t i c u l a r l y e x t e n s i v e l y i n C-4s tumors.  I n f a c t , t h e r e were r a r e l y more t h a n a few c o n t i g u o u s C-4s  c e l l s without intervening connective t i s s u e , while, i n contrast, there seemed t o be a tendency f o r c o n n e c t i v e t i s s u e t o be o r i e n t e d  parallel  t o t h e a d v a n c i n g edge o f t h e C-4c tumors. A f a i r l y w e l l - f o r m e d basement membrane was f r e q u e n t l y , b u t not a l w a y s , a s s o c i a t e d w i t h t h e j u n c t i o n between C-4s c e l l s and c o n n e c t i v e t i s s u e , but was much l e s s f r e q u e n t l y found i n comparable areas o f C-4c tumors ( F i g s . 50, 5 1 ) . I n r e g i o n s o f e p i t h e l i o - m e s e n c h y m a l c o n t a c t where basement membranes were a b s e n t , as w e l l as i n some spaces between canc e r c e l l s , f i n e f i l a m e n t o u s e x t r a c e l l u l a r m a t e r i a l was o f t e n a s s o c i a t e d w i t h t h e t u m o r - c e l l s u r f a c e s i n both c e l l l i n e s ( F i g . 4 8 ) .  C u l t u r e s o f normal c e r v i c a l  epithelium  C u l t u r e s o f normal c e r v i c a l e p i t h e l i u m were examined t o determine which u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s were due t o t h e m a l i g n a n t n a t u r e o f t h e c e l l l i n e s , w i t h t h e added u n d e r s t a n d i n g t h a t t h e d i f f e r e n c e s observed would be those between p r i m a r y and l o n g - t e r m c u l t u r e s , as w e l l as t h o s e between normal and m a l i g n a n t  cells.  In c u l t u r e s o f normal e p i t h e l i u m growing from t i s s u e c e l l s showed p r o g r e s s i v e enlargement,  fragments,  f l a t t e n i n g , and d e c r e a s e i n  n u c l e o - c y t o p l a s m i c r a t i o s w i t h i n c r e a s i n g d i s t a n c e from the e x p l a n t s . They c o n s i s t e d of a t l e a s t t h r e e l a y e r s o f f l a t t e n e d c e l l s , which were s e p a r a t e d by h o r i z o n t a l i n t e r c e l l u l a r s p a c e s .  These spaces were com-  p a r a b l e i n t h e i r s i z e and d i s t r i b u t i o n t o t h o s e " o b s e r v e d i n young C-4  47 c u l t u r e s , although regions  o f c l o s e c e l l a p p o s i t i o n seemed more f r e -  quent i n t h e n o r m a l - e p i t h e l i u m villi ated  cultures.  and by numerous desmosomes.  C e l l s made c o n t a c t  by m i c r o -  The i n t e r c e l l u l a r spaces were s e p a r -  from t h e growth medium by o v e r l a p p i n g  m i c r o v i l l i or c e l l edges and  no t e r m i n a l b a r s were seen ( F i g s . 63, 64, 6 7 ) . The degree o f s t r a t i f i c a t i o n diminished  w i t h d i s t a n c e from t h e e x p l a n t s  and o n l y t h e most  p e r i p h e r a l c e l l s seemed t o grow as t r u e monolayers ( F i g . 5 9 - 6 1 ) . Those c e l l s t h a t were i n d i r e c t c o n t a c t w i t h a t i s s u e fragment and w i t h c u l t u r e s u b s t r a t u m seemed engaged i n i n t e n s e s e c r e t o r y as i n d i c a t e d by numerous G o l g i complexes, s t r a n d s  activity,  o f rough endoplasmic  r e t i c u l u m , c y t o p l a s m i c , v e s i c l e s o f v a r i o u s s i z e s , and l a r g e i n c l u s i o n s t h a t may p o s s i b l y c o n t a i n s e c r e t o r y m a t e r i a l .  .Masses o f f i l a m e n t s ,  o r i e n t e d p a r a l l e l t o t h e s u b s t r a t a , were c o n c e n t r a t e d  i n the p e r i p h e r a l  c y t o p l a s m , p a r t i c u l a r l y a g a i n s t t h e s u r f a c e s a d j a c e n t t o t h e g l a s s , and the c e l l s a l s o c o n t a i n e d  microtubules  and numerous m i t o c h o n d r i a .  the s u r f a c e of such c e l l s , amorphous, or f i n e f i l a m e n t o u s was p r e s e n t both oh t h e s i d e f a c i n g t h e e x p l a n t  On .  material,  and between t h e o p p o s i t e  s i d e and t h e a d j o i n i n g g l a s s ( F i g . 6 5 ) . A c c u m u l a t i o n s o f such m a t e r i a l between t h e c e l l s and t h e g l a s s were f r e q u e n t l y a s s o c i a t e d w i t h densations of the cytoplasmic 68).  f i l a m e n t s i n t o hemidesmosomes  con-  ( F i g s . 63,  E x t r a c e l l u l a r m a t e r i a l o f s i m i l a r appearance, or i n t h e form o f  g r a n u l e s and v e s i c l e s , was d e p o s i t e d out t h e c e l l  between g l a s s and c e l l s t h r o u g h -  s h e e t s i n much g r e a t e r amounts t h a n i n C-4  ( F i g s . 64., '65, 6 8 ) .  cultures  In c o n t r a s t t o squamous e p i t h e l i u m in v i v o ,  the " b a s a l " c e l l s , i . e . t h o s e a d j a c e n t t o the s u b s t r a t u m , were f l a t tened r a t h e r t h a n columnar.  Organelles  i n d i c a t i v e of secretory  activ-  i t y were seen w i t h d i m i n i s h i n g frequency as c e l l s a d j a c e n t t o g l a s s  48  P l a t e IX C u l t u r e s o f Normal C e r v i c a l E p i t h e l i u m F i g u r e s 59-62:  H i s t o l o g y - Epon 812 embedding, a l k a l i n e t o l u i d i n e b l u e ,  F i g u r e 59:  E x p l a n t , m u l t i l a y e r e d e p i t h e l i a l c e l l sheet and e x f o l i a t i n g d i f f e r e n t i a t e d c e l l s . x470  F i g u r e 60:  M u l t i l a y e r e d e p i t h e l i a l c e l l sheet and e x f o l i a t i n g f e r e n t i a t e d c e l l s near e x p l a n t . x470  Figure 61:  One t o two c e l l l a y e r s i n more p e r i p h e r a l p a r t o f e p i t h e l i a l c e l l s h e e t . x470  F i g u r e 62:  C e l l s growing w i t h o u t an e x p l a n t . x470  F i g u r e s 63-68:  dif-  U l t r a s t r u c t u r e o f normal, e p i t h e l i a l c u l t u r e s growing from e x o l a n t s . ( U r a n y l a c e t a t e and l e a d c i t r a t e )  Figure 63:  M u l t i l a y e r e d e p i t h e l i a l c e l l sheet near e x p l a n t . x l 7 0 0 0  F i g u r e 64:  Two c e l l l a y e r s i n more p e r i p h e r a l p a r t o f e p i t h e l i a l c e l l sheet. xl6500  Figure 65:  E p i t h e l i a l c e l l between e x p l a n t and p l a s t i c s h e e t i n g , w i t h basement membranes on both s i d e s . x30000  Figure 66:  E x f o l i a t e d d i f f e r e n t i a t e d squamous c e l l .  F i g u r e 67:  C u l t u r e s u r f a c e f a c i n g growth medium. x32000  Figure 63:  Hemidesrnosome. x48000  xl2000  49 were l o c a t e d f u r t h e r away from e x p l a n t s , and a l s o as c e l l s were d i s p l a c e d from the s u b s t r a t u m towards the c u l t u r e medium.  In g e n e r a l ,  c e l l s became i n c r e a s i n g l y f l a t t e n e d w i t h d i s t a n c e from t i s s u e f r a g m e n t s , o r g a n e l l e s became l e s s numerous and and desmosomes d i m i n i s h e d  l i m i t e d t o the p e r i n u c l e a r a r e a ,  i n number and s i z e .  C e l l sheets  i n areas  a t some d i s t a n c e from t i s s u e fragments were o f t e n i n d i s t i n g u i s h a b l e from young C-4  c u l t u r e s ( F i g . 64  ).  W i t h i n areas of m u l t i i a y e r e d growth a p r o g r e s s i o n o f u l t r a s t r u c t u r a l changes, s u g g e s t i v e of squamous d i f f e r e n t i a t i o n , c o u l d be between the c e l l s a d j a c e n t t o the s u b s t r a t u m and those medium.  observed  f a c i n g the growth  These changes i n v o l v e d t h e l o s s of o r g a n e l l e s , an i n c r e a s e i n  t h e number of f i l a m e n t s and t h e i r arrangement i n t o dense b u n d l e s , the e x f o l i a t i o n of c e l l s w i t h p y k n o t i c n u c l e i and c y t o p l a s m of o r g a n e l l e s other than f i l a m e n t bundles.  There were no  and  devoid microvilli  on the s u r f a c e s of these e x f o l i a t e d c e l l s and t h e i r plasma membranes 'were thickened  ( F i g s . 59, 63, 66  ).  Even at a d i s t a n c e from t i s s u e fragments,  e x f o l i a t i o n of d i f f e r e n t i a t i n g c e l l s from the e p i t h e l i a l sheets still  observed, although  to a l e s s e r extent  ( F i g s . 60, 64  In c o n t r a s t t o the o r g a n i z e d e p i t h e l i a l c e l l from t i s s u e fragments, c o l o n i e s of p r i m a r y  was  ).  outgrowths formed  squamous e p i t h e l i u m growing  i n the absence of e x p l a n t s c o n s i s t e d of c e l l s t h a t seemed randomly o r i e n t e d , l o o s e l y cohesive  or s i n g l e , and  f l a t t e n e d to d i f f e r e n t degrees.  T h i c k p l a s t i c s e c t i o n s of such c o l o n i e s showed g r e a t l y d i m i n i s h e d c o n t a c t and  o v e r l a p between n e i g h b o u r i n g  c e l l s , c o n f i r m i n g the  im-  p r e s s i o n t h a t some of the c e l l s were f l a t w h i l e o t h e r s were roundi n g up and s e p a r a t i n g from the s u b s t r a t u m of from o t h e r c e l l s ( F i g , 6 2 ) . E l e c t r o n microscopy.confirmed  t h a t s t r a t i f i c a t i o n was  greatly.reduced  50  Plate X F i g u r e s 69-72:  U l t r a s t r u c t u r e o f normal c e r v i c a l e p i t h e l i a l c u l t u r e s growing-without e x p l a n t s . ( U r a n y l a c e t a t e and l e a d citrate)  F i g u r e 69;  Flattened c e l l  F i g u r e 70:  Edge o f a c e l l t h a t i s r o u n d i n g and s e p a r a t i n g from c e l l s h e e t . The same c e l l i s shown on t h e extreme r i g h t i n f i g u r e 6 2 . xlSOOO  F i g u r e 71:  Overlapping  Figure 72:  R e t r a c t i n g c e l l edges. x25000  ( m o n o l a y e r ) . x37000  c e l l edges. x27000 "  F i g u r e s 73-74: . F e r r i t i n u p t a k e . (Lead c i t r a t e ) Figure 73:  C-4s c u l t u r e , 15-minute exposure t o f e r r i t i n . Ferr i t i n p a r t i c l e s i n i n t e r c e l l u l a r spaces ( a r r o w s ) and i n p i n o c y t o t i c v e s i c l e (T> ) . X40500  Figure 74:  C-4s c u l t u r e , 7-hour exposure t o f e r r i t i n . Ferritin p a r t i c l e s i n t h r e e lysoscrnes ( a r r o w s ) . X28500  F i g u r e s 75-80:  U l t r a s t r u c t u r a l d i s t r i b u t i o n of a c i d mucosubstances. (Colloidal iron staining)  Figure 75:  C-4s c u l t u r e , e a r l y l o g a r i t h m i c growth phase. x44500  Figure 76:  C-4s c u l t u r e , s t a t i o n a r y growth phase, b a r . X34000  t-terminal  51  Plate XI  F i g u r e 77:  C-4c c u l t u r e . C o l l o i d a l - i r o n s t a i n i n g a f t e r removal from s u b s t r a t u m . x21C00  F i g u r e 78:  C-4s c u l t u r e . C o l l o i d a l - i r o n s t a i n i n g a f t e r removal from s u b s t r a t u m , t - t e r m i n a l b a r . x57500  Figure 79:  C o l l o i d a l - i r o n s t a i n i n g of c e l l - c o a t filaments (arrows). xl26000  F i g u r e 80:  C o l l o i d a l - i r o n stained m a t e r i a l connecting t i p s of ' m i c r o v i l l i . x33000  F i g u r e s 81-82:  D i s t r i b u t i o n of c e l l clurnos superimposed upon c u l t u r e s o f t h e same t y p e . ( V i t a l m i c r o s c o p y , s t a n d a r o p t i c s ; arrows p o i n t a t c e l l c l u m p s ) .  F i g u r e 81:  C-4c c u l t u r e .  x34  F i g u r e 82:  C-4s c u l t u r e . x34  52 ( F i g s . 68, 71  ), and t h a t t h e r e was  jacent c e l l s .  The  thickened  no c o n t a c t at a l l between some ad-  a d j o i n i n g edges of such c e l l s suggested the  p o s s i b i l i t y t h a t t h e y might have s e p a r a t e d (Fig.  72  ).  No e v i d e n c e of squamous d i f f e r e n t i a t i o n was  i n these c u l t u r e s . organized  by r e t r a c t i n g the c e l l  edges  noted anywhere  In c o n t r a s t t o t h e e x f o l i a t i n g c e l l s seen i n t h e  o u t g r o w t h s , c e l l s t h a t were r o u n d i n g up and t h u s  separating  from o t h e r c e l l s were c h a r a c t e r i z e d by an e x t r e m e l y complex c e l l f a c e c o v e r e d w i t h numerous l o n g m i c r o v i l l i  ( F i g . 70  ).  Summary of the h i s t o c h e m i s t r y and u l t r a s t r u c t u r e of the C-4 and  sur-  cell  lines  normal e p i t h e l i u m .  No major d i f f e r e n c e s were found between c u l t u r e s of the two l i n e s by h i s t o c h e m i c a l e x a m i n a t i o n .  In both l i n e s t h e r e was  of a c i d mucosubstances on f r e e c e l l s u r f a c e s o n l y .  In  cell  a coat  aldehyde-fixed  c u l t u r e s , t r a c e s of s i m i l a r m a t e r i a l were observed i n i n t e r c e l l u l a r s p a c e s , and were somewhat more marked i n C-4c s t a i n i n g i n densely  grown areas was  cultures.  more prominent i n C-4s  PAS-positive and  i t is  l i k e l y , i n view of the h i s t o c h e m i c a l r e s u l t s observed i n hamster tumors, t h a t these p o s i t i v e areas were a s s o c i a t e d w i t h c e l l The  most important  degeneration.  o b s e r v a t i o n made by e l e c t r o n m i c r o s c o p y was  the major d i f f e r e n c e s between the c e l l l i n e s were at the l e v e l of sue o r g a n i z a t i o n , r a t h e r t h a n at the c e l l u l a r l e v e l . became: more pronounced as the c u l t u r e s matured and cell  tis-  These d i f f e r e n c e s  appeared t o i n v o l v e  i n t e r a c t i o n s and r e s p o n s e s t o both the medium and. the  substratum.  In the c o u r s e of each passage, the p r o p o r t i o n of c e l l s exposed t o medium i n C-4c  that  the  c u l t u r e s became g r e a t l y reduced as the c e l l s formed an  arrangement r e s e m b l i n g  squamous s t r a t i f i e d  epithelium.  .  5  I n C-4s c u l t u r e s a v e r y l a r g e p r o p o r t i o n o f c e l l s remained w i t h t h e medium through a columnar duced  s t r a t i f i c a t i o n of the c e l l s .  intercellular  arrangement,  3  i n contact  c y s t f o r m a t i o n and r e -  In a s s o c i a t i o n w i t h these  changes,  c o n t a c t and a d h e s i o n t o t h e s u b s t r a t u m were m a i n t a i n e d  i n l i n e C-4c, w h i l e C-4s c e l l s commonly seemed t o s e p a r a t e from each o t h e r as w e l l as from t h e s u b s t r a t u m .  I n crowded C-4c c u l t u r e s ,  inter-  c e l l u l a r spaces were more complex and were s e p a r a t e d from t h e medium by o v e r l a p p i n g m i c r o v i l l i , i n C-4s c u l t u r e s t h e s e spaces were s i m p l e r and s e p a r a t e d from t h e medium by t e r m i n a l b a r s . intercellular  I n both l i n e s , t h e  spaces were open t o t h e s u b s t r a t u m o r were s e p a r a t e d from  i t by l o o s e l y i n t e r d i g i t a t i n g  microvilli.  The C-4c c e l l s had t h e h i g h e r  s u r f a c e - v o l u m e r a t i o , f i r s t , because t h e y were not c u b o i d a l b u t f l a t o r o v o i d , and second, because t h e i r  s u r f a c e s were more i r r e g u l a r ,  due t o  more e x t e n s i v e m i c r o v i l l i . S e v e r a l o f t h e l i g h t m i c r o s c o p i c o b s e r v a t i o n s were c o n f i r m e d and e x p l a i n e d by e l e c t r o n m i c r o s c o p y .  The i n d i s t i n c t  c e l l b o r d e r s observed  w i t h l i g h t m i c r o s c o p y i n C-4c c u l t u r e s c o u l d be accounted f o r by t h e o v e r l a p p i n g o f c e l l s , by t h e i r and p a r t i c u l a r l y  predominantly h o r i z o n t a l  by t h e l a y e r o f v e r y t h i n ,  t h e s e c u l t u r e s from t h e medium.  orientation,  f l a t c e l l s t h a t separated  Because o f t h i s  layer,  intercellular  j u n c t i o n s made up o n l y a v e r y s m a l l p r o p o r t i o n o f t h e s u r f a c e f a c i n g t h e medium, which may be a f a c t o r i n t h e r e s i s t a n c e t o t r y p s i n o f t h i s cell  line.  E l e c t r o n m i c r o s c o p y a l s o c o n f i r m e d t h a t s t r a t i f i c a t i o n was  both more e x t e n s i v e and e a r l i e r  i n onset i n l i n e C-4c. As t h e C-4s  c u l t u r e s matured,  spaces appeared t o become more d i s t i n c t  intercellular  w i t h l i g h t m i c r o s c o p y w h i l e t h e c e l l s seemed t o become s m a l l e r .  This  change was due t o a r e o r i e n t a t i o n o f t h e c e l l s from h o r i z o n t a l t o column-  54 ar,  and t o t h e i r i n c r e a s i n g s e p a r a t i o n by expanding  spaces.  intercellular  The presence o f t e r m i n a l b a r s may be t h e b a s i s f o r t h e l i m i t e d  a c t i o n o f t r y p s i n on m a t e r i a l between t h e c e l l s i n t h i s  line.  Some o f t h e d i f f e r e n c e s i n t h e b e h a v i o u r o f o l d , d e g e n e r a t i n g c u l t u r e s o f t h e two line's were a l s o e x p l a i n e d .  I n o l d C-4c c u l t u r e s , l a r g e  p a r t s of t h e c e l l s h e e t s r e t r a c t e d from t h e s u b s t r a t u m . edges were t h i c k e n e d ,  The r e t r a c t i n g  as though c e l l s were s u b s t i t u t i n g an i n t e r c e l -  l u l a r l o c a t i o n f o r t h e i r p r i o r adhesion t o g l a s s .  I n o l d C-4s c u l t u r e s ,  c e l l d e g e n e r a t i o n was much more e x t e n s i v e , and seemed t o be t h e main r e s p o n s e o f t h e s e c u l t u r e s t o p r o t r a c t e d crowding w i t h o u t  subculture.  O b s e r v a t i o n s on hamster cheek-pouch tumors i n d i c a t e d t h a t many of the c h a r a c t e r i s t i c s of t h e two c e l l l i n e s observed _in v i t r o p e r s i s t e d i n v i v o , such as t h e prominent i n t e r c e l l u l a r spaces w i t h m i c r o v i l l i i n b o t h l i n e s , and t h e more compact growth p a t t e r n o f l i n e C-4c.  Excep-  t i o n s , and t h u s f e a t u r e s which appeared t o be due t o c u l t u r e c o n d i t i o n s , were t h e e x t e n s i v e  f l a t t e n i n g o f s u r f a c e c e l l s i n l i n e C-4c and, i n  l i n e C-4s, t e r m i n a l b a r f o r m a t i o n , stratification.  and d e g e n e r a t i o n a s s o c i a t e d  Also, i n contrast to observations  with  i n c u l t u r e , areas  of c e l l d e g e n e r a t i o n were found almost e x c l u s i v e l y i n C-4c tumors r a t h e r t h a n i n t h e C-4s tumors.  I n a d d i t i o n , i n v i v o growth seemed t o  enhance c o n d e n s a t i o n o f f i l a m e n t bundles and desmosome f o r m a t i o n i n C-4c  tumors and c l o s e r a p p o s i t i o n o f c e l l s i n C-4s tumors, though t h e s e  e f f e c t s were s p o r a d i c a l l y d i s t r i b u t e d . with connective  In both c e l l  lines,  t i s s u e i n i t i a t e d basement membrane f o r m a t i o n ,  contact and t h i s  p r o c e s s was much more i n t e n s i v e i n C-4s tumors, which were a l s o more e x t e n s i v e l y i n f i l t r a t e d by c o n n e c t i v e  tissue.  55 O b s e r v a t i o n s on c u l t u r e s o f normal c e r v i c a l e p i t h e l i u m i n association with explants, separation tures the  showed t h a t c e l l  stratification  l i m i t e d t o the C-4  cells.  the  intercellular  those of  the squamous d i f f e r e n t i a t i o n of the normal c e l l s ,  w h i c h seemed t o p r o g r e s s w i t h d i s t a n c e distance  contacts  cultures.  most s t r i k i n g d i f f e r e n c e between the normal c u l t u r e s and l i n e s was  fea-  A l s o , the h o r i z o n t a l o r i e n t a t i o n of  normal c e l l s , i n c l u d i n g " b a s a l " ones, and  the C-4  and  of c e l l s by c o n s p i c u o u s i n t e r c e l l u l a r spaces were not  on the normal c e l l s , were s i m i l a r t o those of young C-4 The  growing  from c u l t u r e s u b s t r a t a .  from t i s s u e fragments and  Other d i f f e r e n c e s  with  i n the c u l t u r e  of  normal c e l l s , which were q u a n t i t a t i v e r a t h e r t h a n q u a l i t a t i v e , were the more e x t e n s i v e  r e g i o n s of c l o s e c e l l - s u r f a c e a p p o s i t i o n , the more  numerous desrnosomes and and  the more e x t e n s i v e  face.  hemidesmosomes, the g r e a t e r e x t r a c e l l u l a r material  number of  organelles  at the  substratum  F i n a l l y , the morphology of c u l t u r e s of normal  epithelium  grown i n the absence of t i s s u e fragments i n d i c a t e d t h a t c o n t a c t an e x p l a n t i s a p r e r e q u i s i t e f o r o r g a n i z e d growth and e n t i a t i o n i n the p r i m a r y c u l t u r e s . plants resulted  In a d d i t i o n , the  sur-  with  squamous d i f f e r absence of  i n g r e a t l y d i m i n i s h e d c e l l c o h e s i o n and  ex-  stratification  i n the b e n i g n c u l t u r e s , t o a degree where t h e s e c h a r a c t e r i s t i c s were r e d u c e d below the  The  cell  cultures.  o b s e r v a t i o n s summarized i n the above s e c t i o n r a i s e d a number of  questions regarding two  l e v e l s seen i n C-4  l i n e s and  both t h e i r s i g n i f i c a n c e i n the h i s t o g e n e s i s the u n d e r l y i n g  mechanisms i n v o l v e d .  of  Included i n  t h e s e q u e s t i o n s were l ) the r e a s o n f o r the more compact growth of l i n e C-4c  as compared t o l i n e C-4s,  2) the p o s s i b l e r e l a t i o n s h i p  the  56  between c e l l  separation  i n C-4s c u l t u r e s and t h e more d i f f u s e growth  p a t t e r n of t h i s c e l l l i n e , and 3) t h e e f f e c t s of t h e p h y s i c a l ment on t h e o r i e n t a t i o n and s u r f a c e  environ-  s p e c i a l i z a t i o n s of the c e l l s .  These problems were i n v e s t i g a t e d by t h e experiments d e s c r i b e d  i n the  following. (l)  The d i f f e r e n c e i n growth p a t t e r n between t h e c e l l Three p o s s i b l e e x p l a n a t i o n s  were c o n s i d e r e d  lines.  f o r t h e d i f f e r e n c e be-  tween t h e compact, s t r a t i f i e d growth of l i n e C-4c and t h e more d i f f u s e growth o f l i n e C-4s: (a)  A more e f f i c i e n t  (b)  A more complete i s o l a t i o n up o f t h e s e c e l l s ,  (c)  A stronger  (a)  intercellular  intercellular  circulation  i n l i n e C-4c,  of t h e C-4c c e l l s r e s u l t i n g i n a p i l i n g c o h e s i o n o f C-4c c e l l s .  I t was thought p o s s i b l e t h a t t h e d i f f e r e n c e i n s t r a t i f i c a t i o n  might be due t o n u t r i t i o n a l  factors, i . e . that i n t e r c e l l u l a r  circula-  t i o n was more e f f i c i e n t i n C-4c c u l t u r e s because t h e y l a c k e d  terminal  b a r s and had more e x t e n s i v e  intercellular  a l bars could occlude i n t e r c e l l u l a r contact  spaces.  I n C-4s, t h e t e r m i n -  spaces so e f f i c i e n t l y t h a t d i r e c t  w i t h growth medium was n e c e s s a r y f o r adequate n u t r i t i o n .  This  c o u l d e x p l a i n t h e columnar arrangement and l a c k o f s t r a t i f i c a t i o n i n C-4s c u l t u r e s . "To i n v e s t i g a t e t h i s p o s s i b i l i t y t h e r a t e and p a t t e r n of f e r r i t i n uptake were compared i n t h e two c e l l i n the l a t e 10%  Live  cultures  l o g a r i t h m i c or s t a t i o n a r y growth phase were exposed t o  f e r r i t i n / W a y m o u t h ' s medium f o r 15 minutes and f o r 1, 3 and 7 h o u r s .  F e r r i t i n penetrated i n t o the i n t e r c e l l u l a r and  lines.  spaces i n both c e l l  lines  was t a k e n up by C-4s c e l l s as r a p i d l y , and p o s s i b l y i n even l a r g e r  57 amounts, as by the C-4c  cells.  A f t e r 15 m i n u t e s , i n d i v i d u a l  ferritin  p a r t i c l e s were p r e s e n t i n i n t e r c e l l u l a r spaces e x t e n d i n g from t h e m e d i a l s u r f a c e t o the s u b s t r a t u m , and appeared r a t h e r t h a n f r e e i n the medium.  located at the c e l l c o a t s ,  A f t e r one hour, some f e r r i t i n  a l s o found i n p i n o c y t o t i c v e s i c l e s and lysosomes. ferritin  was  A c c u m u l a t i o n of  i n lysosomes c o n t i n u e d over 3 and 7 hours and seemed p a r t i -  c u l a r l y prominent  i n l i n e C-4s.  A f t e r 3 and 7 hours f e r r i t i n  particles  were f r e q u e n t l y a s s o c i a t e d w i t h masses of amorphous m a t e r i a l i n i n t e r c e l l u l a r spaces.  Very few f e r r i t i n p a r t i c l e s were ever seen on t h e  s u r f a c e f a c i n g the growth medium ( F i g s . 73-74 ) . These o b s e r v a t i o n s i n d i c a t e t h a t the r a t e o f i n t e r c h a n g e of p r o t e i n between medium and i n t e r c e l l u l a r spaces does not appear t o be the factor limiting s t r a t i f i c a t i o n i n line (b)  C-4s.  The more e x t e n s i v e s t r a t i f i c a t i o n of C-4c c e l l s c o u l d r e p r e -  s e n t p i l i n g up due t o a l o w e r c o n t a c t i n h i b i t i o n , p o s s i b l y a s s o c i a t e d w i t h h i g h e r c e l l - s u r f a c e charges or t h i c k e r m u c o p r o t e i n  cell-coats.  To t e s t f o r t h e s e p o s s i b i l i t i e s , s u r f a c e charges were determined by . c e l l e l e c t r o p h o r e s i s , and c o l l o i d a l - i r o n s t a i n i n g o f t h e c e l l s u r f a c e s was  examined e l e c t r o n m i c r o s c o p i c a l l y . Repeated  c e l l e l e c t r o p h o r e s i s showed no s i g n i f i c a n t d i f f e r e n c e i n  s u r f a c e charges between the c e l l l i n e s (Table I V ) . C o l l o i d a l i r o n s t a i n i n g r e v e a l e d a c o n t i n u o u s l a y e r of r e a c t i v e m a t e r i a l e x t e n d i n g over the c e l l s u r f a c e s exposed t o growth medium. T h i s l a y e r corresponded i n l o c a t i o n and t h i c k n e s s t o the e x t r a c e l l u l a r c o a t seen i n s t a n d a r d e l e c t r o n m i c r o g r a p h s , and was phases i n both c e l l  lines.  S t a i n i n g of t h e c e l l  present i n a l l  s u r f a c e s extended  growth  T a b l e IV Cell Electroohoresis Experiment Mo.  C e l l Line  Mobility u/sec/volt/cm  C-4c  -0.93  C-4s  -0.85  ,C-4c  -0.93  C-4s  -0.92  Significance of difference *  1  2  Determined  p>0.05  p> 0.20  by means and s t a n d a r d e r r o r s o f t r a v e l l i n g t i m e s o f i n d i v i d u a l  cells.  59 i n t o the i n t e r c e l l u l a r spaces i n young c u l t u r e s of b o t h c e l l (Fig.  75  o f C-4s,  ) and  i n s t a t i o n a r y c u l t u r e s of C-4c  s t a i n i n g was  but i n s t a t i o n a r y c u l t u r e s  l i m i t e d t o the m e d i a l s u r f a c e  d e t e r m i n e whether t h i s l i m i t a t i o n i n C-4s  78  due  ).  t o an absence  i n a b i l i t y of  then s t a i n e d .  Under these c o n d i t i o n s  cell  of desrnosomes and t e r m i n a l b a r s , i n d i c a t i n g  t h a t these c e l l o r g a n e l l e s d i d present b a r r i e r s to c o l l o i d a l (Fig.'78  ).  There was  ).  The  ance of c o l l o i d a l - i r o n p o s i t i v e m a t e r i a l d i f f e r e d on c e l l depending on t h e i r t i s s u e arrangement, i . e . the d i s t a n c e On m e d i a l s u r f a c e s  and  from  other  i n l a r g e i n t e r c e l l u l a r spaces i t had  about 100 A wide ( F i g s . 75, 77-80 ). core and  appear-  surfaces  t h e appearance of s t r a i g h t , h a i r l i k e f i l a m e n t s , up t o 0.5  o f a 30 A u n s t a i n e d  i r o n pene-  no e v i d e n c e of s e c r e t e d e x t r a c e l l u l a r  a c i d m u c o p r o t e i n i n i n t e r c e l l u l a r spaces ( F i g s . 77, 78  cells.  the  i n i n t e r c e l l u l a r spaces s t a i n e d i n b o t h c u l t u r e s ( F i g s . 77,  ), w i t h the e x c e p t i o n  tration  To  beyond t e r m i n a l b a r s , f i x e d c e l l s h e e t s were removed  from the growth s u r f a c e and surfaces  ( F i g . 76  c u l t u r e s was  o f r e a c t i v e groups i n i n t e r c e l l u l a r spaces or t o the s t a i n to penetrate  lines  The  u long  and  f i l a m e n t s seemed t o c o n s i s t  an a c i d m u c o p r o t e i n c o a t ( F i g . 79  ).  c u l t u r e s matured and m i c r o v i l l i became more numerous, the s t a i n e d  As fil-.  arnents o f t e n seemed t o form a f e l t w o r k f i l l i n g the spaces between and above the m i c r o v i l l i  ( F i g . 77  stained material overlayed  ).  O c c a s i o n a l l y , a compact l a y e r of  the t i p s of the m i c r o v i l l i , p o s s i b l y as a  r e s u l t of h a v i n g been l i f t e d o f f a growing and (Fig.  80  ).  m a t e r i a l was and  folding cell  On more c l o s e l y a d j a c e n t c e l l s u r f a c e s , the u s u a l l y more compact and  surface  stained  granular r a t h e r than  filamentous,  p r o b a b l y corresponded t o c e l l c o a t s w i t h i n c o n s p i c u o u s f i l a m e n t s  standard  e l e c t r o n micrographs ( F i g . 7 5 ) .  In g e n e r a l , no  on  consistant  d i f f e r e n c e s c o u l d be demonstrated between the c e l l l i n e s i n the amount,  •  60  appearance or d i s t r i b u t i o n of c o l l o i d a l - i r o n r e a c t i v e m a t e r i a l . Therefore,  on the b a s i s of c e l l e l e c t r o p h o r e s i s and  colloidal-iron  s t a i n i n g , the d i f f e r e n c e s i n growth p a t t e r n between the c e l l  lines  c o u l d not be accounted f o r by an i n c r e a s e d i s o l a t i o n of C-4c  cells  due  t o a d i f f e r e n c e i n s u r f a c e charges or m u c o p r o t e i n c o a t s . (c)  The  t h i r d approach was  t o c o n s i d e r whether the more compact  growth and more e x t e n s i v e s t r a t i f i c a t i o n of C-4c t o a d i f f e r e n c e between the c e l l c e l l u l a r cohesion,  c u l t u r e s c o u l d be  l i n e s i n the s t r e n g t h of e i t h e r i n t e r -  or of c e l l - t o - g l a s s a d h e s i o n , or b o t h .  C e l l s were  grown i n s u s p e n s i o n t o demonstrate e f f e c t s of i n t e r c e l l u l a r a l o n e , w h i l e the r a t e of s p r e a d i n g between the c e l l  cohesion  of s i n g l e c e l l s on g l a s s was  l i n e s as an i n d i c a t i o n of c e l l - t o - g l a s s  compared  adhesiveness.  F u r t h e r , c e l l s of each l i n e were seeded upon p a r t i a l l y c o n f l u e n t s h e e t s of the same type t o compare t h e i r tendency t o p i l e In s u s p e n s i o n ,  due  cell  up.  the c e l l s formed aggregates w i t h d i a m e t e r s i n the  o r d e r of 0.1-0.3 mm.  The  C-4c  aggregates were round, compact, u s u a l l y  c o v e r e d w i t h f l a t c e l l s , and  o f t e n t h e y fused i n t o l a r g e r , i r r e g u l a r  masses ( F i g s . 95, 97  C-4s  ).  The  were o f i r r e g u l a r shapes, and l o o s e l y arranged and  aggregates tended t o be  f r e q u e n t l y formed c y s t s .  not f l a t t e n e d at the s u r f a c e  smaller,  The  c e l l s were  ( F i g s . 96, 98  No new  f e a t u r e s were observed e l e c t r o n m i c r o s c o p i c a l l y ( F i g s .  105).  Therefore,  of C-4c  i t appears t h a t the more c o h e s i v e  c e l l s p e r s i s t e d i n the absence of any  ). 103-  growth p a t t e r n  cell-substratum  inter-  action. The was  attachment and  more r a p i d i n C-4s  between the c e l l  spreading cultures.  on g l a s s of s u b c u l t u r e d  single cells  T h i s i s shown by the d i f f e r e n c e  l i n e s i n the p r o p o r t i o n s of round and  flattened single  Table V S p r e a d i n g o f S i n g l e S u b c u l t u r e d C e l l s on G l a s s C e l l shape  round flattened  C-4c  C-4s  Number o f c e l l s mean ( r a n g e )  %  192.2 (154-257)  84  81.2 (49-118)  35  36.7 (27-61)  16  148.1 (96-225)  65  The d a t a a r e based on 10 c u l t u r e s p e r c e l l  Number o f c e l l s mean ( r a n g e )  l i n e , 24 hours a f t e r  subculture  62 c e l l s observed 24 hours a f t e r s u b c u l t u r e compact growth and more e x t e n s i v e  of spreading  Thus, the more  s t r a t i f i c a t i o n i n l i n e C-4c  f l u e n c e d both by s t r o n g e r c o h e s i o n adhesion to g l a s s .  (see T a b l e V ) .  seem i n -  between c e l l s and by l e s s r a p i d  I t s h o u l d be n o t e d , however, t h a t the slower  oh g l a s s i n C-4c  rate  does not n e c e s s a r i l y imply t h a t t h e r e i s  a d i f f e r e n c e , once the p r o c e s s of a d h e s i o n i s completed, between the cell  lines i n cell-substratum i n t e r a c t i o n . When c e l l clumps were superimposed upon c u l t u r e s of the same t y p e ,  the g r e a t m a j o r i t y of C-4c  c e l l s attached  t o the p r e - e x i s t i n g c e l l  s h e e t w i t h i n .24 hours and became i n c o r p o r a t e d  into i t .  In C-4s  cul-  t u r e s , a much s m a l l e r p r o p o r t i o n of added c e l l s became i n c o r p o r a t e d , w h i l e many others, remained i n s u s p e n s i o n d u r i n g the 3-day e x p e r i m e n t . There a l s o appeared t o be a d i f f e r e n c e between t h e c e l l l i n e s i n the p r e f e r e n t i a l s i t e s of attachment of superimposed c e l l s . l i n e , t h e s e s i t e s were, i n d e c r e a s i n g c o l o n y - g l a s s ; w h i l e the o r d e r glass - c e l l  In the  C-4c  o r d e r , c e l l sheet - edge of  i n the C-4s  sheet ( F i g s . 81, 82 ).  l i n e was,  edge of c o l o n y  These o b s e r v a t i o n s  -  indicate that  c e l l s of b o t h l i n e s have the a b i l i t y t o p i l e up, but t h a t t h i s tendency i s more prominent i n C-4c, of i n t e r c e l l u l a r c o h e s i o n Since  i t was  which a g a i n p o i n t s t o a more dominant r o l e in this cell  e v i d e n t t h a t the c e l l  line. lines differed in intercellular  a d h e s i o n , as w e l l as i n c e r t a i n phases of c e l l - s u b s t r a t u m  adhesion,  the r o l e s of c e l l - s u r f a c e p r o t e i n s and d i v a l e n t c a t i o n s i n the maintenance of c u l t u r e morphology were s t u d i e d . observations media:  l)  T a b l e VI summarizes  on l i v e c u l t u r e s grown f o r 24 hours i n f o u r d i f f e r e n t B a l a n c e d s a l i n e , 2)  trypsin/balanced  s a l i n e , and 4)  Ca" " ',  Mg  0.12%  t r y p s i n / C a , Mg"^  1  1  ++  - f r e e s a l i n e , 3) + +  0.12%  -free saline  Table V I The Length o f exposure  Balanced saline  E f f e c t s o f T r y p s i n D i g e s t i o n and D i v a l e n t - C a t i o n D e p l e t i o n on t h e C u l t u r e Morphology o f t h e C-4 C e l l L i n e s Ca  -H++ , Mg -free saline  0.12% t r y p s i n i n balanced s a l i n e  Ca  0.12% t r y p s i n i n , Mg^ -free saline  15 minutes  no chanae  more prominent i n t e r c e l l u l a r spaces  edges o f c o l o n i e s r e t r a c t ing  edges o f c o l o n i e s r e t r a c t i n g , number o f c y s t s increased  30 minutes  no change  wider i n t e r c e l l u l a r spaces, c y s t - w a l l s b r e a k i n g down  edges r e t r a c t i n g , some c o l o n i e s off g l a s s as sheets  edges r e t r a c t i n g , some c o l o n i e s o f f g l a s s as sheets, i n c r e a s i n g i n t e r c e l l u l a r spaces  1 hour  no change  wider i n t e r c e l l u l a r s p a c e s , no c y s t s  f u r t h e r r e t r a c t i o n and tearing  cysts breaking w i s e as above  2-3 hours  no change  c e l l s separating but attached t o glass  most c o l o n i e s as s h e e t s i n suspension  a l l colonies i n suspension, some c e l l s round w i t h i n sheets  4 hours  no change  most c e l l s s i n g l e , round b u t a t t a c h e d to glass  as above  as above  24 hours  c e l l s granular  some c e l l s i n suspens i o n , some c e l l l y s i s  a l l c o l o n i e s as s h e e t s i n suspension  c e l l s round, smaller  up, o t h e r -  sheets  ON  64  ( F i g s . 83-89 ). The  cell  l i n e s d i d not d i f f e r q u a l i t a t i v e l y i n t h e i r response  t h e s e media, but t h e r e , was  to  c o n s i d e r a b l e v a r i a t i o n i n the t i m i n g and  degree o f r e s p o n s e s , presumably due t o u n c o n t r o l l e d v a r i a b l e s i n the. e x p e r i m e n t a l system. cell  l i n e s may  T h e r e f o r e , q u a n t i t a t i v e d i f f e r e n c e s between the  e x i s t , but were obscured  i n T a b l e V I , t h e r e was  As shown  no change i n c o l o n y morphology a f t e r 24 hours  o f exposure t o b a l a n c e d ular.  by t h i s v a r i a t i o n .  s a l i n e , but the c e l l s seemed damaged and  In s a l i n e w i t h o u t d i v a l e n t c a t i o n s t h e r e was  gradual  gran-  widening  o f i n t e r c e l l u l a r spaces i n i t i a l l y , then t h e c e l l s seemed t o s e p a r a t e from one  another and became rounded, but most o f t h e s e c e l l s remained  a t t a c h e d t o the g l a s s up t o 24 h o u r s .  At t h a t t i m e , some of the  had gone i n t o s u s p e n s i o n , and t h e r e was T r y p s i n i n balanced  a g a i n evidence  of c e l l damage.  s a l i n e d i d not v i s i b l y a f f e c t i n t e r c e l l u l a r  but caused r e l e a s e of the i n t a c t c e l l  cells  sheets from t h e g l a s s .  adhesion,  This  p r o c e s s seemed t o s t a r t a t the f r e e edges of c o l o n i e s , which g r a d u a l l y r e t r a c t e d from the g l a s s . of f o l d s w i t h i n the c e l l c e l l s , suggested  The  appearance of t e a r s and the d i s t r i b u t i o n  s h e e t s , as w e l l as t h e d i s t o r t i o n of i n d i v i d u a l  t h a t the c o l o n i e s were r e t r a c t i n g a l o n g l i n e s of t e n -  s i o n which extended over l a r g e p a r t s of the c u l t u r e s and were more prominent i n l i n e C-4c.  A f t e r 24 hours i n t h i s medium, a l l c e l l s were  i n s u s p e n s i o n , but t h e g r e a t m a j o r i t y were s t i l l cell  sheets.  Trypsin i n C a , Mg + +  + +  seen p r e v i o u s l y w i t h each substance  incorporated i n intact  - f r e e s a l i n e combined the alone.  I t caused the r e l e a s e of  the c e l l s h e e t s from the g l a s s , as w e l l as the d i s r u p t i o n of c e l l u l a r cohesion.  T h i s d u a l e f f e c t was  effects  inter-  i l l u s t r a t e d by the appearance  6 5  Plate  XII  The E f f e c t s o f D i v a l e n t - C a t i o n D e p l e t i o n and o f T r y p s i n D i g e s t i o n on C u l t u r e Morphology ( F i g u r e s 83-89:  v i t a l microscopy, standard  Figure  83:  C-4c  c u l t u r e , balanced  Figure  84:  C-4s  c u l t u r e , Ca  Ii  , Mg  optics) •  s a l i n e f o r two h o u r s . -H-free  xl40  s a l i n e f o r four hours.  xl40  F i o u r e -85:  C-4s c u l t u r e , 0.12% h o u r . x34  t r y p s i n i n balanced  saline for  one  Figure  86:  C-4s c u l t u r e , 0.12% hour. xl40  t r y p s i n i n balanced  saline for  one  Figure  87:  Figure  88:  C-4c c u l t u r e , 0.12% 24 h o u r s . x i 4 0  t r y p s i n i n balanced  Fioure  89:  C-4c c u l t u r e , 0.1?% f o r 24 h o u r s . xl40  trypsin in Ca  F i g u r e 90:  +4- '  C-4s c u l t u r e , 0.12?o t r y p s i n i n 6a f o r one h o u r . x l 4 0  , Mg  + +  ++  -free  saline  saline for  , M g_++^ - f r e e  saline  C-4c c u l t u r e ; two minutes of t r y p s i n t r e a t m e n t f o l l o w e d by Carnoy's' f i x a t i o n and colloidal-iron/hematoxylin s t a i n i n g . Arrows p o i n t a t c o l l o i d a l - i r o n s t a i n e d m a t e r i a l , x660  66  i n l i n e C-4s hesion  of new  cysts, i . e . interference with cell-substratum  i n i t i a l l y , and  ad-  subsequent breakdown of t h e s e c y s t s , i . e . i n t e r -  ference with i n t e r c e l l u l a r cohesion ( F i g . 87).  Trypsin retarded,  rather 4-4-  t h a n a c c e l e r a t e d , the d i s s o c i a t i o n i n t o s i n g l e c e l l s e f f e c t e d by Ca  ,  44Mg  - free s a l i n e alone.  On the o t h e r hand, the l a c k of d i v a l e n t c a t i o n s  appeared t o reduce the t e n s i o n e v i d e n t  i n cultures treated with t r y p s i n  i n balanced s a l t s o l u t i o n . These r e s u l t s were s u p p o r t e d by those o b t a i n e d  by t r e a t m e n t of  t u r e s w i t h r u t h e n i u m r e d coupled w i t h a 15-minute exposure t o the media noted above. with calcium  e f f e c t of Ca  This  four  Ruthenium r e d , a c a t i o n i c dye expected t o compete  f o r n e g a t i v e l y charged s i t e s , p a r t i c u l a r l y augmented the +4-  and  cul-  44, i-ig  -free saline in disrupting i n t e r c e l l u l a r  connections,  g r e a t l y a c c e l e r a t e d the s e p a r a t i o n of c u l t u r e s i n t o s i n g l e c e l l s . i m p l i e s t h a t c e l l s e p a r a t i o n d i d not n e c e s s a r i l y i n v o l v e  appearance on the c e l l  s u r f a c e of an i n c r e a s e d  number of  the  negative  c h a r g e s , but r a t h e r a f u n c t i o n of c a l c i u m t h a t c o u l d not be  performed  by r u t h e n i u m r e d . The  observed e f f e c t s of t r y p s i n on c e l l - s u b s t r a t u m a d h e s i o n suggested  t h a t t h i s a d h e s i o n might i n v o l v e masking of a c i d mucoproteins by trypsin-sensitive basic proteins.  To t e s t f o r such a r e l a t i o n s h i p ,  l i v e and Carnoyr-fixed  c u l t u r e s were t r e a t e d w i t h t r y p s i n and  with c o l l o i d a l  Colonies  iron.  f i x e d w i t h Carnoy's w i t h o u t t r y p s i n  t r e a t m e n t d i d not s t a i n w i t h c o l l o i d a l i r o n at a l l . exposed t o 0.25%  stained  trypsin/balanced  s a l i n e f o r 2-3  I f c u l t u r e s were  minutes and  then  f i x e d , c o l l o i d a l - i r o n p o s i t i v e m a t e r i a l appeared where c e l l s were separa t i n g from the g l a s s , i . e . at the edges of the c o l o n i e s A f t e r 5 minutes of t r y p s i n t r e a t m e n t b e f o r e  ( F i g . 90).  f i x a t i o n , the s t a i n i n g  67 was  a g a i n absent or g r e a t l y d i m i n i s h e d ,  a l t h o u g h the c o l o n i e s  had  s e p a r a t e d f u r t h e r from the g l a s s , p r o b a b l y as the r e l e a s e of f u r t h e r e x t r a c e l l u l a r material continued. m i n u t e s of t r y p s i n t r e a t m e n t and some c e l l s u r f a c e s loidal iron.  I f the c e l l s were s t a i n e d a f t e r 15 complete s e p a r a t i o n from the  as w e l l as e x t r a c e l l u l a r m a t e r i a l s t a i n e d w i t h  col-  I f the c e l l s were exposed t o t r y p s i n a f t e r f i x a t i o n  i n s i t u , the s t a i n i n g r e a c t i o n was now  glass,  much more i n t e n s e and  i t occurred  on the s u r f a c e of the c e l l s t h r o u g h o u t the c o l o n i e s , p r o b a b l y  to protein denaturation increased  and b e t t e r r e a g e n t p e n e t r a t i o n .  i n i n t e n s i t y between 1-6  c o l l o i d a l - i r o n s t a i n i n g occurred b a l a n c e d s a l i n e was  This  reaction  minutes of t r y p s i n t r e a t m e n t .  i n any  With e i t h e r  p r e - f i x a t i o n or p o s t - f i x a t i o n enzyme d i g e s t i o n , c o l l o i d a l appeared somewhat more i n t e n s i v e i n C-4c  No  c o n t r o l c u l t u r e s where t r y p s i n /  r e p l a c e d by b a l a n c e d s a l i n e o n l y .  These o b s e r v a t i o n s  due  iron staining  cultures.  would i n d i c a t e t h a t t h e r e were, a c i d mucosubstanc  on the c e l l s u r f a c e s of the C-4  c o l o n i e s , but t h a t a d h e s i v e p r o c e s s e s  i n v o l v e d masking of t h e s e groups by t r y p s i n - s e n s i t i v e p r o t e i n s .  It  would seem a l s o t h a t the mechanisms of i n t e r c e l l u l a r c o h e s i o n and cell-substratum  adhesion d i f f e r e d .  of  I n t e r c e l l u l a r c o h e s i o n and the main  tenance of c h a r a c t e r i s t i c non-round shapes depended p r i m a r i l y on d i v a l e n t c a t i o n s , while c e l l - g l a s s adhesion r e q u i r e d i n t a c t c e l l - c o a t p r o t e i n , which l i k e l y i n c l u d e d the b a s i c p r o t e i n s t h a t are  involved  i n the masking of a c i d mucosubstances.  I t can be concluded t h e r e f o r e ,  t h a t the b a s i s f o r the more compact and  s t r a t i f i e d growth p a t t e r n  C-4c  c e l l l i e s , at l e a s t i n p a r t , i n s t r o n g e r  intercellular  and r e l a t i v e l y , or a b s o l u t e l y , weaker c e l l - s u b s t r a t u m  of  cohesion  adhesion,  and  Table V I I V i a b i l i t y o f C e l l s Shed i n t o Growth Medium over 48-hour P e r i o d s Growth Phase  Early  Viability  logarithmic  <=/  Late  logarithmic 0/ A>  Early  stationary O/  Late s t a t i o n a r y  Number o f C e l l s p e r C u l t u r e x 10 C-4c C-4s  Total Dead Alive Alive  22.5 - 1.6 12.6 ± 1.3 9.9 0.9 41.1  t  31.8 ± 2.6 13.6 ± 1.9 18.2 1 1.2 59.0  Total Dead A.live Alive  44.1 1 5.1 26.6 ± 2.6 17.5 + 2.8 37.7  77.2 ± 5.9 28.2 + 2.0 49.0 ± 4.4 62.9 167.0 I 12.6 62.9 1 6.3 104.1 ± 7.1 62.8  Total Dead . Alive Alive Total •Dead Alive Alive  91.4 1 9.9 51.8 ± 5.3 39.6 1 5.1 40.4  Means - s t a n d a r d e r r o r s .  214.5 1 12.9 57.9 - 7.0 156.6 10.5 76.8  t  . 69 t h a t t h i s d i f f e r e n c e may i n v o l v e d i f f e r e n c e s i n t h e amounts o r d i s t r i b u t i o n o f d i v a l e n t c a t i o n s and/or p r o t e i n s on t h e c e l l  The r e l a t i o n s h i p o f " c e l l s e p a r a t i o n  and growth p a t t e r n  surface.  i n l i n e C-4s  One of t h e major h i s t o g e n e t i c d i f f e r e n c e s between t h e c e l l l i n e s was t h e g r a d u a l i n c r e a s e ing  i n t h e i n t e r c e l l u l a r spaces observed i n matur-  C-4s c u l t u r e s , i n c o n t r a s t t o t h e p e r s i s t e n t c l o s e a s s o c i a t i o n o f  c e l l s observed i n comparable C-4c c u l t u r e s ( F i g s . 19-22, 31-33). separation  Such  o f C-4s c e l l s c o u l d be o f s i g n i f i c a n c e as a means o f c e l l  d i s p e r s i o n i n r e s p o n s e t o crowding, and might be- r e l a t e d t o more d i f f u s e patternsof  i n v a s i o n of t h e s e c e l l s in v i v o .  I f the c e l l  separation  observed i n C-4s c u l t u r e s was p a r t of such a mechanism, c o n c u r r e n t c e l l d i s p e r s a l s h o u l d be demonstrable i n v i t r o .  To t e s t f o r t h i s  i t y , t h e number of c e l l s shed i n c u l t u r e , t h e i r v i a b i l i t y c a p a c i t y t o form c o l o n i e s lines. did  possibil-  and t h e i r  i f r e c u l t u r e d were compared between t h e c e l l  The r e s u l t s , as shown i n T a b l e V I I , i n d i c a t e t h a t C-4s c u l t u r e s  r e l e a s e more c e l l s i n t o t h e growth medium i n a l l growth p h a s e s .  T h i s d i f f e r e n c e between t h e c e l l mic phase, but i n c r e a s e d  l i n e s was s m a l l  i n the e a r l y l o g a r i t h - .  s e v e r a l f o l d as c u l t u r e s matured, and c o u l d  be seen t o be due t o an i n c r e a s i n g number of l i v e c e l l s r e l e a s e d  into  the medium by t h e C-4s c u l t u r e s , as t h e number o f dead c e l l s was s i m i l a r i n both l i n e s a t each growth phase. Since c e l l v i a b i l i t y ,  as d e f i n e d  by dye e x c l u s i o n , does not n e c e s s a r  i l y imply r e t e n t i o n of p r o l i f e r a t i v e c a p a c i t y , c e l l s r e l e a s e d  into  growth medium and v i a b l e by t r y p a n b l u e e x c l u s i o n were r e c u l t u r e d t o examine t h e i r a b i l i t y  t o form c o l o n i e s .  As shown i n T a b l e V I I I ,  the number o f c o l o n i e s formed by C-4c c e l l s was v e r y s m a l l on e i t h e r g l a s s or c o l l a g e n g e l . T h e r e f o r e , under t h e c o n d i t i o n s t e s t e d , even t h e  Table V I I I Number o f C o l o n i e s Formed by C e l l s R e c o v e r e d from Growth Medium and R e c u l t u r e d Substratum  Days i n Culture  Glass  Collagen Gel  Cell C-4C  Line C-4s  3  0.7 (0-2)  18.5 (2-37)  6  0.5 (0-2)  19.8 (4-45)  3  0.3 (0-1)  44.7 (13-72)  6  0 (0)  34.2 (11-59)  Means and r a n g e , s i x c u l t u r e s per g r o u p .  71 C-4c  c e l l s t h a t were v i a b l e by dye  colonies.  e x c l u s i o n were r a r e l y a b l e t o form  In c o n t r a s t , a f a i r number of r e c u l t u r e d C-4s  to p r o l i f e r a t e .  The  number of c o l o n i e s formed by C-4s  c e l l s was c e l l s was  able  c.uite  s m a l l c o n s i d e r i n g the f a c t t h a t 60,000-100,000 v i a b l e c e l l s were p l a t e d i n each c u l t u r e but s e v e r a l f a c t o r s p r o b a b l y  artifically  lowered  the  t r u e f r a c t i o n of c e l l s w i t h p r o l i f e r a t i v e c a p a c i t i e s . C e l l s f o r r e c u l t u r e were h a r v e s t e d and  from growth medium at 48-hour  intervals,  i t i s t h e r e f o r e p o s s i b l e t h a t i n b o t h c e l l l i n e s some of them  became i r r e v e r s i b l y damaged d u r i n g the p r o l o n g e d s t a y i n  suspension,  w h i l e under p h y s i o l o g i c a l c o n d i t i o n s they would have been c a p a b l e of proliferation.  A l s o , the much higher- y i e l d of c o l o n i e s i n l i n e  C-4s  on c o l l a g e n g e l , as compared t o g l a s s , suggests the p o s s i b i l i t y  that  many c e l l s of t h i s l i n e which d i d not p r o l i f e r a t e on g l a s s , or even on c o l l a g e n , would have been a b l e t o do so under m o r e p h y s i o l o g i c a l  con-  ditions.  S u b s t i t u t i o n of c o l l a g e n g e l f o r g l a s s as the s u b s t r a t u m i n  l i n e C-4c  d i d not produce any  improvement i n c o l o n y  f u r t h e r r e d u c t i o n i n the number of C-4s  c o l o n i e s was  formation.  caused by the  g r e g a t i o n of many s i n g l e c e l l s p r i o r t o t h e . a t t a c h m e n t t o the Each of t h e s e c e l l aggregates formed a l a r g e c o l o n y hundreds of c e l l s w h i c h , a l t h o u g h counted as "one" absent i n the C-4c  i n Table V I I I .  c o n s i s t i n g of  not d e r i v e d from a s i n g l e c e l l , Such c e l l a g g r e g a t i o n  was  was  essentially small  line.  r e s u l t s shown i n T a b l e s V I I and V I I I support the  t h a t crowding i n C-4s  ag-  substratum.  c u l t u r e s and t h e r e f o r e c o u l d not account f o r the  number of c o l o n i e s formed i n t h i s The  A  c u l t u r e s , but not i n C-4c  consideration  c u l t u r e s , induces  the r e l e a s e i n t o the environment o f . l a r g e numbers of v i a b l e c e l l s which are c a p a b l e of p r o l i f e r a t i o n and  colony  formation.  The  separation  of  72  s t a t i o n a r y C-4s c e l l s observed e l e c t r o n m i c r o s c o p i c a l l y , and d e s c r i b e d above ( p p . 31-35) l i k e l y , i s p a r t o f t h i s p r o c e s s of c e l l  dispersal,  w h i c h c o u l d be a f a c t o r i n f l u e n c i n g t h e more d i f f u s e growth p a t t e r n observed i n v i v o w i t h C-4s tumors. The mechanism o f c e l l s e p a r a t i o n i n C-4s c u l t u r e s c o u l d  involve  p r e s s u r e from i n t e r c e l l u l a r spaces p u s h i n g a p a r t c e l l s , or a l t e r n a t i v e l y s e p a r a t i o n o f c e l l s by a change i n t h e i r shape o r o r i e n t a t i o n . The l i m i t a t i o n o f i n t e r c e l l u l a r spaces i n C-4s c u l t u r e s by t e r m i n a l b a r s suggested t h a t p r e s s u r e i n t h e s e spaces c o u l d be i n c r e a s e d by a c c u m u l a t i o n o f m a t e r i a l s s e c r e t e d by t h e c e l l s .  The f o r m a t i o n o f  f a i r l y prominent basement membranes by C-4s tumors i n v i v o  further  suggested t h a t such a p r o c e s s might i n v o l v e s e c r e t i o n o f mucoproteins i n t o i n t e r c e l l u l a r spaces and c y s t s .  However, i n c u l t u r e , no such  m a t e r i a l c o u l d be demonstrated i n t h e s e spaces e i t h e r h i s t o c h e m i c a l l y or e l e c t r o n m i c r o s c o p i c a l l y (see p p  0  29, 5 9 ) , and no evidence of i n -  c r e a s e d e l e c t r o n d e n s i t y i n expanding i n t e r c e l l u l a r spaces c o u l d be obtained.  In a d d i t i o n , t h e r a p i d p e n e t r a t i o n o f f e r r i t i n i n t o  inter-  c e l l u l a r spaces (see pp. 56-57), does not s u p p o r t t h e reo,uirement t h a t i n t e r c e l l u l a r spaces i n t h e s e c u l t u r e s were s u f f i c i e n t l y i s o l a t e d t o p e r m i t t h e b u i l d u p of p r e s s u r e which would s e p a r a t e c e l l s .  To d e t e r -  mine more d i r e c t l y whether p r e s s u r e was expanding i n t e r c e l l u l a r spaces, stationary intact live cell  s h e e t s were c a r e f u l l y removed from g l a s s  by p o l i c e m a n and f i x e d f o r e l e c t r o n m i c r o s c o p y w i t h i n one m i n u t e . I f t h e s e p a r a t i o n of t h e c e l l s w i t h i n t h e sheets was due t o p r e s s u r e , t h e i n t e r c e l l u l a r , spaces would be expected t o c o l l a p s e i m m e d i a t e l y upon removal from t h e g l a s s . of t h e c e l l  However, t h e i n t e r c e l l u l a r  organization  s h e e t s remained, e s s e n t i a l l y u n d i s t u r b e d upon removal from  73 the g l a s s , a g a i n p r o v i d i n g no e v i d e n c e f o r i n c r e a s e d pressure  as the b a s i c mechanism o f c e l l  intercellular  s e p a r a t i o n i n C-4s  I t t h e r e f o r e seems l i k e l y , t h a t t h i s s e p a r a t i o n p r o c e s s  cultures.  involved a l t e r -  a t i o n s i n c e l l shape due t o i n t r a c e l l u l a r changes, p o s s i b l y a s s o c i a t e d w i t h the d e s c r i b e d r e o r i e n t a t i o n o f c y t o p l a s m i c a diminution i n c e l l  The  f i l a m e n t s and  with  adhesiveness.  r o l e o f . s u b s t r a t u m - v i s c o s i t y i n the h i s t o g e n e s i s of the C-4 The  lines  appearance of s p e c i f i c m o d i f i c a t i o n s i n c e l l - o r i e n t a t i o n  c e l l surface s t r u c t u r e i n r e l a t i o n to p l a s t i c sheeting, other  and  cells,  or growth medium, suggested t h a t some of these c h a r a c t e r i s t i c s might a c t u a l l y be r e s p o n s e s t o changes i n v i s c o s i t y of the immediate environment o f the c e l l s . s o l i d and  To examine s p e c i f i c a l l y the e f f e c t s of l i q u i d , semi-  s o l i d s u b s t r a t a on the c e l l s , the growth p a t t e r n s o f c u l t u r e s i n  t h e l a t e l o g a r i t h m i c phase, when on p l a s t i c s h e e t i n g or i n e i t h e r i n l i q u i d or s e m i s o l i d (agar-containi<ng) The  t y p i c a l morphology of the c e l l  medium, were compared.  l i n e s , when grown on a s o l i d  s u b s t r a t u m i n l i q u i d medium has been d e s c r i b e d above.  In summary,  the c h a r a c t e r i s t i c m o r p h o l o g i c f e a t u r e s found i n the c e l l s to  growth medium were the f l a t t e n i n g of C-4c  formation  i n C-4s  c e l l s , and,  suspension,  c e l l s , the  adjacent  terminal-bar  i n both c e l l l i n e s , a f i l a m e n t o u s  cell  c o a t , m i c r o v i l l i , and numerous f i l a m e n t s but few o r g a n e l l e s i n the p e r i p h e r a l cytoplasm.  When a d j a c e n t t o the p l a s t i c s h e e t i n g , the  were not f l a t t e n e d , t h e r e were no t e r m i n a l bars c e l l u l a r j u n c t i o n s i n both c e l l interdigitating microvilli.  i n C-4s,  and the  l i n e s were open or o n l y l i m i t e d  cells inter-  by  M i c r o v i l l i on these c e l l - s u r f a c e s were  74  absent or p a r a l l e l t o the s u b s t r a t u m , and the e x t r a c e l l u l a r coat not p r o m i n e n t .  was  C y t o p l a s m i c f i l a m e n t s and o r g a n e l l e s were not as  s e g r e g a t e d as i n t h o s e p o r t i o n s of c e l l s c l o s e t o the medium. Some s p e c i f i c e f f e c t s on c u l t u r e morphology of the e x p e r i m e n t a l s o l i d and l i q u i d phases were determined by suspending c e l l s i n l i q u i d medium under d i f f e r e n t c o n d i t i o n s .  In one e x p e r i m e n t a l s e r i e s ,  ments of c e l l sheets were removed from g l a s s by p o l i c e m a n , and for  e l e c t r o n microscopy l )  s u s p e n s i o n , and 3)  w i t h i n one minute, 2)  fragfixed  a f t e r one hour i n  f o r t h r e e days, t o observe some immediate  l a y e d e f f e c t s of e l i m i n a t i o n o f the s o l i d s u b s t r a t u m .  and  There was  delittle  change i n c o l o n y morphology i m m e d i a t e l y upon removal from the g l a s s , i n d i c a t i n g t h a t the o r g a n i z a t i o n of the c e l l s w i t h i n . c o l o n i e s , once e s t a b l i s h e d , c o u l d p e r s i s t a t l e a s t f o r a s h o r t time w i t h o u t c o n t i n u e d c o n t a c t w i t h the s o l i d , s u b s t r a t u m , and t h e r e f o r e d i d not seem t o r e q u i r e e i t h e r d i r e c t i n t e r a c t i o n of the c e l l s w i t h the g l a s s , or c o n t a c t of  i n t e r c e l l u l a r spaces w i t h a d j o i n i n g s o l i d s u r f a c e s .  No change i n  u l t r a s t r u c t u r e from t h a t of c o l o n i e s f i x e d _in s i t u c o u l d be i n such c e l l  sheets ( F i g s . 91, 92, 99, 100.).  removal from the g l a s s , c e l l c o n f i g u r a t i o n a l changes. long a x i s .  demonstrated  However, one hour  after  sheets of both l i n e s had undergone major  Each c e l l sheet was  now r o l l e d up a l o n g i t s  E x a m i n a t i o n by l i g h t - and e l e c t r o n m i c r o s c o p y showed t h a t  i n t h i s p r o c e s s , c e l l s had become e l o n g a t e d a l o n g t h e a x i s  perpen-  d i c u l a r t o the former growth s u r f a c e w i t h c o r r e s p o n d i n g n a r r o w i n g a l o n g the o t h e r a x i s , i . e . p a r a l l e l t o the growth s u r f a c e . of  The e x t e n t  t h i s n a r r o w i n g was uneven i n d i f f e r e n t p a r t s of the c e l l s ; i t was  g r e a t e s t near t h e s i d e f o r m e r l y i n c o n t a c t w i t h the g l a s s , and d i m i n i s h e d toward the s i d e t h a t had been exposed t o growth medium i n s i t u .  This  uneven r e d u c t i o n of the c e l l d i a m e t e r s r e s u l t e d i n the r o l l i n g up of  75 the c e l l  sheets.  In t h i s way, t h e same s i d e o f each sheet t h a t had  f o r m e r l y been i n c o n t a c t w i t h t h e medium was a g a i n on t h e s u p e r f i c i a l surface.  T h i s p r o c e s s was accompanied i n both c e l l  l i n e s by a r e d u c -  t i o n i n t h e i n t e r c e l l u l ar spaces, w h i c h was most s t r i k i n g i n l i n e C-4s, by t h e appearance i n both l i n e s of complex f o l d s on those  surfaces  f o r m e r l y i n c o n t a c t w i t h g l a s s , and, i n l i n e C-4c, by t h e appearance o f a dense l a y e r o f c y t o p l a s m i c the same s u r f a c e .  filaments adjacent  and p a r a l l e l t o  A s i m i l a r f i l a m e n t band c o u l d not be d e f i n i t e l y  i d e n t i f i e d i n C-4s c e l l s ( F i g s . 9 3 , 94, 101, 102, 106, 1 0 7 ) . A f t e r suspension  i n growth medium over t h r e e - d a y  appearance o f t h e c e l l  sheets had a g a i n changed.  now formed r o u n d , compact clumps covered aggregates c o n t a i n e d  periods, the  The C-4c c e l l s had  w i t h f l a t c e l l s , w h i l e C-4s  c y s t s , were s m a l l e r , i r r e g u l a r , more l o o s e l y c o -  h e s i v e , and f r e q u e n t l y formed t e r m i n a l bars a t t h e c e l l j u n c t i o n s i n c o n t a c t w i t h t h e medium. ing  These o b s e r v a t i o n s  i n d i c a t e that the f l a t t e n -  o f s u r f a c e c e l l s i n C-4c, as w e l l as t e r m i n a l b a r f o r m a t i o n ,  limited  s t r a t i f i c a t i o n and c y s t f o r m a t i o n i n C-4s, are f e a t u r e s independent of a s o l i d substratum.  A l s o , t h e o r i e n t a t i o n and u l t r a s t r u c t u r e o f c e l l s  i n these aggregates d i d not d i f f e r from t h a t of c e l l s grown on p l a s t i c s h e e t i n g , i n d i c a t i n g t h a t t h e f a c t o r s r e s p o n s i b l e f o r t h e morphology a t one hour i n s u s p e n s i o n  were no l o n g e r e f f e c t i v e ( F i g s . 95-98, 104, 1 0 5 ) .  To reduce t h e e f f e c t s of a l i q u i d on t h e c e l l s , t h e y were c u l t u r e d i n growth medium rendered  s e m i s o l i d by t h e a d d i t i o n of a g a r .  pended clumps of c e l l s grew g r a d u a l l y and tended t o fuse i n t o  The susirregular  masses, o f t e n becoming surrounded by d e b r i s a f t e r about a week. c o n t r a s t t o the suspension  In  c u l t u r e s i n l i q u i d medium, C-4s aggregates  i n agar d i d not form c y s t s , and appeared as compact a s , and o f s i m i l a r  76  Plate XIII The H i s t o l o g y o f C-4 c o l o n i e s i n s u s p e n s i o n . ( F i g u r e s 91-94 and 97-98: Epon 812 embedding, a l k a l i n e t o l u i d i n e b l u e s t a i n i n g . F i g u r e s 95-96: v i t a l m i c r o s c o p y , s t a n d a r d o p t i c s )  F i g u r e 91:  C-4c c o l o n y , f i x e d w i t h i n one minute a f t e r x470  suspension.  F i g u r e 92:  C-4s c o l o n y , f i x e d w i t h i n one minute a f t e r x470  suspension.  F i g u r e . 93:  C-4c c o l o n y , one hour i n s u s p e n s i o n , area shown i n f i g u r e 101. x470  F i g u r e 94:  C-4s c o l o n y , one hour i n s u s p e n s i o n . x470  F i g u r e 95 :  C-4c c o l o n y , two s u s p e n s i o n - c o l o n i e s a f t e r t h r e e d a y s . xl40  F i g u r e 96:  C-4s c o l o n y , a s u s p e n s i o n - c o l o n y  F i g u r e 97:  C-4c c o l o n y , t h r e e days i n s u s p e n s i o n . x470  F i g u r e 98:  C-4s c o l o n y , t h r e e days i n s u s p e n s i o n .  Arrow points at  a f t e r t h r e e days. x l 4 0  x470  77  P l a t e XIV The U l t r a s t r u c t u r e o f C-4 c o l o n i e s i n s u s p e n s i o n ( F i g u r e s 99-1C7) ( u r a n y l a c e t a t e and l e a d c i t r a t e s t a i n i n g )  F i g u r e 99:  C-4c c o l o n y , f i x e d w i t h i n one minute a f t e r s u s p e n s i o n . S i d e f o r m e r l y a d j a c e n t t o s u b s t r a t u m . xlOOOO.  F i g u r e 100:  C-4s c o l o n y , f i x e d w i t h i n one minute a f t e r x7000  F i g u r e 101:  C-4c c o l o n y , one hour i n s u s p e n s i o n . arrow i n f i g u r e 93. x3600  Area shown by  F i g u r e 102:  C-4s c o l o n y , one hour i n s u s p e n s i o n . i n t e r c e l l u l a r s p a c e s . x24000  Narrowing o f  F i g u r e 103:  C-4s c o l o n y , 3 days i n s u s p e n s i o n . X21C00  Terminal bar.  F i g u r e 104:  C-4c c o l o n y , 3 days i n s u s p e n s i o n . x6000  Cell  F i g u r e 105:  C-4s c o l o n y , 3 days i n s u s p e n s i o n . and c e l l d e g e n e r a t i o n , x8000  Cyst formation (c)  suspension.  flattening.  78  P l a t e XV  F i g u r e 106:  C-4c c o l o n y , one hour i n s u s p e n s i o n . S i d e f o r m e r l y a d j a c e n t t o s u b s t r a t u m , f - f i l a m e n t band. x20000  F i g u r e 107:  C-4s c o l o n y , one hour i n s u s p e n s i o n . a d j a c e n t t o s u b s t r a t u m . x26000  Side formerly  • 79 s i z e t o , t h e C-4c a g g r e g a t e s .  In C-4c c u l t u r e s , t h e c h a r a c t e r i s t i c  f l a t t e n i n g o f c e l l s i n d i r e c t c o n t a c t w i t h t h e medium was completely absent.  almost  The r o u g h l y c u b o i d a l o r i e n t a t i o n i n s u p e r f i c i a l  C-4s c e l l s , which had been observed i n l i q u i d medium, was e s s e n t i a l l y unchanged.  In s p i t e o f t h e i r r e l a t i v e l y l a r g e s i z e , t h e s e C-4s ag-  g r e g a t e s showed l i t t l e  c e l l d e g e n e r a t i o n ( F i g s . 108, 1 0 9 ) .  Electron  m i c r o s c o p i c e x a m i n a t i o n c o n f i r m e d t h e change i n o r i e n t a t i o n o f the superf i c i a l C-4c c e l l s , and a l s o showed t h a t the i n t e r c e l l u l a r spaces were o r i e n t e d more p e r p e n d i c u l a r t o the s u r f a c e .  The i n t e r c e l l u l a r  junctions  c o n s i s t e d o f i n t e r d i g i t a t i n g m i c r o v i l l i , which were o f t e n o r i e n t e d a t a n g l e s or p a r a l l e l t o the c e l l  surfaces.  I n C-4s c u l t u r e s , t h e most  s t r i k i n g change ch the s u r f a c e of c e l l aggregates-was of  t h e absence  t e r m i n a l b a r s , and t h e i r r e p l a c e m e n t , as i n C-4c a g g r e g a t e s , by  c e l l j u n c t i o n s c l o s e l y r e s e m b l i n g those seen a d j o i n i n g p l a s t i c s h e e t i n g ( F i g s . 110, 1 1 1 ) . changes.  In both c e l l l i n e s t h e r e were a l s o o t h e r l e s s d e f i n i t e  There seemed t o be l e s s e v i d e n c e of an e x t r a c e l l u l a r c o a t ,  p a r t i c u l a r l y on t h e t i p s of m i c r o v i l l i , and o c c a s i o n a l l y t h e s e t i p s were connected by a t h i n l a y e r of e l e c t r o n - d e n s e m a t e r i a l , which s i b l y r e p r e s e n t e d some d e p o s i t i o n a t the agar s u r f a c e .  pos-  P i n o c y t o s i s on  the s u r f a c e s exposed t o the agar medium seemed more a c t i v e t h a n i n c u l t u r e s grown i n l i q u i d and t h e r e seemed t o be a r e o r i e n t a t i o n w i t h the c e l l s o f c y t o p l a s m i c f i l a m e n t s away from the s u r f a c e f a c i n g t h e medium and of o r g a n e l l e s toward t h e same s u r f a c e ( F i g . 1 1 0 ) . C o l o n i e s of b o t h l i n e s grew w e l l on p l a s t i c s h e e t i n g i n t h e presence of  agar, b u t . a f t e r a few days t h e i r edges r e t r a c t e d from the substratum  and formed  f o l d s and clumps.  Other changes i n c u l t u r e s grown on p l a s t i c  s h e e t i n g i n agar were s i m i l a r t o those observed i n a g a r - s u s p e n s i o n  80 but t h e y seemed i n c o m p l e t e and t e n i n g of s u r f a c e  c e l l s i n C-4c  i n many a r e a s and,  less evenly d i s t r i b u t e d . c u l t u r e s was  Thus, f l a t -  a g a i n reduced or absent  i n c o n t r a s t t o c u l t u r e s i n l i q u i d medium, the  w e r e ' f r e q u e n t l y o r i e n t e d s i m i l a r l y i n a l l l a y e r s of the cultures. terminal poorly  In c o r r e s p o n d i n g C - 4 s . c u l t u r e s ,  cells  stratified  t h e r e were no c y s t s  and  b a r s i n most i n t e r c e l l u l a r j u n c t i o n s were e i t h e r absent or  formed, but. the change was  a g a i n not as complete as i n agar  s u s p e n s i o n c u l t u r e s ( F i g s . 112-114). To determine whether the e f f e c t s of agar on the h i s t o g e n e s i s C-4  of  c e l l s were l i m i t e d t o i_n v i t r o c o n d i t i o n s , c e l l s grown i n hamsters  were compared t o t h o s e i n c u l t u r e , w i t h the c o n n e c t i v e t i s s u e i n the cheek pouches t a k e n t o r e p r e s e n t the s e m i s o l i d s u b s t r a t u m . v i o u s l y mentioned, the o r i e n t a t i o n , c e l l j u n c t i o n s and f i c a t i o n s of the C-4  c e l l s i n contact  i n contact tures.  surface  the p l a s t i c s h e e t i n g  modi-  rather  They a l s o were seen t o resemble t h o s e of the C-4  with, a g a r , p a r t i c u l a r l y as observed i n the  s t r a t e t h e i r _in v i v o response t o a l i q u i d phase, was  t o demon-  unsuccessful,  because of immunologic i n t o l e r a n c e , f o r i n s p i t e of  sone t r e a t m e n t no p e r i t o n e a l tumors grew and i n t h e s e a n i m a l s were r e j e c t e d .  cells  suspension c u l -  An attempt t o grow c e l l s as p e r i t o n e a l i m p l a n t s ,  apparently  pre-  w i t h c o n n e c t i v e t i s s u e resembled  those seen i n v i t r o i n c e l l s c o n t a c t i n g t h a n the medium.  As  corti-  a l l cheek pouch i m p l a n t s  However, some i n c i d e n t a l  observations  i n o t h e r cheek-pouch tumors gave an i n d i c a t i o n of the p o s s i b l e e f f e c t of a l i q u i d phase on the h i s t o g e n e s i s In n e c r o t i c p s e u d o c y s t s of C-4c  of the C-4  c e l l l i n e s i_n v i v o .  tumors, the c e l l s f a c i n g the  center  were f l a t t e n e d , s i m i l a r l y t o c e l l s f a c i n g l i q u i d medium i n c u l t u r e (Fig,  115).  In C-4s  tumors, c e l l s were j o i n e d by t e r m i n a l . b a r s  around  81  P l a t e XVI The e f f e c t o f e n v i r o n m e n t a l v i s c o s i t y on t h e morphology o f C-4  cells  F i g u r e 108:  C-4c a g a r - s u s p e n s i o n c u l t u r e . a l k a l i n e t o l u i d i n e b l u e . x470  Epon 812 embedding,  F i g u r e 109:  C-^4s a g a r - s u s p e n s i o n c u l t u r e . a l k a l i n e t o l u i d i n e b l u e . x470  Epon 812 embedding,  F i g u r e 110:  C-4c a g a r - s u s p e n s i o n c u l t u r e ; c e l l s a d j a c e n t t o a g a r . U r a n y l a c e t a t e and l e a d c i t r a t e . x l 2 0 0 0  F i g u r e 111:  C-4s a g a r - s u s p e n s i o n c u l t u r e ; c e l l s a d j a c e n t t o a g a r . U r a n y l a c e t a t e and l e a d c i t r a t e . x l l 5 0 0  F i g u r e 112:  C-4c c u l t u r e grown on p l a s t i c s h e e t i n g i n a g a r . Epon 812 embedding, a l k a l i n e . t o l u i d i n e b l u e . x470  F i g u r e s 113-114:  F i g u r e 115:  C-4s c u l t u r e grown on p l a s t i c s h e e t i n g . I n t e r c e l l u l a r j u n c t i o n a t the,growth medium. U r a n y l a c e t a t e and l e a d c i t r a t e . x3500Q  C-4c hamster cheek-pouch tumor, w a l l of p s e u d o - c y s t . H e m a t o x y l i n and e o s i n . x640  82 some i n t e r c e l l u l a r spaces ( F i g . 5 2 ) . These o b s e r v a t i o n s C-4c  i n d i c a t e t h a t f l a t t e n i n g of s u r f a c e c e l l s i n  c u l t u r e s and t e r m i n a l b a r f o r m a t i o n  i n C-4s c u l t u r e s are r e s p o n s e s  t o a l i q u i d environment, and t h a t b o t h a r e r e p l a c e d w i t h a more "open" system o f l o o s e l y o v e r l a p p i n g m i c r o v i l l i and p e r p e n d i c u l a r i n t e r c e l l u l a r spaces i n t h e presence o f a s o l i d o r s e m i s o l i d medium, i . e . p l a s t i c sheeting, agar.or connective  tissue.  Further,  increase  i n t h e v i s c o s i t y o f the c u l t u r e medium of c e l l s growing on p l a s t i c s h e e t i n g a l t e r s t h e i r o r i e n t a t i o n and reduces a d h e s i o n t c t h e s o l i d substratum.  F i n a l l y , i n c r e a s e d v i s c o s i t y of t h e growth medium may  a l s o a l t e r t h e appearance o f t h e e x t r a c e l l u l a r s u r f a c e coat and cause a r e d i s t r i b u t i o n of c y t o p l a s m i c  f i l a m e n t s and o r g a n e l l e s , which would  i m p l y t h a t t h e s e c h a r a c t e r i s t i c s were a l s o i n f l u e n c e d by changes i n microenvironment v i s c o s i t y .  83  Discussion Growth p a t t e r n s  of carcinoma c u l t u r e s depend on the i n t e r a c t i o n  t h o s e p r o p e r t i e s t h a t are a s s o c i a t e d w i t h the d i f f e r e n t i a t i o n of tissue  of o r i g i n and t h o s e p r o p e r t i e s t h a t r e s u l t  the c e l l s .  In the p r e s e n t study each of the two  have r e t a i n e d p r e d o m i n a n t l y c h a r a c t e r i s t i c s squamous d i f f e r e n t i a t i o n histogenetic variation when C-4s  was  the  i n the m a l i g n a n c y of cell  of one  (Ashworth e t a l . , . 1960;  of  l i n e s seemed t o  s p e c i f i c phase of  S n e l l , 1967).  This  seen most c l e a r l y i n l a t e r growth phases  cultures exhibited characteristics  of b a s a l c e l l s w h i l e  C-4c  c u l t u r e s r e t a i n e d p r o p e r t i e s of the more s u p e r f i c i a l s t r a t u m spinosum. Thus, the C-4s  c e l l s were columnar, w i t h a h i g h e r n u c l e o c y t o p l a s m i c  p o l a r i t y of o r g a n e l l e s , l i t t l e c e l l s and  dispersed  cytoplasmic  s t r a t i f i c a t i o n , no f l a t t e n i n g filaments.  In c o n t r a s t , C-4c  o v a l , s t r a t i f i e d and w i t h s u p e r f i c i a l c e l l s f l a t t e n e d , w i t h a lower n u c l e o c y t o p l a s m i c condensed i n t o bundles and two  cell  r a t i o and  l i n e s seemed t o be a r r e s t e d at one  particular  l o s t the f u l l h i s t o g e n e t i c p o t e n t i a l  l o s t the a b i l i t y t o undergo any  considered one  tissue  types  s t i m u l i , i . e . they  (Evans, 1966).  changes u s u a l l y Possibly then,  the observed _in v i t r o changes i n the two  population.  How-  of s t r a t i f i e d squamous e p i t h e l i u m  as the b e h a v i o u r .of a m a l i g n a n t b a s a l - c e l l spinous-cell  the  stage of squa-  i n d i c a t e d t h a t both c e l l  of the p r o g r e s s i v e  as squamous d i f f e r e n t i a t i o n  could i n t e r p r e t  Thus each of  able to p r o l i f e r a t e w i t h i n that stage.  t y p i c a l l y e x h i b i t e d i n response to connective had  polarity,  abundant f i l a m e n t s t h a t were  ever a l l in v i v o and j l n v i t r o o b s e r v a t i o n s had  superficial  c e l l s were  without  a s s o c i a t e d w i t h desmosomes.  mous, d i f f e r e n t i a t i o n but was  of  ratio,  population  and  cell  lines  of a m a l i g n a n t  84 I t i s p o s s i b l e , f o r example, t h a t t h e more e x t e n s i v e  adhesion t o  s u b s t r a t a , observed i n l i n e C-4s, c o u l d be due t o g r e a t e r c e l l deforma b i l i t y , as compared t o l i n e C-4c, a d i f f e r e n c e t h a t c o u l d be r e l a t e d t o t h e s t a g e s o f d i f f e r e n t i a t i o n a t which t h e two t y p e s o f C-4 c e l l s seemed f i x e d .  U l t r a s t r u c t u r a l observations  i n t h i s study and i n o t h e r s  ( S n e l l , 1967; Cooper e t a l . , 1967) showed t h a t squamous d i f f e r e n t i a t i o n i s a s s o c i a t e d w i t h i n t r a c e l l u l a r changes, v i z . , t h i c k e n i n g o f t h e c e l l membrane and r e o r i e n t a t i o n o f c y t o p l a s m i c  filaments, that  c o n t r i b u t e t o the i n c r e a s i n g r i g i d i t y of the c e l l s . from o b s e r v a t i o n s  likely  A l s o , i t i s known  on e x f o l i a t e d squamous c e l l s t h a t , i n s u s p e n s i o n ,  b a s a l c e l l s assume round shapes w h i l e t h e shapes o f o t h e r c e l l s become i n c r e a s i n g l y f i x e d as they d i f f e r e n t i a t e ( T a y l o r , 1967).. F u r t h e r , i t would seem r e a s o n a b l e t h a t under normal _in v i v o c o n d i t i o n s ,  contact  guidance (Weiss, 1958) would be a p r o p e r t y most i m p o r t a n t t o b a s a l s i n c e t h e s e c e l l s must e f f i c i e n t l y cover u n d e r l y i n g The  connective  c e l l s of t h e s p i n o u s l a y e r , however, s t r a t i f y , i . e . , t h e y  i s t i c a l l y move away from t h e c o n n e c t i v e  tissue. character  t i s s u e o r s u b s t r a t u m i n t o a pos  i t i o n o f a s s o c i a t i o n o n l y w i t h c e l l s o f t h e i r own k i n d .  I t i s evident  t h a t i n l i n e C-4c i t was t h i s l a t t e r k i n d o f a s s o c i a t i o n t h a t p r e f e r a b l y assumed, w h i l e  cells  cells  i n l i n e C-4s a s s o c i a t i o n w i t h t h e s u b s t r a t u m  was more dominant. Among u l t r a s t r u c t u r a l d i f f e r e n c e s between the c e l l  lines that  could  be s i m i l a r l y i n t e r p r e t e d where t h e more prominent i n t e r c e l l u l a r spaces i n l i n e C-4c and t h e d i f f e r e n t i n t e r c e l l u l a r j u n c t i o n s found a d j a c e n t t o growth medium i n t h e two c e l l l i n e s .  I t i s known t h a t  intercellular  spaces widen as c e l l s d i f f e r e n t i a t e from t h e b a s a l t o t h e more s u p e r ficial  l a y e r s o f squamous e p i t h e l i u m  (Ashworth e t ' a l . , I 9 6 0 ) .  Terminal  85 b a r s , or t i g h t j u n c t i o n s , are t h e t y p i c a l response  o f many c e l l s t o a  c a v i t y , i n c l u d i n g immature c u b o i d a l v a g i n a l e p i t h e l i u m (Cooper e t a l . , 1967), but they are not f o u n d . i n most squamous s t r a t i f i e d (Bloom and F a w c e t t ,  1962;  ent c e l l d e g e n e r a t i o n compared t o C-4c  Farquhar  and P a l a d e , 1963,  epithelia  1965).  and l i m i t e d s t r a t i f i c a t i o n of C-4s  The  promin-  cultures,  as  c u l t u r e s , c o u l d a l s o be r e l a t e d t o t h e d i f f e r e n c e s  between normal b a s a l and s p i n o u s c e l l s i n t h e i r p r o x i m i t y t o the o f m e t a b o l i c i n t e r c h a n g e , i . e . the c o n n e c t i v e t i s s u e .  site  I t would be of  i n t e r e s t t o a s c e r t a i n whether the d i f f e r e n c e i n g l y c o l y s i s p r e v i o u s l y observed ted  between t h e s e c e l l  l i n e s ( A u e r s p e r g , 1963)  and u s u a l l y a s s o c i a -  w i t h d i f f e r e n t l e v e l s of a n a p l a s i a (Burk e t a l . , 1967), c o u l d a l t e r *  n a t i v e l y be r e l a t e d t o a d i f f e r e n c e i n metabolism and spinous c e l l s .  In t h i s c o n n e c t i o n , the h i g h e r surface/volume  i n the s p i n o u s - l i k e C-4c  of C-4c  ratio  c e l l s s h o u l d f a c i l i t a t e exchanges w i t h the  environment and p e r m i t s u r v i v a l under more adverse t i o n s , which may  between normal b a s a l  n u t r i t i o n a l condi- .  w e l l be a f a c t o r i n the more e x t e n s i v e  stratification  cultures.  An i l l u s t r a t i o n o f a p o s s i b l e i n t e r a c t i o n i n h i s t o g e n e s i s of m a l i g nancy and. d i f f e r e n t i a t i o n was C-4 i.e.  p r o v i d e d by the morphologic  changes i n o l d  c u l t u r e s . . Crowding d i d not a r r e s t c e l l m u l t i p l i c a t i o n i n the C-4 t h e y d i d not e x h i b i t c o n t a c t i n h i b i t i o n of growth (Eagle and  1967), thus assuming a c h a r a c t e r i s t i c of m a l i g n a n c y . which t h e c e l l  l i n e s developed  Levine,  However, the means  t o c o n t i n u e m u l t i p l i c a t i o n under crowded  c o n d i t i o n s , i . e . t o escape from a c o n t r o l mechanism e f f e c t i v e i n ben i g n c e l l p o p u l a t i o n s , were d i f f e r e n t i n each c e l l l i n e and  could  a g a i n be i n t e r p r e t e d i n terms of stages of squamous d i f f e r e n t i a t i o n . The C-4s  lines,  c e l l s responded t o crowding  by s p r e a d i n g .  They s e p a r a t e d  86  from each o t h e r and from t h e s u b s t r a t u m .  _In v i v o , t h i s change r e s u l t e d  i n an i n c r e a s e d p o t e n t i a l area o f c o n t a c t w i t h u n d e r l y i n g c o n n e c t i v e t i s s u e s , which was e x p l o i t e d by t h e s p r e a d i n g o f f r e e d cancer c e l l s  into  new a d j a c e n t a r e a s o f c o n n e c t i v e t i s s u e as w e l l as by p e n e t r a t i o n o f c o n n e c t i v e t i s s u e i n t o spaces between cancer c e l l s .  Thus, t h e b a s i c  means o f c o n t i n u i n g growth i n t h i s tumor was by an i n c r e a s e i n c e l l s u b s t r a t u m a s s o c i a t i o n , a p r o c e s s i n which c o n t a c t guidance may be o f considerable importance. as e f f i c i e n t . had formed  In c u l t u r e , however, t h i s mechanism was not  C e l l s e p a r a t i o n here was impeded by t e r m i n a l b a r s t h a t .  i n response t o t h e l i q u i d medium, and thus c y s t s were d e v e l o p e d .  B u t , more i m p o r t a n t , c e l l s e p a r a t i o n d i d not r e s u l t i n t h e appearance o f a d d i t i o n a l s u b s t r a t u m so t h a t o n l y more c r o w d i n g , a s s o c i a t e d w i t h c e l l d e g e n e r a t i o n , took p l a c e i f C-4s c u l t u r e s were m a i n t a i n e d beyond t h e s t a t i o n a r y phase.  On t h e o t h e r hand, C-4c c e l l s responded t o  crowding by s t r a t i f i c a t i o n .  _In y i y o . t h i s growth p a t t e r n r e s u l t e d i n  the f o r m a t i o n o f compact expanding tumor masses, i n which c o n t a c t w i t h , c o n n e c t i v e t i s s u e became r e l a t i v e l y reduced as t h e tumors i n c r e a s e d i n s i z e , and i n which i n v a s i o n l i k e l y f o l l o w e d p l a n e s o f l e a s t tance t o p r e s s u r e .  I f , i_n v i v o , c e l l rearrangements  resis-  analogous t o t h e  r e t r a c t i o n of o l d C-4c c u l t u r e s s h o u l d t a k e p l a c e , t h e n t h e s e would a l t e r t h e c o n f o r m a t i o n of t h e tumor masses and t h e d i s t r i b u t i o n o f pressures a g a i n s t the surrounding host t i s s u e s . f i c a t i o n o f C-4c c e l l s produced  In c u l t u r e , the s t r a t i - '  c o h e s i v e c e l l sheets w i t h c o n s i d e r a b l e  t e n s i l e s t r e n g t h , thus r e s e m b l i n g d i f f e r e n t i a t e d squamous e p i t h e l i u m . The  P A S - p o s i t i v e , basement-membrane-like m a t e r i a l d e p o s i t e d more  e x t e n s i v e l y JLn v i v o around C-4s tumors t h a n around C-4c tumors,  would  appear t o be another d e m o n s t r a t i o n of t h e more " b a s a l - c e l l " l i k e c h a r -  •  87  a c t e r of the former l i n e , as would the prominent c o n n e c t i v e infiltration  i n C-4s  tumors.  The  tissue  o t h e r observed h i s t o l o g i c a l  ences between hamster cheek pouch tumors of the two  cell  differ-  l i n e s may  t h e secondary r e s u l t s of the d i f f e r e n c e s i n growth p a t t e r n s ,  be  more ex-  t e n s i v e c o n t a c t w i t h h o s t t i s s u e s in.C-4s tumors c o u l d have been r e s p o n s i b l e f o r b e t t e r n u t r i t i o n and this cell  line.  v/ere l i k e l y distance  thus f o r absence of n e c r o s i s i n tumors of  C o n v e r s e l y , the c e n t r a l f o c i of n e c r o s i s i n C-4c  the r e s u l t of m e t a b o l i c  from c o n n e c t i v e  f o c i were due  tissue.  d e f i c i e n c i e s a r i s i n g from i n c r e a s i n g I t may  be t h a t w h i l e t h e s e n e c r o t i c  to n u t r i t i o n a l f a c t o r s , c e l l degeneration associated  i n f l a m m a t o r y r e a c t i o n s i n both c e l l l i n e s was by the  tumors  with  p a r t of the r e j e c t i o n p r o c e s s  host.  A l t h o u g h many of the d i f f e r e n c e s observed between the two C-4 l i n e s c o u l d be  i n t e r p r e t e d as v a r i a t i o n s i n d i f f e r e n t i a t i o n ,  cell  other  u l t r a s t r u c t u r a l and h i s t o g e n e t i c c h a r a c t e r i s t i c s observed i n t h i s t i g a t i o n were a s s o c i a t e d w i t h m a l i g n a n c y . morphological  While there  inves-  i s no s i n g l e  f e a t u r e t y p i c a l of c a n c e r , tumor c e l l s do t e n d t o  c h a r a c t e r i z e d by a c o m b i n a t i o n of f a c t o r s , p r e s e n t t o v a r i o u s  be  degrees,  which g e n e r a l l y seem t o be more t y p i c a l i n more a n a p l a s t i c tumors ( O b e r l i n g and B e r n h a r d , 1961). nucleocytoplasmic c h r o m a t i n and  These f e a t u r e s i n c l u d e an i n c r e a s e  r a t i o , abnormal n u c l e a r shapes w i t h clumping of  prominent n u c l e o l i , a g e n e r a l  p a u c i t y of o r g a n e l l e s , a  p a r t i c u l a r l y n o t i c e a b l e decrease i n the"extent and  u s u a l l y i n the  free ribosomes.  in  s i z e and  The  C-4  of endoplasmic r e t i c u l u m  number of m i t o c h o n d r i a ,  and  an i n c r e a s e  in  c e l l s of b o t h l i n e s showed most of t h e s e f e a -  t u r e s , w i t h the p a u c i t y of o r g a n e l l e s , and p o s s i b l y b e i n g more d e f i n i t e i n l i n e C-4c.  of m i t o c h o n d r i a  In p a r t i c u l a r ,  More s p e c i f i c a l l y ,  experimental  88  and  spontaneous i n v a s i v e squamous carcinomas i n animals  and man  are  c h a r a c t e r i z e d by i m p e r f e c t or absent basement membranes, i n c r e a s e d i n t e r c e l l u l a r spaces and m i c r o v i l l u s f o r m a t i o n , and a r e d u c t i o n i n the number and r e g u l a r i t y of•desmosomes (Ashworth e t a l . , 1960; Webber, 1966; 1967).  Luibel et a l . ,  A l l of t h e s e  1960;  Borland  S c h r o d t and Foreman, 1965;  f e a t u r e s were found i n the C-4  Tarin,  l i n e s , but p o s s i b l y  more s i g n i f i c a n t l y , t h e y were a l s o a l l more prominent i n l i n e Thus, on the b a s i s of b o t h u l t r a s t r u c t u r e and l i n e C-4c  c o u l d be c o n s i d e r e d  t o be b o t h more a n a p l a s t i c and more the i n c r e a s e i n  i n t e r c e l l u l a r s p a c e s , c o u l d be i n t e r p r e t e d as e i t h e r due  present  The  C-4c.  intercellular organization,  "squamous", so t h a t a. number of o b s e r v a t i o n s , e.g.,  t i o n , or t o a n a p l a s i a .  and  d i f f e r e n c e s between the C-4  to d i f f e r e n t i a l i n e s might r e -  an example o f tumor p r o g r e s s i o n which i s a c h i e v e d  not by l o s s of  t i s s u e s p e c i f i c p r o p e r t i e s but by s e l e c t i o n of c e l l p o p u l a t i o n s a t d i f f e r e n t s t a g e s of d i f f e r e n t i a t i o n . The  observations  i n the C-4  c u l t u r e s were a l s o a n a l y s e d  in relation  t o the in v i t r o h i s t o g e n e s i s o f normal squamous e p i t h e l i u m (Gey  et . a l . ,  1952;  1967;  Matolsky,  1960;  Prose e t a l . , 1967;  Flaxman e t a_l., 1968).  P r e v i o u s o b s e r v a t i o n s , which were c o n f i r m e d  s t r u c t u r a l l y i n the p r e s e n t e p i t h e l i u m i n primary  J e p s e n and T h e i l a d e ,  i n v e s t i g a t i o n , had  ultra-  shown t h a t normal c e r v i c a l  c u l t u r e undergoes m o r p h o l o g i c changes i n d i c a t i v e  of squamous d i f f e r e n t i a t i o n , and t h a t c o n t a c t w i t h an e x p l a n t i s a p r e r e q u i s i t e f o r such o r g a n i z e d Worth, 1966).  The  growth and d i f f e r e n t i a t i o n (Auersperg  resemblance of the b e n i g n squamous e p i t h e l i a l  growths t o young C-4  c u l t u r e s i n the o r i e n t a t i o n of c e l l s ,  spaces and o r g a n e l l e s , i n d i c a t e d t h a t a l l these  and out-  intercellular  f a c t o r s are due  t o the  sue c u l t u r e environment and are not s p e c i f i c f o r the tumor c e l l s .  In  tis-  89 a d d i t i o n , s i n c e s t r a t i f i c a t i o n was observed i n d i f f e r e n t i a t i n g epithelium  i n c u l t u r e , i t appears t h a t t h e more e x t e n s i v e  normal .  stratification  i n l i n e C-4c, as compared t o l i n e C-4s, can not be t a k e n as e v i d e n c e o f greater anaplasia. a normal p r o p e r t y  On t h e c o n t r a r y ,  t h e r e t e n t i o n of  o f squamous e p i t h e l i u m i n c u l t u r e , more prominent i n  C-4c c e l l s because of t h e i r l i n e C-4s, and a g a i n cells.  i t may r e p r e s e n t  g r e a t e r c o h e s i v e c a p a c i t y as compared t o  suggestive  o f t h e more squamous c h a r a c t e r  o f C-4c  This i n t e r p r e t a t i o n of d i f f e r e n c e s i n the s t r a t i f i c a t i o n of  m a l i g n a n t c u l t u r e s as b e i n g due t o d i f f e r e n t h i s t o g e n e t i c p o t e n t i a l s may be c o n s i d e r e d  as an a l t e r n a t i v e t o t h e hypotheses o f s t r a t i f i c a t i o n  b e i n g due t o t h e l o s s o f c o n t a c t  inhibition,  or due t o d i m i n u t i o n o f  a d h e s i o n t o s u b s t r a t a , (Abercrombie, .1966), as has been observed i n "spontaneous" t r a n s f o r m a t i o n cells  i n t i s s u e c u l t u r e and v i r u s t r a n s f o r m e d tumor  ( S t o k e r , 1964; S a n f o r d e t aJL., 1967; S a n f o r d and Hoeman, 1967;  Ternin, 1966) as w e l l as i n c e l l s t r a n s f o r m e d by c a r c i n o g e n s and S a c h s , 1965).  In r e l a t i o n  t o t h e o f t e n remarked g e n e r a l i z a t i o n  t h a t m a l i g n a n t c e l l s are l e s s c o h e s i v e t h a n normal c e l l s it  should  be p o i n t e d  (Eerwald.  (Ambrose, 1966)  out t h a t i n t h e p r e s e n t i n v e s t i g a t i o n b e n i g n squamous  c e l l s growing w i t h o u t e x p l a n t s were l e s s c o h e s i v e than e i t h e r C-4c c e l l s or C-4s c e l l s .  Finally,  s i n c e some c e r v i c a l  carcinomas do  differentiate  i n p r i m a r y c u l t u r e (Gey e t a l . , 1952; A u e r s p e r g and Worth, 1966) and s i n c e squamous d i f f e r e n t i a t i o n  of normal c e l l s i n c u l t u r e can be p r e v e n t -  ed by removal of t h e e x p l a n t s ,  i t follows that lack of  i n t h e C-4 c u l t u r e s cannot be s i m p l y t h e i r malignant nature.  differentiation  i n t e r p r e t e d as an e x p r e s s i o n o f  I t appears t h e n , t h a t t h e o n l y major d i f f e r e n c e  observed between b e n i g n squamous c e l l s and t h e C-4 c e l l s i n c u l t u r e i n the a b i l i t y of the l a t t e r t o maintain  an i n t e r c e l l u l a r  lies  organization  90 i n the absence of o t h e r s u p p o r t i n g The  tissues.  h i s t o g e n e t i c changes i n the two  cell  l i n e s c o u l d be  analysed  c  f u r t h e r from the v i e w p o i n t of i n t e r a c t i n g f o r c e s d e t e r m i n i n g the o r g a n i z a t i o n of c e l l s i n c u l t u r e .  C e l l surface tension, c e l l u l a r  and c e l l u l a r r i g i d i t y are f o r c e s which r e s i s t d e f o r m a t i o n c e l l s and o f c e l l groups, w h i l e c e l l a d h e s i o n associated with c e l l deformation the c o m p e t i t i v e p r o c e s s e s adhesion.  processes  elasticity  of  individual  are u s u a l l y  and they can be f u r t h e r d i v i d e d i n t o  of i n t e r c e l l u l a r adhesion  and c e l l - s u b s t r a t u m  Many of the m o r p h o l o g i c v a r i a t i o n s observed  i n the C-4  cul-  t u r e s c o u l d be i n t e r p r e t e d i n terms of changes i n the i n t e r a c t i o n of t h e s e f o r c e s , as m o d i f i e d by the s p e c i f i c p r o p e r t i e s i n h e r e n t i n each cell  line. The C-4  c e l l s , i n common w i t h o t h e r s , were round and had  m i c r o v i l l i on t h e i r s u r f a c e s when s i n g l e and  i n suspension.  numerous This  may  r e p r e s e n t the most s t a b l e c o n f i g u r a t i o n of these c e l l s i n i s o l a t i o n , and c o n t a c t w i t h o t h e r systems, beyond i n t e r d i g i t a t i o n of p r e - e x i s t i n g m i c r o v i l l i , would t h e r e f o r e r e q u i r e d e f o r m a t l o n  of t h i s c o n f i g u r a t i o n .  I t has been shown i n systems as d i v e r s e as c e l l a g g r e g a t i o n i n embryonic development ( L e s s e n s , 1963), c e l l  f u s i o n by v i r u s e s (Schneeberger  H a r r i s , 1966), i n b l a s t o c y s t i m p l a n t a t i o n (Mayer et al., c e l l attachment _in v i t r o  ( F i s h e r and Cooper, 1967)  1967)  i s thought t o f a c i l i t a t e i n i t i a l  and i n  that contact i s i n i t i a t -  ed by m i c r o v i l l i and then extended over a d j a c e n t c e l l s u r f a c e This process  and  areas.  c l o s e c o n t a c t between  c e l l s , by r e d u c i n g the r e p u l s i v e energy which i s p r o p o r t i o n a l t o the r a d i u s of c u r v a t u r e of approaching I960).  cell  s u r f a c e s (Bangham and P e t h i c a ,  I t has been shown a l s o t h a t , w h i l e i n i t i a l c o n t a c t i n embryonic  development i s based on c a l c i u m b o n d i n g , subsequent, more e x t e n s i v e  and  91 permanent c e l l a d h e s i o n i n v o l v e s s y n t h e s i s of e x t r a c e l l u l a r ( S t a b l e f o r d , 1967).  protein  The e x p e r i m e n t s i n the p r e s e n t i n v e s t i g a t i o n  showed, t h a t i n both C-4  l i n e s t r y o s i n - s e n s i t i v e p r o t e i n s were p r e -  dominantly involved i n c e l l  substratum a d h e s i o n , w h i l e d i v a l e n t  were r e s p o n s i b l e f o r the maintenance  of i n t e r c e l l u l a r  cations  organization.  T h i s d i f f e r e n c e between the two t y p e s o f c e l l a d h e s i o n was  shown not  o n l y by the e f f e c t s of t r y p s i n d i g e s t i o n and of d i v a l e n t c a t i o n d e p l e t i o n , but a l s o by the o b s e r v a t i o n t h a t a t a l l t i m e s a f t e r c e l l  dis-  p e r s i o n , a g g r e g a t i o n o f c e l l s would occur i m m e d i a t e l y , but a d h e s i o n to  the s u b s t r a t u m r e q u i r e d one or two days, s u g g e s t i n g t h a t  synthetic  p r o c e s s e s may  be i n v o l v e d i n the l a t t e r but not i n the former t y p e  of  The dependence on d i v a l e n t i o n s f o r the maintenance  adhesion.  t h e i n t e r c e l l u l a r o r g a n i z a t i o n of C-4  of  c o l o n i e s c o n c u r s w i t h the e l e c t r o n  m i c r o s c o p i c o b s e r v a t i o n s t h a t most of the i n t e r c e l l u l a r , c o n t a c t s , p a r t i c u l a r l y i n l i n e C-4c,  d i d not proceed beyond a d h e r i n g m i c r o v i l l i ,  s u g g e s t i n g t h a t the c e l l s were not c a p a b l e of a change i n c e l l ation.  The C-4s  configur-  c e l l s d i d seem t o have a c a p a c i t y t o form somewhat  more e x t e n s i v e i n t e r c e l l u l a r a s s o c i a t i o n s , but a d j a c e n t C-4c a s s o c i a t e d almost e x c l u s i v e l y by m i c r o v i l l i , as i f s t i l l  cells  in isolation.  T h e r e f o r e , a l t h o u g h t h e s e c a n c e r c e l l s were not i s o l a t e d from one  another  by h i g h n e g a t i v e charges or a m u c o p r o t e i n coat (Ambrose, 1966; Temin, 1966), t h e y s t i l l may of  have been i s o l a t e d , as compared t o normal c e l l s , because  t h e i r i n a b i l i t y t o expand i n t e r c e l l u l a r c o n t a c t s beyond those of  m i c r o v i l l i , , i . e . because of a d e f e c t i n d e f o r m a b i l i t y . between t h e c e l l  The  variation  l i n e s i n the c a p a c i t y t o deform would e x p l a i n such  o b s e r v a t i o n s as the more r a p i d a d h e s i o n and s p r e a d i n g of s u b c u l t u r e d C-4s  c e l l s and. the more c o n s p i c u o u s e f f e c t of c o n t a c t guidance i n t h i s  92 same l i n e . o f C-4c  The  more e x t e n s i v e m i c r o v i l l u s f o r m a t i o n on the  c e l l s may  surfaces  a l s o e x p l a i n the s t r o n g e r c o h e s i o n between t h e s e  since t h i s cohesion  c o u l d be the r e s u l t of the l a r g e r area of  cells  contact  r a t h e r t h a n n e c e s s a r i l y t h e r e s u l t of any q u a l i t a t i v e d i f f e r e n c e between c e l l surface p r o p e r t i e s .  Such a d i f f e r e n c e i n c e l l s u r f a c e  conformation  c o u l d a l s o account f o r the apparent d i s c r e p a n c y between the somewhat more i n t e n s e h i s t o c h e m i c a l s t a i n i n g of mucosubstances of C-4c  cell  surfaces,  and the v e r y s i m i l a r s t a i n i n g of b o t h l i n e s on the u l t r a s t r u c t u r a l R e s u l t s i n t h i s i n v e s t i g a t i o n and  level.  others i n d i c a t e that t r y p s i n - s e n s i - •  t i v e p r o t e i n s of the c e l l s u r f a c e mask a c i d groups, p a r t i c u l a r l y  those  a s s o c i a t e d w i t h s i a l i c a c i d , and t h a t the e x t e n t of the masking i n cancer c e l l s can v a r y from p a r t i a l t o complete ( G a s i c and Baydak, 1962; e t a l . , 1965).  The  Gasic  absence of s t a i n i n g f o r a c i d mucosubstances on  C-4  c e l l s u r f a c e s i n c o n t a c t w i t h g l a s s or w i t h o t h e r c e l l s suggests t h a t a d h e s i o n i n v o l v e d a m o d i f i c a t i o n of a c i d groups s i n c e such s t a i n i n g was present  on unattached  p r e s e n t was  cell  surfaces.  That masked a c i d groups were  a l s o i n d i c a t e d by the s t a i n i n g of a l l c e l l s u r f a c e s i n  a l d e h y d e - f i x e d , but not i n Carnoy's f i x e d , c e l l s , and a f t e r t r y p s i n digestion.  More d i r e c t m o r p h o l o g i c e v i d e n c e of t h i s v a r i a t i o n was  i n the e l e c t r o n m i c r o s c o p i c where i t was  examination  found t h a t on f r e e c e l l  s p a c e s , s t a i n i n g was  of c o l l o i d a l  s u r f a c e s , and  seen  iron staining  i n large  intercellular  d i s t r i b u t e d along s t r a i g h t h a i r l i k e f i l a m e n t s ,  w h i l e i n spaces between c l o s e l y a d j a c e n t c e l l s , the s t a i n e d m a t e r i a l was  more f l a t , i r r e g u l a r and g r a n u l a r .  of the c e l l coat f i l a m e n t s c o u l d be due  On f r e e s u r f a c e s the  t o t h e i r h i g h e r charge d e n s i t y  w h i l e t h e i r c o l l a p s e i n l i m i t e d spaces c o u l d be due charges,  extension  p o s s i b l y by d i f f u s i b l e p r o t e i n s .  t o a masking of the  93 The  f a s t e r r a t e of spreading  i n C-4s c e l l s , c o u l d between t h e c e l l  on g l a s s , a f t e r t r y p s i n d i g e s t i o n ,  imply t h a t t h e e x t r a c e l l u l a r p r o t e i n i s s i m i l a r  l i n e s but l e s s m a t e r i a l i s removed i n C-4s d u r i n g t h e  t r y p s i n t r e a t m e n t , or t h a t C-4s c e l l s r e s y n t h e s i z e  the p r o t e i n at a  f a s t e r r a t e , or t h a t C-4s c e l l s r e q u i r e l e s s e x t r a c e l l u l a r p r o t e i n because t h e y are more deformable p e r s e . The s i m i l a r i t y i n t h e p a t t e r n and  r a t e s o f a c t i o n o f t r y p s i n i n the two c e l l  l i n e s , and u l t r a s t r u c -  t u r a l s i m i l a r i t y o f t h e c e l l s u r f a c e s do not l e n d s u p p o r t t o t h e f i r s t p o s s i b i l i t y , a l t h o u g h i t cannot be e x c l u d e d .  A more r a p i d r a t e o f  r e s y n t h e s i s might be suggested by t h e more prominent G o l g i complexes i n t h e l i n e C-4s, w h i l e g r e a t e r  i n t r i n s i c d e f o r m a b i l i t y o f C-4s c e l l s  c o u l d be t h e r e s u l t of v a r i o u s changes, some of w h i c h were i n t h i s study.  considered  Thus C-4s c e l l s c o u l d be more deformable i f they had  a lower c e l l s u r f a c e c h a r g e , or a lower c e l l s u r f a c e c a l c i u m  concen-  t r a t i o n (Weiss, 1967), or i f t h e y were more p l i a b l e t h a n C-4c c e l l s because o f d i f f e r e n c e s i n i n t r a c e l l u l a r s t r u c t u r e . ' I t was shown i n t h e p r e s e n t study t h a t s u r f a c e charges between t h e two c e l l  lines  were not d i f f e r e n t b u t , a l t h o u g h i t was f u r t h e r shown t h a t d i v a l e n t c a t i o n s were i n v o l v e d i n t h e maintenance of non-round shapes i n b o t h cell  l i n e s , a d i f f e r e n c e i n e f f e c t s o f these i o n s between t h e two c e l l  l i n e s was n e i t h e r demonstrated nor r u l e d o u t . I t i s p o s s i b l e t h a t one of t h e reasons f o r t h e decreased deforma b i l i t y o f C-4c c e l l s can be found i n t h e d i f f e r e n t c y t o p l a s m i c t u r e of t h e s e c e l l s , i n p a r t i c u l a r i n t h e more prominent  struc-  cytoplasmic  f i l a m e n t s which c o u l d be expected t o i n c r e a s e e l a s t i c i t y and t h u s reduce d e f o r r n a b i l i t y . C y t o p l a s m i c f i l a m e n t s , thought f o r a long time (Lewis and L e w i s , 1924; Bang and Gey, 1948) t o have, s u p p o r t i v e ,  tensile  94 or c o n t r a c t i l e f u n c t i o n s  (Buckley  and P o r t e r , 1967; Clermont and  P e r e i r a , 1966; C l o n e y , 1966), were c h a r a c t e r i s t i c a l l y o r i e n t e d a l o n g t h e main a x i s o f t h e C-4 c e l l s .  T h i s should r e s u l t i n t e n s i o n along  this  p l a n e o f o r i e n t a t i o n i n c e l l s t h a t were adherent, b u t i n s h o r t e n i n g and  eventual  r o u n d i n g o f c e l l s t h a t were f r e e o f a t t a c h m e n t s .  Some o f t h e  changes observed i n t h e l a t e r growth phases o f C-4 c u l t u r e s c o u l d be i n t e r p r e t e d as an i n t e r a c t i o n between t h e t e n s i l e s t r e n g t h due t o f i l a ments and i n t e r c e l l u l a r and c e l l - s u b s t r a t u m  adhesion.  When C-4s c e l l s  became crowded, they s e p a r a t e d from each o t h e r and then from t h e s u b s t r a tum,  u n t i l they were a t t a c h e d  t o t h e medium.  o n l y by t e r m i n a l b a r s , o r were r e l e a s e d i n -  I t had not been p o s s i b l e t o show e x p e r i m e n t a l l y  that the  s e p a r a t i o n o f c e l l s was due t o a c c u m u l a t i o n o f m a t e r i a l s i n i n t e r c e l l u l a r spaces.  The a l t e r n a t i v e p o s s i b i l i t y was t h a t , i n t h e s t a t i o n a r y phase,  i n t r a c e l l u l a r t e n s i o n began t o exceed a d h e s i o n and r e s u l t e d i n a rounding  of t h e C-4s c e l l s , which should be l e a s t e f f e c t i v e a t t h e s u r f a c e  f a c i n g t h e medium s i n c e these a p i c a l r e g i o n s were connected b a r s w i t h f i l a m e n t s arranged p a r a l l e l t o t h e c e l l s u r f a c e . e x a c t p a t t e r n observed i n t h e c u l t u r e s . cess of c e l l  separation  by.terminal T h i s was t h e  I t i s not known whether t h i s  i n v o l v e d a decrease i n c e l l a d h e s i o n , an i n c r e a s e  i n i n t r a c e l l u l a r tension, or both.  The r e t r a c t i o n o f o l d C-4c c u l t u r e s  a l s o i n v o l v e d a process i n which t h e o r g a n i z a t i o n o f c e l l  colonies,  based on an e q u i l i b r i u m between a d h e s i v e f a c t o r s and i n t r a c e l l u l a r was a l t e r e d by an excess o f t e n s i l e s t r e n g t h .  a g a i n as a t i s -  sue r a t h e r t h a n as s i n g l e c e l l s . observations  tension,  B u t t h e main d i f f e r e n c e  was t h a t , i n c o n t r a s t t o C-4s c e l l s , t h e C-4c c e l l s acted  The  pro-  on changes i n t h e c o n f i g u r a t i o n o f c e l l  f o l l o w i n g removal from g l a s s gave f u r t h e r supoort t o t h e i d e a  sheets that  95 t e n s i o n due  to cytoplasmic  f i l a m e n t s p l a y s an i m p o r t a n t r o l e i n the.  maintenance of c u l t u r e morphology.  The  c c n f i g u r a t i o n a l change of  c e l l s h e e t s d u r i n g the f i r s t hour a f t e r r e l e a s e from the g l a s s l i k e l y the e f f e c t of a sudden excess of i n t r a c e l l u l a r t e n s i l e  the  was forces  which i n s i t u i n t e r a c t w i t h e x t r a c e l l u l a r a d h e s i v e f a c t o r s i n the maintenance of c e l l o r g a n i z a t i o n w i t h i n c o l o n i e s .  The  shape and u l t r a s t r u c -  t u r e of the c e l l s h e e t s i n d i c a t e d t h a t these i n t r a c e l l u l a r f o r c e s peared t o be most i n t e n s e near the s o l i d s u b s t r a t u m and ward the l i q u i d medium, t h a t they acted  ap-  diminished  p r e d o m i n a n t l y a l o n g the  to-  axis  p a r a l l e l t o the growth s u r f a c e , and t h a t t h e y l i k e l y i n v o l v e d the t i o n or rearrangement of c y t o p l a s m i c axis.  The  f i l a m e n t s o r i e n t e d a l o n g the same  appearance of dense f i l a m e n t bands i n r e g i o n s  n a r r o w i n g of C-4c  of  greatest  c e l l s p a r t i c u l a r l y bore a s t r i k i n g resemblance t o  s i m i l a r s t r u c t u r e s seen i n a c t i v e l y c o n t r a c t i n g e p i t h e l i a l c e l l s 1966), was  That t h i s c o n t r a c t i o n was  l i k e l y due  the g l a s s .  contrac-  (Cloney,  not permanent a l s o suggests t h a t :'t  t o an imbalance produced by the r e l e a s e of the c e l l s  I t may  from  be t h a t the arrangement of f i l a m e n t s i n response t o  a d h e s i o n t o the s u b s t r a t u m i s i n f a c t a response t o the l a y e r of e x t r a c e l l u l a r material.deposited  by the c e l l s i n the p r o c e s s of a d h e s i o n  r e t a i n e d , at l e a s t i n p a r t , d u r i n g removal of c e l l s from the  and  glass.  As t h i s m a t e r i a l i s g r a d u a l l y l o s t f o l l o w i n g s u s p e n s i o n of the  cells,  an imbalance between the d e f o r m i n g e f f e c t of t h i s m a t e r i a l and  the  t e n s i l e e f f e c t of the c e l l sheets. c e l l s had  The  f i l a m e n t s i s e x p r e s s e d i n the r o l l i n g up of  observation  t h a t , a f t e r t h r e e days i n c u l t u r e , the  resumed t h e i r r e g u l a r morphology and  t h i s imbalance can be  the  o r i e n t a t i o n suggests t h a t  .corrected.  E x p e r i m e n t s w i t h agar showed t h a t s u r f a c e m o d i f i c a t i o n s and  intercel-  96 l u l a r o r g a n i z a t i o n of e p i t h e l i a l c e l l s can be m o d i f i e d by p h y s i c a l environmental f a c t o r s .  In g e n e r a l , t e r m i n a l b a r f o r m a t i o n and f l a t t e n i n g  of s u p e r f i c i a l squamous c e l l s a r e t h e two a l t e r n a t i v e r e s p o n s e s t o a c a v i t y . I t was demonstrated  i n t h e p r e s e n t study t h a t the f o r m a t i o n _in v i t r o o f  t h e s e m o d i f i c a t i o n s o f c e l l form and s u r f a c e can be e x p e r i m e n t a l l y  con-  t r o l l e d by v a r i a t i o n s i n t h e v i s c o s i t y of t h e medium and t h a t c e l l s can r e t a i n t h e c a p a c i t y t o form t e r m i n a l b a r s over y e a r s i n c u l t u r e .  In the  C-4 c u l t u r e s , a d d i t i o n o f agar t o t h e medium changed t h e response o f t h e c e l l s from t h e u l t r a s t r u c t u r a l m o d i f i c a t i o n s seen a t a c a v i t y t o those found a t s u b s t r a t a , and t h e growth p a t t e r n of t h e C-4s c e l l l i n e was modi f i e d by t h i s change of e n v i r o n m e n t a l d e n s i t y t o a more compact with l e s s c e l l degeneration. observation  This l a t t e r observation,  growth  t o g e t h e r w i t h the  t h a t C-4s c e l l s i n hamster tumors a l s o c o u l d a c h i e v e  consid-  e r a b l e s t r a t i f i c a t i o n w i t h o u t e v i d e n c e of c e l l d e g e n e r a t i o n suggested  that  the t e r m i n a l b a r s formed i n l i q u i d growth medium must p r e s e n t a b a r r i e r t o m e t a b o l i c exchange i n C-4s c u l t u r e s as they do i n o t h e r systems (Farquhar and P a l a d e , 1965).  T h i s idea was f u r t h e r supported by t h e o b s e r v -  a t i o n t h a t p i n o c y t o s i s i n both c e l l more e x t e n s i v e  l i n e s , i n l i q u i d medium, was much  i n i n t e r c e l l u l a r spaces than on t h e s u r f a c e  f a c i n g the  medium, b u t t h a t t h i s apparent p o l a r i t y of exchanges w i t h t h e environment c o u l d be e l i m i n a t e d by a d d i t i o n of a g a r .  However, t h e comparison  of the  r a t e s of f e r r i t i n d i f f u s i o n i n t o i n t e r c e l l u l a r spaces d i d not s u p p o r t the r o l e of t e r m i n a l bars i n l i n e C-4s as b a r r i e r s t o d i f f u s i o n . s i b l e explanation  f o r t h i s d i s c r e p a n c y may be t h a t t h e t e r m i n a l  A posbars  were i n c o m p l e t e but whatever t h e mechanisms i n v o l v e d , t h e r e s u l t s of t h e f e r r i t i n experiments do not p e r m i t t h e c o n c l u s i o n t h a t t h e l i m i t e d s t r a t i f i c a t i o n i n l i q u i d c u l t u r e medium i n l i n e C-4s was due t o r e s t r i c t e d  97 intercellular circulation.  An a l t e r n a t i v e e x p l a n a t i o n f o r t h i s  limit-  a t i o n and f o r t h e more compact growth o f C-4s c e l l s i n agar and i n v i v o may l i e i n t h e f a c t t h a t , i n a l i q u i d medium, t h e m e t a b o l i c r a t e of c e l l s would be expected t o be h i g h e r because o f l o s s of m e t a b o l i t e s by leakage to  ( E a g l e and P i e z , 1962) and t h a t C-4s c e l l s might be more s e n s i t i v e  t h i s f a c t o r than C-4c c e l l s .  of extensive c e l l degeneration  T h i s c o u l d a l s o e x p l a i n why, i n s p i t e i n o l d C-4s c u l t u r e s , concomitant  growth  o f h e a l t h y c e l l s c o n t i n u e d as such growth would be t a k i n g p l a c e i n a h i g h l y c o n d i t i o n e d medium.  An - a d d i t i o n a l e f f e c t of agar was i t s r e d u c -  t i o n o f adhesion o f t h e c e l l s t o t h e substratum when i n c e l l  sheets,  r e s u l t i n g i n a pronounced tendency o f the c e l l s t o l e a v e t h e g l a s s surface.  C o n v e r s e l y , t h e e f f e c t s of agar on s u p e r f i c i a l c e l l s were  a l s o reduced  under these c o n d i t i o n s . These two o b s e r v a t i o n s  t h a t agar and s o l i d substratum  suggest  c o u n t e r a c t e d each o t h e r , p r o b a b l y because  a d d i t i o n of agar i n c r e a s e s t h e s i m i l a r i t y o f t h e  microenvironments  on t h e two s i d e s of t h e c e l l s h e e t s , thus d i m i n i s h i n g t h e d i f f e r e n t i a l p r o p e r t i e s o f t h e two opposing for  c e l l s u r f a c e s t h a t are l i k e l y  c e l l p o l a r i t y and subsequent  necessary  adhesion.  F i n a l l y , t h e q u e s t i o n of t h e r e l e v a n c e of t h e s e f i n d i n g s t o h i s t o g e n e s i s i n g e n e r a l and t o t h e u n d e r s t a n d i n g  of t h e h i s t o g e n e s i s of  cancer  I t would seem t h a t h i s t o -  i n p a r t i c u l a r s h o u l d be c o n s i d e r e d .  g e n e t i c d i f f e r e n c e s bet/ween normal and m a l i g n a n t  t i s s u e s a r e due t o  m o d i f i c a t i o n s of t h e same b a s i c mechanisms and t h a t t h e o b s e r v a t i o n s made i n t h e p r e s e n t s t u d y , p a r t i c u l a r l y those r e g a r d i n g  environmental  i n f l u e n c e s on c e l l s u r f a c e s t r u c t u r e s and c e l l o r i e n t a t i o n and t h o s e concerned  w i t h the i n t e r a c t i o n of i n t r a c e l l u l a r and e x t r a c e l l u l a r  f a c t o r s i n e p i t h e l i a l h i s t o g e n e s i s may a l s o be a p p l i c a b l e t o o t h e r  98 systems.  The  p o s s i b l e s i g n i f i c a n c e of the r e s u l t s of t h i s study  in  the i n t e r p r e t a t i o n of growth p a t t e r n s of carcinomas i s suggested  by  the s i m i l a r i t y of the h i s t o c h e m i c a l and u l t r a s t r u c t u r a l obtained  i n the p r e s e n t  i n v e s t i g a t i o n with that reported  tumors (Ashworth e t a l . , I960; B o r l a n d 1961; The  L u i b e l et a l . . , 1960;  S c h r o d t and Foreman, 1965;  f o r other Dougherty,.  T a r l n , 1967).  r e l a t i o n s h i p of t h i s study t o carcinomas of the c e r v i x can a l s o be  evaluated C-4  and Webber, 1966;  information  by a comparison of the in v i v o and  in v i t r o h i s t o g e n e s i s of the  l i n e s t o t h a t of o t h e r c e r v i c a l carcinomas under somewhat s i m i l a r  conditions.  In such a p r e v i o u s  study  (Auersperg  c o r r e l a t i o n between o r i g i n a l tumor h i s t o l o g y and ogy was  and Worth, 1966)  p r i m a r y c u l t u r e morphol-  demonstrated, i.e-. groups of tumors w i t h s p e c i f i c  histologic  p r o p e r t i e s produced c h a r a c t e r i s t i c growth p a t t e r n s in v i t r o and c o r r e l a t i o n s c o u l d be shown w i t h c y t o g e n e t i c and of the tumors.  o n i e s , thus c l o s e l y resembling  i n v i v o and  clinical  further  properties  I t i s of i n t e r e s t t o note t h a t among those tumors  group grew i n - v i v o as compact clumps and  r e s e m b l e d C-4s  a  l i n e C-4c,  one  in v i t r o as v e r y c o h e s i v e and  col-  another group of t h o s e tumors  i n t h a t c e l l s l e f t the main tumor mass i n s m a l l groups  the c o l o n i e s formed jjn v i t r o were l e s s c o h e s i v e  irregular outlines.  I t seems t h e r e f o r e t h a t the C-4  and had more  cell lines  are  r e p r e s e n t a t i v e , i n some of t h e i r p r o p e r t i e s , of a f a i r p r o p o r t i o n  of  i n v a s i v e c e r v i c a l carcinomas and t h a t f a c t o r s s i m i l a r t o those demons t r a t e d h e r e i n may  p l a y a r o l e i n the h i s t o g e n e s i s of the  latter.  99 SUMMARY 1..  A comprehensive  e x a m i n a t i o n of two c l o s e l y r e l a t e d carcinoma  cell  l i n e s i_n v i t r o and _in v i v o was u n d e r t a k e n t o d i s t i n g u i s h c e l l u l a r and e n v i r o n m e n t a l f a c t o r s t h a t may  p l a y a r o l e i n turnor h i s t o g e n e -  sis. 2.  A histochemical  and u l t r a s t r u c t u r a l a n a l y s i s of the d i f f e r e n t growth  phases w i t h i n a passage _in v i t r o , t o observe the e f f e c t s of changing growth r a t e and c e l l c r o w d i n g , was  conducted.  were more c o h e s i v e , l e s s deformable than C-4s  C e l l s of l i n e  C-4c  c e l l s and responded  t o crowding by s t r a t i f i c a t i o n , while' C - 4 s ' c e l l s responded t o crowding  by a change from a h o r i z o n t a l t o a columnar o r i e n t a t i o n and by  cell dispersal.  C-4s  c e l l s had a h i g h e r n u c l e o c y t o p l a s m i c r a t i o ,  p o l a r i t y of o r g a n e l l e s , d i s p e r s e d bars.  C-4c  terminal  c e l l s had no p o l a r i t y o f o r g a n e l l e s but showed c y t o p l a s -  mic f i l a m e n t s condensed cells.  c y t o p l a s m i c f i l a m e n t s and  i n t o bundles and f l a t t e n i n g o f s u p e r f i c i a l  In b o t h l i n e s , but p a r t i c u l a r l y i n C-4c,  interdigitating  m i c r o v i l l i i n l a r g e i n t e r c e l l u l a r spaces p r o v i d e d the main c e l l contact. 3.  An a n a l y s i s of tumors grown from the same c e l l s i n hamster  cheek-  pouches, conducted t o compare _in v i t r o and i n v i v o h i s t o g e n e t i c c h a r a c t e r i s t i c s , showed t h a t most in v i t r o c h a r a c t e r i s t i c s of the cell  l i n e s were r e t a i n e d in v i v o .  In a d d i t i o n , i n c o m p l e t e basement  membranes were formed i n both l i n e s , though more e x t e n s i v e l y i n l i n e C-4s. 4.  Normal c e r v i c a l e p i t h e l i u m , grown in v i t r o t o d i s t i n g u i s h m a l i g n a n t c h a r a c t e r i s t i c s from r e s p o n s e s t o the i n v i t r o  environment,  demonstrated t h a t t h e o n l y major in v i t r o d i f f e r e n c e between b e n i g n squamous c e l l s and t h e carcinoma c e l l s l a y i n the a b i l i t y of t h e l a t t e r t o m a i n t a i n an i n t e r c e l l u l a r o r g a n i z a t i o n i n t h e complete absence of any s u p p o r t i n g 5.  E x p e r i m e n t s designed  tissue.  t o t e s t t h e r e a s o n f o r more compact growth  i n l i n e C-4c r e v e a l e d no d i f f e r e n c e i n i n t e r c e l l u l a r  circulation  of p r o t e i n and no e v i d e n c e of v a r i a t i o n i n c e l l s u r f a c e charges or c e l l  s u r f a c e a c i d mucosubstances between t h e c e l l  c e l l u l a r c o h e s i o n depended p r e d o m i n a n t l y was s t r o n g e r i n l i n e C-4c.  lines.  Inter-  on d i v a l e n t c a t i o n s and  These and o t h e r d e s c r i b e d  observations  suggested a d e f e c t i n d e f o r m a b i l i t y of these c e i l l i n e s w i t h t h e p r o b a b i l i t y that the stronger cohesion  i n l i n e C-4c'was due t o the  more e x t e n s i v e . m i c r o v i l l u s p o p u l a t i o n . 6.  I n examining t h e r e l a t i o n s h i p between c e l l  s e p a r a t i o n and t h e d i f f u s e  growth p a t t e r n i n l i n e C-4s i t was found t h a t these c u l t u r e s , when crowded, shed l a r g e numbers of v i a b l e c e l l s i n t o t h e growth medium that subsequently  were able t o p r o l i f e r a t e .  Such a process d i d  not t a k e p l a c e i n C-4c c u l t u r e s . 7.  I n v e s t i g a t i n g t h e r e l a t i o n of v i s c o s i t y o f t h e medium t o c e l l o r i e n t a t i o n and s u r f a c e s p e c i a l i z a t i o n r e v e a l e d t h a t s p e c i f i c m o d i f i c a t i o n s , such as c e l l , f l a t t e n i n g and t e r m i n a l - b a r f o r m a t i o n o f s u p e r f i c i a l c e l l s _in v i t r o , c o u l d be shown t o be r e s p o n s e s o f t h e c e l l s t o t h e v i s c o s i t y of t h e immediate environment and t h u s  experimentally  modifiable. 8.  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