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The role of neutrophils in the response of solid cancers to photodynamic therapy Cecić, Ivana F. K. 1999

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T H E R O L E OF N E U T R O P H I L S IN T H E R E S P O N S E OF SOLID C A N C E R S T O PHOTODYNAMIC THERAPY by Ivana F . K . Cecic B . S c , The University of British Columbia, 1995  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF  M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology)  W e accept this thesis as conforming to the required standard  The University of British Columbia A p r i l , 1999 © I v a n a F . K . Cecic, 1999  In  presenting  degree  this  thesis  in  at the University of  partial  fulfilment  British Columbia,  of  the  requirements  for  of  department  this or  thesis by  publication of this  for scholarly  his thesis  or  her  may  representatives.  be  granted  is  understood  It  for financial gain shall not  permission.  Department The University of British Columbi; Vancouver, Canada  DE-6 (2/88)  purposes  advanced  I agree that the Library shall make it  freely available for reference and study. I further agree that permission copying  an  for extensive  by the head that  be allowed without  of  my  copying  or  my written  ABSTRACT  Photodynamic Therapy ( P D T ) has been employed as a treatment for the eradication o f solid neoplastic lesions over the last decade.  W i t h the F D A approval o f Photofrin for use i n  P D T , this therapy has been clinically established for the treatment o f several cancers i n the Netherlands, Japan, France, the U S A , and Canada.  P D T involves the administration o f a  photosensitive drug, excited by light o f appropriate wavelength, causing localized tumor cell death i n the presence o f oxygen.  The mechanism o f cell death by P D T is complex, yet is  increasingly understood, with accumulating evidence suggesting that the host response to P D T plays a major role i n the success o f this treatment. A marked feature o f P D T is the induction o f strong, acute inflammation characterized by edema formation, and a wave o f infiltrating inflammatory cells, first o f which is the neutrophil.  It has been documented that activated  neutrophils sequester i n tumors during and following P D T , and their role is indispensable for the effectiveness o f this treatment modality. For instance, as shown i n this thesis, i n the absence o f circulating neutrophils (achieved by the administration o f 5 mg/kg o f the monoclonal a n t i - G R L antibody) long-term tumor control by P D T is diminished for the murine squamous  cell  carcinoma S C C V I I grown in syngeneic C3Ff/HeN mice. Using this tumor model and also the murine E M T 6 mammary sarcoma, we further examined the systemic response o f neutrophils to PDT.  Based on Wright stain analysis, it was determined that P D T induced a rise i n relative  circulating neutrophil content up to 2.3 times normal levels, from approximately 2 5 % to 60% o f nucleated cells i n blood. Consequently, total neutrophil numbers i n circulation rose from 2.95 ± 1.1 x 10 to 18.3 ± 5 x 10 per m l o f blood at the peak interval 3 hours following P D T light 6  6  treatment o f subcutaneous back tumors.  These increases were significantly higher than the  effect on neutrophil levels by any stress-related reaction the animals experienced i n handling  ii  during treatment (tail bleeding for blood collection, i.v. injection, immobilization i n lead holders, P D T light only). N o changes were observed in the total numbers o f other white blood cell types, such as lymphocytes and monocytes. Identifying neutrophils by their high level o f G R 1 expression, flow cytometry analysis was used to compare the neutrophil cell content i n tumors, blood, lungs, and bone marrow o f tumor-bearing mice over a 24-hour interval following P D T . A temporal rise i n the levels o f these cells i n tumors, blood, and lungs, corresponded with a 50% drop i n the granulocyte cell content o f bone marrow. It therefore appears that a release o f neutrophils from bone marrow occurred i n response to P D T . L-selectin ( C D 6 2 L ) expression was analyzed i n these neutrophil populations, as an indicator o f age and activation state. F l o w cytometry analysis detected a significant increase in L-selectin expression i n the neutrophil populations o f bone marrow and tumors from 2 and 8%, to 33 and 53%, respectively.  In  circulation, the majority o f neutrophils expressed medium concentrations o f L-selectin, with a very small fraction expressing high levels. There was an increase i n L - s e l e c t i n corresponding decrease i n L - s e l e c t i n marked rise i n L - s e l e c t i n  hlgh  low  with a  populations 10 hours after P D T treatment, while a  neutrophils was detected 24 hours post P D T .  changes were observed i n the lungs.  m e d l u m  N o significant  Collectively, these results suggest that localized P D T  induced a systemic response characterized by the release o f younger, less mature, L - s e l e c t i n  hlgh  neutrophils from bone marrow. Hence, tumor-infiltrating neutrophil populations may consist o f both mature and young neutrophils (present due to their accelerated release from bone marrow) which together contribute to tumor eradication by P D T . Therefore,  PDT-induced inflammation  partly  characterized  by  the  continuous  sequestration o f neutrophils into P D T treated mouse tumors during the first day following P D T treatment, is causing a systemic response dominated by neutrophilia and an increased activation status o f these cells. This condition reflects a massive mobilization o f neutrophils from their  iii  storage pools and myeloid precursors, as they are recruited to participate i n the destruction o f P D T treated tumor tissue.  iv  T a b l e of Contents  Abstract  ii  Table o f contents  v  List o f Tables  viii  List o f Figures  ix  Abbreviations  xi  Acknowledgments  xiii  Introduction  1  Section 1: History and present day clinical use o f photodynamic therapy. I. History o f Photodynamic Therapy. II. Advantages o f P D T III. Clinical use and studies involving Photofrin® -based P D T IV. Second generation sensitizers i n clinical trials Section 2: Mechanism o f PDT-induced cell death  c  1 1 2 2 4 5  Part 1: Primary cell death  5  I.  6  Light delivery  II. Photosensitizers III. Cellular drug uptake I V . D r u g uptake and localization in plasma membranes, lysosomes, and mitochondria.  6 7  Part 2: Secondary cell death  10  I.  10  P D T induced inflammatory response  Section 3: Activity o f neutrophils i n inflammation I.  Function o f neutrophils i n inflammation  II. Interplay o f inflammatory signals and adhesion molecules promote neutrophil extravasation. III. Mechanism o f tissue destruction by neutrophils IV. Neutrophil clearance from an inflamed site Apoptosis i n resolving acute inflammation.  v  7  12 12 13 14 15 16  Section 4: Activity o f neutrophils i n cancer I. Neutrophil-mediated damage o f cancerous tissue II. Activity o f neutrophils i n ischemia-reperfusion injury III. P D T and apoptosis Section 5: Neutrophil response to P D T o f solid tumors I. Accumulation o f neutrophils i n PDT-treated tumors II. The role o f neutrophils i n the induction o f a tumor-specific immune response to PDT-treated lesions III. Modulation o f blood flow i n PDT-treated tumors by alteration o f neutrophil activity I V . Influence o f modifications i n neutrophil activity on the response o f tumors to P D T  17 17 18 19 20 20 21 21 22  Hypothesis  24  Specific aims o f this project  24  Materials and Methods  25  I. Tumor models  25  II. P D T 1 .Photosensitizers 2. Light treatment 3. Light dose III. G R 1 antibody. I V . B C G vaccine V . Growth delay V I . Relative blood neutrophil levels VII. Obtaining total leukocyte numbers i n circulation. VIII. Control groups i n blood neutrophil analysis. I X . Systemic response o f neutrophils to photodynamic therapy 1. Tumor model and PDT. 2. Harvest o f tumor, lung, blood, and bone marrow. 3. Antibody staining and flow cytometry 4. G R 1 and L-selectin analysis X . Statistical analysis  26 26 27 27 28 28 28 29 29 30 30 30 31 32 33 33  Results  3  Section 1  4  34  vi  I. Growth delay following P D T is shortened with administration o f G R 1 antibody. II. Photofrin and mTHPC-based P D T stimulate an increase i n circulating neutrophil levels i n S C C V I I tumor-bearing mice III. P D T induces a marked increase i n circulating neutrophil levels IV. P D T induces a rise in circulating neutrophil levels i n EMT6-tumor bearing mice, but is suppressed i n a combined treatment with B C G vaccine Section 2  36 38  40 ;  I. Intravenous drug administration and tail bleeding capable o f inducing an increase in circulating neutrophil levels II. Changes i n relative circulating neutrophil levels i n non-PDT treated mice a.) i.v. injection o f D 5 W or 20 minute restraint, or light alone o f Balb/c tumor-free footpad  Section 3  34  ;  42  42 45 45  47  I. Increase in circulating neutrophil levels is PDT-specific Section 4  47 50  I. Analysis o f total circulating leukocyte numbers following P D T treatment o f tumors and normal skin II. Control treatments also induced a change i n total circulating leukocyte and neutrophil numbers Section 5: Systemic response o f G R l - p o s i t i v e cells to P D T I. Tumors II. Lungs III. B l o o d I V . Bone marrow  50 54 57 59 60 62 63  Section 6: L-selectin expression on neutrophils following P D T  64  Discussion  70  Future directions  79  References  81  vii  List of Tables Table 1: Total circulating leukocyte numbers per m l o f blood following Photofrin-based P D T of: i) tumor-free footpad, ii) s.c. E M T 6 back tumor, i i i ) E M T 6 foot tumor, in Balb/c mice 51 Table2: Total circulating neutrophil numbers following Photofrin-based P D T of: i) Balb/c footpad, ii) s.c. E M T 6 back tumor, and iii) E M T 6 foot tumor 52 Table 3: Total circulating lymphocyte numbers per m l o f blood following Photofrin-based P D T of: i) tumor-free footpad, ii) s.c. E M T 6 foot tumor, iii) E M T 6 foot tumor, in Balb/c mice 53 Table 4: Total leukocyte and neutrophil numbers in mice following either i.v. injection o f D 5 W or 20 minute restraint 54 Table 5: Total circulating leukocyte and neutrophil numbers i n mice following a light dose o f 60 J/cm , in the absence o f photosensitizer 55 2  viii  L i s t of Figures  Figure 1: Neutrophil depletion reduces delay for tumor recurrence following P D T  35  Figure 2: Photofrin- and mTHPC-based P D T o f s.c. S C C V I I back tumors can induce an increase i n circulating neutrophil levels  37  Figure 3: P D T induces an increase in circulating neutrophil levels  39  Figure 4: P D T induces an increase i n circulating neutrophil levels i n both E M T 6 tumor and tumor-free Balb/c mice, whereas, B C G vaccine has a suppressive effect  41  Figure 5: Increase i n circulating neutrophil levels after i.v. administration o f 10 mg/kg Photofrin.  43  Figure 6: Relative changes i n circulating neutrophil levels over a 24 hour period o f multiple blood sampling per animal  44  Figure 7: Changes i n blood neutrophil levels after: i) i.v. injection o f 0.2 ml/20g mouse 5% dextrose i n H 0 ( D W ) , ii) 20 minute restraint, or iii) 60 J/cm , 630 nm o f light on tumor-free footpad  45  Figure 8: Changes i n circulating neutrophil levels following P D T on the foot  48  2  2  5  Figure 9: Normalized changes i n circulating neutrophil levels following P D T on the E M T 6 back tumor  49  Figure 10: Systemic response o f G R 1 -positive cells to P D T  58  Figure 11: Changes i n cell content o f GR1-positive cell population i n E M T 6 tumors following P D T  59  Figure 12: Change i n the levels o f G R 1 -positive cells i n lungs following P D T .  61  Figure 13: Changes i n G R l - p o s i t i v e cell content o f blood following treatment with PDT  62  Figure 14: Changes i n G R l - p o s i t i v e cell content o f bone marrow following P D T  63  Figure 15: Changes contentexpression o f granulocytes high ilevels o f L-selectin Figure 16: Changes ii n n the L-selectin o f G Rexpressing l - p o s i t i v e cells n tumors following in bone marrow and lungs PDT  64  ix  6  4  Figure 17: Changes i n L-selectin expression o f neutrophils in circulation following PDT  67  Figure 18: F l o w cytometry analysis dot-plots o f PDT-induced changes i n L-selectin expression on G R 1 cells in peripheral blood  68  Figure 19: F l o w cytometry analysis dot-plots o f PDT-induced changes i n L-selectin expression on G R 1 cells i n tumor and bone marrow.  69  Abbreviations  (ADCC ) antibody-dependent cell-mediated cytotoxicity (ALA) aminolevulinic acid (AMD) age-related macular degeneration (ATP) adenosine triphosphate (BCG) Bacillus of Calmette and Guerin (BPD-MA) Benzoporphyrin derivative mono-acid (CO2) carbon dioxide (D W) 5 % dextrose in H 0 5  2  (EDTA) ethylenediaminetetraacetic acid (FDA) Food and Drug Association (FITC) fluorescein isothiocyanate (G-CSF) granulocyte colony-stimulating factor (HpD) hematoporphyrin derivative (HBSS) Hank's buffered salt solution (HOC1) hypochlorous acid (H2O2) hydrogen peroxide (ICAM-1) intercellular adhesion molecule-1 (IL-1) interleukin-1 (IL-8) interleukin-8 (LDL) low-density lipoprotein (LFA-1) lymphocyte function-associated antigen (Lu-Tex) Lutetium Texaphrin  xi  ( M H C ) major histocompatibility complex ( m T H P C ) meta-tetrahydroxyphenyl chlorin (Npe6) N-aspartyl chlorin e6 (O2") superoxide anion ( O H ) hydroxyl radical (PBS ) phosphate buffered saline (PDT) Photodynamic therapy (PEG-400) polyethylene glycol-400 (PE) phycoerythrin ( P M N ) polymorphonuclear leukocyte (s.c.) subcutaneous (SnET2) T i n Etiopurpurin ( T A M ) tumor-associated macrophages ( T N F - a ) tumor necrosis factor-alpha (TPPS4) tetraphenylporphine  Acknowledgments  To the staff o f Cancer Imaging, Medical Biophysics, and Advanced Therapeutics, for their unending advice and friendship, I am thankful. I would like to acknowledge the receptiveness, support and confidence I received from the members o f m y supervisory committee. In particular, I thank Dr. Minchinton for unlimited use o f laboratory space and helpful advice. I am indebted to Dr. Matisic, for her encouragement and willingness to make time for m y questions. To my supervisor, Dr. Korbelik, I express deepest gratitude for the opportunity to be a member o f his research team. H i s constant guidance led me to persevere through this project. M y family has above all given me the foundation o f strength to diligently work through obstacles.  True knowledge comes in realizing how little we know." -Josip Cecic  xiii  INTRODUCTION SECTION 1; HISTORY AND PRESENT DAY CLINICAL USE OF PHOTODYNAMIC THERAPY  I. History of Photodynamic Therapy  Documentation for the practice of combining light and photosensitive substances for medicinal use dates back several thousand years to ancient cultures of India and Egypt. The condition visiligo was treated in both areas using plants with photosensitive properties combined with sunlight (1). Photochemical sensitization with the intent to induce cell death was also found in the time of ancient Greece when the Greek scholar Herodotus described the healing aspects of light (2). However, it was in 1903 that the first use of Photodynamic Therapy (PDT) was reported for the treatment of neoplastic lesions, when Jesionek et al combined eosin and light to treat skin cancer (3). By 1911, Hausman pioneered the use of the photosensitive drug hematoporphyrin in PDT, which led to a specific interest in porphyrin-based photosensitizers (4). It was in the 1940s that hematoporphyrin was discovered to accumulate with greater specificity in malignant tumors compared to normal tissue (5).  As time went on, tumor-  localizing properties were improved with the formation of hematoporphyrin derivative (HpD), a mixture of porphyrin monomers, dimers, and oligomers, which was selectively retained in a large percentage of squamous cell carcinomas and adenocarcinomas (6). In the 1970s Dr. T. Dougherty began to study the application of HpD in combination with light for treatment of different animal and human malignancies (7-11). This work led to the development of the most widely used photosensitizer to date, Photofrin, and, in 1993, the approval of Photofrin-based PDT for clinical use. Following the usual practice with new modalities, PDT was introduced into clinics  1  primarily for palliative use where other more conventional treatments had failed (12). In recent years, clinical use o f P D T for the treatment o f bladder, skin, lung, and other cancers has been regulatory approved i n many countries including the Netherlands, U K , Japan, U S A , and Canada. Clinical trials at present include those for Photofrin-based P D T o f early stage head and neck cancers, certain types o f skin cancer, and also i n conjunction with surgery for brain and intrathoracic tumors (pleural mesothelioma) (12).  II. Advantages of PDT P D T has certain advantages over standard cancer treatments such as chemotherapy and radiotherapy. P D T does not induce resistance and can be used repeatedly (13). This therapy does not wear-out the patient, as it is a localized form o f treatment not systemic (12). F e w unwanted side effects are associated with this treatment modality, with the exception o f skin photosensitivity (14). Quick recovery and cosmetic healing, thereby healing o f normal tissue is favorable, and this therapy can be used i n cases where other more conventional therapies have failed (15).  III. Clinical use and studies involving Photofrin®-based PDT Although initial studies focused on treating patients with cutaneous or subcutaneous malignancies, many different tumors at various sites have now been treated with P D T . P D T treated  malignancies include basal  and squamous  cell  carcinomas o f skin,  malignant  melanomas, mycosis fungoides, recurrent metastatic breast carcinoma, and A I D S associated Kaposi's sarcomas.  Complete responses lasting up to 4 years have been achieved (16), and the  response o f managing basal cell carcinoma is equal or superior to other forms o f therapy (17,18). Although problems can arise with pigmented melanomas for example that virtually do  2  not respond to Photofrin-based P D T , since melanin absorbs light efficiently, clinical use o f P D T shows exciting progress. P D T may become a treatment o f choice for head and neck cancers. Long-term control o f ear, nose, and throat carcinoma in situ has been reported with a 70% response rate (19). A s P D T was found to be effective in eradicating papillomas i n an animal model (20), the eradication o f laryngeal papillomatosis by P D T is currently being evaluated clinically. In 1995, F D A approval was granted for P D T use i n the treatment o f advanced stage esophageal tumors (21). Photofrin-based P D T for lung cancers was originally used as palliative care i n patients with endobronchial obstruction (22). In 1998, F D A approval was obtained for early stage lung cancer (12). Tumor localized fluorescence o f photosensitizers is also being studied as potential means for detecting and/or delineating small carcinomas (in situ lesions) or superficial lung tumors covering a large endobronchial area (23). The treatment o f advanced non-small cell lung cancer with P D T has been compared with ablation using N d - Y A G laser therapy alone. Tumor response to P D T was superior as shown i n studies conducted i n both Europe and Canada/USA (13).  In addition, encouraging results comparing the use o f P D T plus radiotherapy and  radiotherapy alone for the opening o f obstructed airways has been reported b y L a m (24). P D T is also investigated i n clinical studies for gastrointestinal malignancies (25), colon cancer (26), and i n gynecologic (27) and intra-abdominal malignancies (28), results for which show promise i n the future use o f P D T in palliation, an alternative to surgery, or as an intraoperative procedure, respectively.  In murine and human cases sensitizer is selectively  retained i n bladder tumors compared to normal bladder mucosa (29).  With  specific  combinations o f photosensitizer and light delivery, superficial transitional cell cancers not involving the muscularis o f the bladder, can be destroyed successfully (30).  3  IV. Second generation sensitizers in clinical trials A l l o f these studies mentioned above have been conducted using the F D A approved Photofrin-based P D T . N e w sensitizers have also entered clinical trials for various types o f malignancies.  T i n Etiopurpurin (AnET2) is in phase II trials for the treatment o f cutaneous  metastatic breast cancer and Kaposi's sarcoma (31). phasell/III trial for certain skin lesions (12).  Lutetium Texaphrin (Lu-tex) is into a  Benzoporphyrin Derivative M o n o - A c i d ( B P D -  M A ) has undergone phase I/II trials for the treatment o f skin cancers, psoriasis, and age-related macular degeneration ( A M D ) (32). The latter is a common cause o f blindness whose current treatments using thermal lasers damages overlying retina. Not only does B P D - M A - b a s e d P D T manage to avoid damage to overlying retina, close to 50% o f patients treated experienced improved vision (33).  The most potent sensitizer to date, tetrametahydroxyphenyl chlorin  ( m T H P C ) is undergoing clinical trials for head and neck cancers in Europe and the U . S . Results indicate that all o f the patients i n this study treated for early stage squamous cell carcinoma  o f the upper  aerodigestive tract,  and Barrett's  esophagus  with  superficial  adenocarcinoma, were tumor-free after a follow-up o f 3-35 months (34). P D T efficacy depends on the depth o f light penetration, drug and light doses, as well as the mode o f light delivery, combinations o f which often differ depending on the type and site o f malignancy. It is important therefore, that all o f these parameters are taken into consideration when designing specific protocols.  4  S E C T I O N 2: M E C H A N I S M O F P D T - I N D U C E D C E L L D E A T H P A R T 1: P R I M A R Y C E L L D E A T H P D T involves the use o f three main components to induce cell death: the administration o f a photosensitive drug, light o f appropriate wavelength, and molecular oxygen.  Following  selective accumulation o f drug i n malignant tissue, the tumor is illuminated with light o f appropriate wavelength for absorption by the respective sensitizer used.  U p o n absorption o f  light energy the photosensitizer molecule, in an excited, triplet state, can undergo two types o f reactions designated as Type I and Type II (35). The Type I reaction occurs when the drug reacts directly with a substrate b y a mechanism involving hydrogen or electron transfer to form radicals which, in the presence o f oxygen, can form oxygenated products. The Type II reaction, which predominates in P D T , results in the formation o f singlet oxygen b y the transfer o f energy from the excited triplet state o f the drug to molecular oxygen. Singlet oxygen can then react with substrates causing oxidative damage to cell components such as plasma membranes, liposomes, mitochondria, and D N A (35, 36). Singlet oxygen is accepted as the major free radical formed b y P D T , inducing tumor cell death by oxidative damage (14,15). Due to the extremely short radius o f action (<0.02 ujn) o f singlet oxygen, localized damage inflicted on a cell by the photodynamic effect depends on the relative accumulation o f photosensitizer in a particular organelle at the time o f light treatment. Damage inflicted b y P D T is highly localized at the cellular level.  5  I. Light delivery Most photosensitizers are excited by tissue-penetrating wavelengths o f the red light spectrum. Photofrin for example absorbs light o f 630nm, whereas m T H P C absorbs at 652nm. Light can be delivered b y a number o f different light sources including a 2 5 0 W metal halide lamp coupled with a light guide at its distal end, a 300W short arc plasma discharge or xenon arc lamp, diode lasers, and N d - Y A G lasers (12). Reasons for choosing a particular mode o f photoillumination may depend on the wavelength o f light sought, and the fluence requirements o f the sensitizer used (37,38).  Other issues would encompass power, practicality, cost, and  minimal heat emittance (39,40).  II. Photosensitizers The first-generation photosensitizer hematoporphyrin derivative ( H p D ) is a mixture o f porphyrin monomers, dimers and oligomers, formed from the treatment o f hematoporphyrin with acetic and sulphuric acids (41).  The heterogeneity o f H p D however led to unpredictable  localization i n different parts o f a cell. The need for a more purified form o f sensitizer with less heterogeneity, led to the production o f the most widely used photosensitizer to date, Photofrin® (42).  Although much progress has been made i n Photofrin-based P D T , there do exist some  limitations such as prolonged skin photosensitivity and that it is excited by light o f relatively short wavelength limiting depth o f treatment. Therefore, intense research i n developing new and improved second- generation sensitizers is well underway. Criteria for developing new photosensitive drugs include: (1) selective uptake by neoplastic cells/tissue, (2) low skin photosensitivity, (3) to be excited by light o f longer wavelengths to increase treatment depth, and (4) reduced photobleaching and destruction (41). A number o f compounds have arose including tin etiopurpurin (SnET2), lutetium texaphrin ( L u -  6  tex),  benzoporphyrin  derivative  monoacid  ring  A  (BPD,  verteporfin),  tetrametahydroxyphenylchlorin ( m T H P C ) , N-aspartyl chlorin e6 (Npe6), and aminolevulinic acid ( A L A ) protoporphyrin I X pro-drug (12).  These new drugs are pure forms, not mixtures as  is H p D . Compared to H p D , these newly formed sensitizers are more easily targeted towards specific organelles or cell components such as mitochondria, liposomes, and plasma membranes (12). Tumor vasculature has also been targeted (43).  III. Cellular drug uptake Uptake o f the drug, prior to light treatment, into cells is crucial for effective P D T (44). W h y porphyrin-based sensitizers are more selectively taken up b y neoplastic cells/tissues compared to normal cells has long been debated.  Cancer cells seem to have upregulated  expression o f the low-density lipoprotein ( L D L ) receptor (45). This may enhance binding and entry o f circulating lipoprotiens carrying lipophilic porphyrins, such as B P D - M A (46).  Poor  lymphatic drainage may result in the build-up o f porphyrins i n the interstitial space (35). Rapidly dividing tumor cells may have an increased ability to phagocytose porphyrin aggregates or v i a pinocytosis (36).  Recent studies have shown that it is not necessarily malignant cells  involved i n selective drug uptake, instead it may be stromal elements o f the tumor. Korbelik et al have shown that a population o f tumor-associated macrophages ( T A M ) collect the large concentration o f sensitizer i n malignant tissue (47,48,49).  Interestingly, experimental tumors  can have up to 80% T A M o f cellular content and human cancers generally comprise between 20 and 50% o f these cells (50).  IV. Drug uptake and localization in plasma membranes, lysosomes. and mitochondria Localization into specific cellular components varies among photosensitizers, depending  7  on their lipophilicity, hydrophobicity, and other properties. T o date it is well accepted that most sensitizers do not accumulate in nuclei, resulting i n little potential for D N A damage or for mutations to occur.  Some damage to D N A has been reported including strand breaks and  chromosome aberrations.  Recovery from the latter however can occur, suggesting that this  effect may not necessarily be lethal (51,52).  Direct cell damage does nonetheless vary from plasma membrane, to lysosomes, and to mitochondria, initiating different forms o f cell injury and death. Damage by HpD-based P D T seems to concentrate to the plasma membrane (53). Porphyrin uptake begins with binding at the level o f plasma membrane then migrating with time to other cellular components (54). M a n y reports have described the damage induced b y P D T on plasma membranes. Observations described include swelling, bleb formation, shedding o f vesicles containing plasma membrane marker enzymes, cytosol and lysosomal enzymes (55,56). transport is also affected.  Membrane  There is a reduction o f active transport, depolarization, increased  uptake o f a photosensitizer, and increased permeability to lactate dehydrogenase (57-60). The activity o f numerous plasma membrane enzymes is inhibited including that o f N a K - A T P a s e +  +  and M g - A T P a s e , and damage to multidrug transporters occurs (61, 62). Furthermore, P D T 2+  induces a rise i n C a  2 +  concentrations, up- and down-regulation o f surface antigens, and lipid  peroxidation (63-65). The cumulative effect is a halt in cell division, followed b y cell lysis (14). Other membranes also affected b y P D T include that o f mitochondria, G o l g i apparatus, lysosomes, and the endoplasmic reticulum. Aggregated and also hydrophilic sensitizers, such as T P P S (tetraphenylporphines), are likely to be taken up by pinocytosis and endocytosis, thereby 4  accumulating in lysosomes and endosomes (12).  The membranes o f these vesicles become  permeabilized upon light exposure, resulting in the release o f hydrolytic enzymes into the cytosol (66). 8  Photofrin and A L A , a naturally occurring substance converted into the photosensitive protoporphyrin I X by the heme biosynthetic pathway, have been shown to localize in mitochondria (12). P D T damage to this organelle has been described as causing aberrations in functional oxidative phosphorylation and electron transport chain activity, i n addition to a decrease i n cellular adenosine triphosphate ( A T P ) levels (67,68). O f particular interest is the induction o f cell death b y apoptosis induced upon photoillumination, with a rapid release o f cytochrome C from mitochondria into the cytoplasm.  Cytochrome C binds with A P A F - 1  activating apoptotic caspases, inducing cell death by apoptosis (69). Therefore, depending on the site o f PDT-induced photo-damage, cells can undergo cell death b y necrosis as a result o f membrane damage, or apoptosis believed to be associated primarily with mitochondrial damage (70-72). This is an important consideration, for malignant cells often lack the ability to undergo apoptosis, and therefore chemotherapy (73).  escape the effects  of  In this respect, P D T may prove to be an effective treatment against  otherwise drug-resistant cell types.  9  PART 2 : SECONDARY CELL DEATH It is generally accepted that the anti-tumor effect o f P D T combines both direct and indirect cell death processes (12,74).  The direct lethal effect is a result o f irreparable  photooxidative injury o f vital cellular structures. The indirect killing o f cancer cells results from a series o f events triggered by the formation o f phototoxic lesions (not necessarily lethal) i n cellular and acellular tumor constituents, including parenchymal and host immune cells, tumor vasculature, and extracellular matrix.  Secondary tumor cell death is associated with the  induction o f a strong, acute inflammatory response mediated b y the combined release o f histamine and serotonin, as well as lipid degradation products, and metabolites o f arachidonic acid from photooxidative lesions o f lipids (75). The complex interplay o f secondary anti-tumor effects induced by P D T is dominated by three major responses: the breakdown o f tumor vasculature, an inflammatory reaction, and an immune response. A series o f events that lead up to secondary cell k i l l include ischemic death from vascular damage and blood flow stasis, ischemia-reperfusion injury, cell death mediated by resident and infiltrating inflammatory cells, and a tumor specific immune reaction (76).  I. P D T induced inflammatory response The inflammatory response induced by P D T includes the massive infiltration o f activated neutrophils into the treated site and emerging evidence suggests that these cells are major contributors to the rapid ablation o f PDT-treated cancers (12,77). In addition to inflicting substantial direct damage to the tumor vasculature and malignant parenchyma, neutrophils are the initiators o f inflammatory/immune processes involving the mobilization o f various types o f non-specific and specific immune effector cells (78).  These developments encompass an  immune recognition o f PDT-treated cancer and the induction o f antitumor immunity mediated  10  by T-lymphocyte populations (74,79,80).  Interestingly, P D T represents a rare example o f an  anti-cancer modality whose therapeutic outcome relies on the exploitation o f the destructive capabilities o f neutrophils. This particular approach to the treatment o f cancer, thus involves the mobilization o f host neutrophils, the most powerful immune cell regarding their tissue destructive potential. This aspect o f P D T w i l l be examined i n detail i n this thesis.  11  S E C T I O N 3: A C T I V I T Y O F N E U T R O P H I L S I N I N F L A M M A T I O N  I. Function of neutrophils in inflammation In order to show how it is that neutrophils can have anti-tumor activity, it is helpful to understand their origin and function i n inflammatory responses. A l l circulating blood cells are derived from a common progenitor stem cell in the bone marrow. The hematopoietic pathway o f white blood cell differentiation can be separated into two lineages: lymphoid and myeloid. Neutrophils are phagocytic myeloid cells that serve as the first line o f (innate) cellular defense against invading microorganisms and are the principal mediators o f acute inflammation (81). Hereditary deficiencies in neutrophil function for example, can lead to bacterial infections causing death i f left untreated (82). Inflammation can be described as the response to infection or physical injury that has evolved to eliminate invading microorganisms and promote repair o f damaging tissues.  Four  classical signs o f inflammation include redness and heat which occur as a response to vascular dilatation, swelling as a result o f increased vascular permeability to plasma and leukocytes, and pain (81).  In the first phase, inflammatory activity concentrates on preventing the spread o f  damage from an affected site, inactivation o f microbes ( i f present) and dissolution o f irreparably injured tissue.  The induction o f pro-inflammatory damage triggers an almost instantaneous  release o f various chemotactic  factors  that promote,  along a path  o f their  increasing  concentration, the migration o f inflammatory cells from the bloodstream to an inflamed site. Neutrophils are the first cells engaged in the inflammatory response (81,83).  These cells  normally remain contained to the bloodstream and do not migrate into healthy tissue. However, when elicited by inflammatory signals, neutrophils arrive rapidly i n large numbers at an affected site, and become activated as powerful mediators i n the destructive phase o f an inflammatory  12  response.  Their activity is associated with the release o f additional stimuli that propagate the  inflammatory process with continuing waves o f an influx o f neutrophils and other types o f inflammatory cells, including mast cells and monocytes/macrophages. There is also evidence that signals released from extravasated neutrophils can directly attract lymphocytes to the affected site (84). Once extravasated from the blood vasculature, neutrophils, characterized by a short constitutive half-life (6 h in human blood), w i l l never emigrate back into circulation but w i l l shortly after invading inevitably meet their fate (85).  II.  Interplay of inflammatory signals and adhesion molecules promote neutrophil  extravasation Intravascular stimulus generated from the complement, coagulation or kinin-generating system o f the plasma, phospholipid metabolites, cytokines (IL-1, I L - 8 , T N F - a ) or other substances, attract the migration o f neutrophils to an inflammatory site (78). These stimuli also activate or up-regulate adhesion molecules on both neutrophils and endothelial cells i n the vascular lining o f blood vessels i n the inflamed area (86).  Upon arrival, neutrophils w i l l  marginate and roll along the surface o f the endothelium, a process mediated b y a class o f adhesion molecules called selectins.  L-selectin is expressed on the surface o f leukocytes, E -  selectin and P-selectin on endothelial cells (87). Transendothelial migration is mediated b y the (32-integrin family o f adhesion molecules, which includes L F A - 1 and Mac-1 expressed on the surface o f neutrophils, and I C A M - 1 on the endothelium.  These adhesion molecules help to  anchor the neutrophil to endothelial cells initiating diapedesis across the vessel wall toward a site o f injury (88). Therefore, three major interactions between neutrophils and endothelial cells must unfold i n order for extravasation to occur: cell rolling, anchoring, and diapedesis.  13  III. Mechanism of tissue destruction by neutrophils The neutrophil has been identified as the primary mediator o f tissue destruction i n a variety o f inflammatory diseases, including rheumatoid arthritis, myocardial reperfusion injury, blistering skin disorders, and ulcerative colitis (89).  In these diseases and various acute  inflammatory disorders, neutrophils are triggered to release a complex mixture o f destructive agents that normally defend the host against invading microbes. Because these cells have no inherent ability to differentiate between foreign and host antigens (this is left to other arms o f the immune system), i n the above mentioned disorders its destructive agents destroy normal cells and dissolve connective tissues (78). The neutrophil's inventory contains over 50 different toxic agents for mediating inflammatory tissue damage (89,90). These agents are usually grouped into those localized i n the plasma membrane and others that are found i n the intracellular granules.  The enzyme  N A D P H oxidase located i n the plasma membrane enables the activated neutrophils to generate a family o f reactive oxidative species. The granules contain microbicidal peptides, proteins and enzymes.  When specifically triggered by proinflammatory stimuli, the N A D P H  (otherwise dormant  oxidase  i n unstimulated neutrophils) starts to generate and release  oxygen  metabolites, while almost simultaneously the granules fuse with the plasma membrane and discharge their contents into the phagocytic vacuole and/or extracellular medium (89,91). The membrane-associated N A D P H oxidase system is capable o f producing at least three oxygen metabolites: superoxide anion (0 ~)> hydrogen peroxide (H2O2), and the hydroxyl 2  radical ( O H ) . The bulk o f generated oxygen metabolites appears to be processed as hydrogen peroxide by myeloperoxidase into HOC1 (hypochlorous acid, known as household bleach), an extremely powerful oxidant that rapidly inactivates a wide range o f biologically relevant molecules (91,92).  It is noteworthy to point out that neutrophils contain large quantities o f  14  myeloperoxidase i n their granules (up to 5% o f the  cell's dry weight), so that substantial  amounts o f this enzyme are released b y activated cells into extracellular fluids (93).  This  enables neutrophils to generate impressive high quantities o f HOC1: 2 x l 0 " m o l per m i l l i o n cells 7  in two hours, an amount high enough to destroy 150 million microbes i n milliseconds (93). A m o n g over 20 enzymes contained in neutrophil granules, three proteinases - elastase, collagenase and gelatinase - appear to have the greatest potential for mediating tissue destruction (91).  These particular enzymes are specialized i n selective degradation o f key  components o f the extracellular matrix, the structure with an indispensable role in orderly function and repair o f tissues. Normally, the destructive potential o f such enzymes would be very limited because o f the abundance o f powerful antiproteinases (proteinase inhibitors) both in the plasma and interstitial fluid. However, HOC1 and other oxidants produced b y neutrophils destroy this antiproteinase shield (91,92).  Hence, in order to utilize its ultimate destructive  potential, the neutrophil uses both the N A D P H oxidase system and proteolytic granule contents in a cooperative and synergistic fashion (91).  IV. Neutrophil clearance from an inflamed site Inflammation has long thought to be a beneficial process i n which the host can defend itself against foreign invaders (81).  However, it is also understood that the functions o f  activated neutrophils i n chronic inflammation can unfortunately be involved i n the pathogenesis o f certain diseases such as myocardial infarction/reperfusion injury, atherosclerosis, and chronic bronchitis (85). Chronic inflammation is difficult to resolve, posing a major hindrance to the normal healing process o f surrounding healthy tissue.  Acute inflammation, as is induced by  P D T , can however, under normal conditions, clear out completely, with cell death by apoptosis playing a very important role (94).  15  Apoptosis in resolving acute inflammation Apoptosis is a form o f cell death i n which a cell receives a signal to die from within, and is a natural process i n development. Apoptosis contrasts with necrosis b y the well described characteristics o f internucleosomal D N A cleavage, nuclear degeneration and condensation, the formation o f apoptotic bodies, and phagocytosis o f cell residua (81). Cells dying b y apoptosis do not release their cytosolic contents into circulation and their immediate surroundings (94). Apoptosis therefore diminishes the induction or propagation o f an inflammatory process and its associated neutrophilic damage. A s discussed, neutrophils are the first inflammatory cell to arrive at a site o f perturbation creating chemotactic fragments from proteolytic cleavage o f matrix proteins, amplifying the inflammatory response.  Therefore, i n order to resolve an inflammatory state, the first step  would be to remove activated neutrophils. In acute inflammation activated neutrophils undergo cell death with morphological changes characteristic o f apoptosis (85). Apoptosis i n neutrophils appears to occur constitutively most likely due to a short half-life o f six hours (95). Apoptotic neutrophils become senescent, with a shutdown o f secretory processes, and are recognized and ingested intact b y macrophages through the recognition o f cell surface changes (96). In this process, pro-inflammatory neutrophil granular contents are not released nor are those o f macrophages, such as thromboxane, proteolytic enzymes, and cytokines. These inflammatory macrophages which have ingested apoptotic neutrophils in a site o f injury, are cleared via draining lymph nodes, which could lead to antigen presentation to lymphocytes (97).  16  S E C T I O N 4; A C T I V I T Y O F N E U T R O P H I L S I N C A N C E R  I. Neutrophil-mediated damage of cancerous tissue The importance o f neutrophils i n host rejection o f malignant tumors has only recently received quality attention.  It is increasingly clear that these cells can become activated as  potent tumoricidal effectors, while, on the other hand, they may have a critical role in the initiation o f an immune response to cancer cells (98-100). Neutrophils have been shown to destroy cancer cells in vitro by releasing oxygen metabolites or v i a antibody-dependent cellmediated cytotoxicity ( A D C C ) (101,102). Direct involvement o f neutrophils was suggested to be responsible for endotoxin-mediated tumor necrosis (103).  Using transduction o f the  granulocyte colony-stimulating factor ( G - C S F ) gene into mouse  adenocarcinoma  cells,  Colombo and co-workers presented direct evidence o f neutrophil-mediated tumor inhibition in vivo (104). G-CSF-releasing cancer cells i n tumors, attract neutrophils that became engaged i n direct contact with the cytokine-releasing cells.  Anti-tumor effects o f neutrophils have also  been described i n a clinical study, i n which peritoneum-infiltrating neutrophils, activated b y the streptococcal agent O K - 4 3 2 , mediated the destruction o f cancer ascites (105). Close contact between neutrophils and macrophages was shown to be essential for cancer cell cytostasis exhibited by cells present i n inflammatory granulomas (98).  The  interaction o f neutrophils with other host immune cells may be o f critical importance i n the development o f a host immune response against malignant tumors.  W h e n infiltrating into  tumor tissue, neutrophils produce chemotactic factors for other immune cells, including macrophages and lymphocytes, as well as releasing cytokines such as interleukin-1 and tumor necrosis factor alpha (TNF-oc) through which they can exert a variety o f immunoregulatory functions (100,106).  Thus, the depletion o f neutrophils was demonstrated to abrogate the  17  immune rejection o f transplanted tumors (100). However, i n some cancer variants, malignant cells subvert neutrophils to produce factors that are stimulatory for malignant growth and their elimination results i n the inhibition o f tumor growth (107).  II. Activity of neutrophils in ischemia-reperfusion injury Ischemia-reperfusion  injury is a well-known physiological insult that can cause  considerable damage to the affected tissue. The sudden re-introduction o f oxygen at the time o f reperfusion results i n the induction o f oxidative stress at the level o f vascular endothelium and is associated with a massive recruitment and activation o f neutrophils.  These cells are largely  responsible for the damage inflicted by ischemia/reperfusion injury (108). events lead to  irreversible neutrophil-induced damage  Although such  in myocardial infarction, these  occurrences can be beneficial i f incurred i n tumor tissue (109,110).  It was shown that the  induction o f ischemia-reperfusion injury by transiently clamping the feeding blood vessels to subcutaneous tumors could cause their ablation (111).  18  III. P D T and apoptosis The ability o f a host to resolve an acute inflammatory response i n a process inhibiting long-term inflammatory damage, can also be advantageous i n the response to P D T . It is now known that neutrophils sequestered in tumors treated with mTHPC-based P D T undergo apoptosis in vivo (Korbelik, unpublished results). Furthermore, the induction o f an acute, not chronic, inflammatory host response may explain why the P D T effect tends to be so passive towards normal tissue. It could perhaps be advantageous to enhance apoptosis i n the later stages o f inflammation following P D T to reduce any long-term, i l l effects o f neutrophil function on normal tissue, and augment the killing o f remaining cancerous cells.  19  S E C T I O N 5: N E U T R O P H I L R E S P O N S E T O P D T O F S O L I D T U M O R S I. Accumulation of neutrophils in PDT-treated tumors Insult to tumor vasculature by P D T contributes to the induction o f an inflammatory response (12,60,75).  Vascular effects o f P D T are manifested as the breakdown o f vessel  basement membrane, contraction o f endothelial cells, platelet aggregation, and vasoconstriction (8,42,44). Inflammation induced by P D T is further propagated b y the release o f potent inflammatory mediators such as arachidonic acid metabolites, extracellular matrix components, and the cytokine interleukin (IL)-8 into the immediate surroundings and circulation, and promotes the adherence o f polymorphonuclear leukocytes to vessel walls (60,75,112,113). In response to inflammatory chemotactic signals, neutrophils rapidly and i n large numbers accumulate in PDT-treated tumors. For instance, the neutrophil content o f S C C V I I squamous cell carcinoma murine tumors increases 200-fold within five minutes o f the start o f P D T light treatment (77).  In an activated state, sequestered  neutrophils presumably release  the  chemoattractant leukotriene B 4 maintaining an influx o f neutrophils, and other inflammatory cells including mast cells and monocytes/macrophages  i n the PDT-treated tumor (77).  Similarly, Gollnick et al observed a change i n the relative content o f various cellular components o f the PDT-treated E M T 6 mammary sarcoma murine tumor.  They reported that  tumor cell numbers were diminished 24 hours after treatment as they succumbed to treatment, whereas the macrophage and granulocyte content increased.  The granulocyte population  increased to 50% o f all cells retrieved compared to 3% in the untreated controls (114). Similar results were obtained with the E M T 6  tumor treated with P D T mediated b y  another  photosensitizer, m T H P C (76). Activated neutrophils release from their cytosolic granules the enzyme myeloperoxidase that can be utilized as a qualitative indicator o f neutrophil presence and activation (93).  20  Myeloperoxidase activity in S C C V I I and E M T tumors has been observed to increase with time following mTHPC-based P D T and was PDT-dose dependent (Cecic, Parkins, and Korbelik, unpublished results).  II. The role of neutrophils in the induction of a tumor-specific immune response to P D T treated lesions A s described earlier, P D T inflicts direct cell damage to cells.  Thus, P D T results i n  inflammation, characterized i n part by the sequestration o f inflammatory cells to an affected site in response to chemotactic signals (12). Neutrophils are the first cells to arrive to the site o f perturbation.  These cells play a dual role o f inflicting cell damage and attracting other  inflammatory/immune cells, such as phagocytic macrophages, as well as T-lymphocytes with the capability to become tumor-specific immune memory cells, to the P D T treated lesion (12,74). P D T generates a large amount o f cellular debris at a tumor site, which can be taken up by macrophages and dendritic cells recruited to the site o f P D T damage. This material is then processed and presented on the surface o f these professional antigen-presenting cells in the context o f M H C (major histocompatibility complex) molecules, to T-lymphocytes (12). These tumor-specific T lymphocytes may then contribute to both, the eradication o f PDT-treated lesions, and long-term tumor control, since it has been shown that functional T-lymphocytes are crucial for the curative outcome o f P D T (80).  III. Modulation of blood flow in PDT-treated tumors by alteration of neutrophil activity The activity o f sequestered neutrophils has an impact on the blood flow i n PDT-treated  21  tumors, observed to decrease i n a P D T dose-dependent manner (13,115).  Sluiter and co-  workers demonstrated in vitro that neutrophils adhere to PDT-treated endothelial cells, and that the pVintegrin molecule C D 18 plays a crucial role i n this event (116).  B l o c k i n g leukocyte  adhesion to endothelial cells in vivo with the administration o f monoclonal antibodies to the C D 18 molecule before P D T light treatment, completely abolished the induction o f blood flow decrease in S C C V I I tumors (78). However, this effect o f anti-CD 18 was not seen i n the E M T 6 tumor model implicating the involvement o f other factors i n addition to the obstruction o f blood flow b y adherent and aggregated blood cells, for the modulation o f blood flow i n PDT-treated rumors.  Interestingly, the PDT-induced decrease i n blood flow o f both S C C V I I and E M T 6  tumors was markedly enhanced i n mice whose circulating pool o f neutrophils was depleted b y the administration o f monoclonal antibody to the myeloid differentiation marker G R - 1 .  A  possible explanation for this finding is that invading neutrophils release nitric oxide that could act as a vasodilatory mediator in the vasculature o f PDT-treated tumors (78,117). Reduction i n tumor blood flow during photodynamic light delivery results i n decreased tumor oxygenation, having a negative impact on the therapeutic effect due to a decreased production o f cytotoxic oxygen species. O n the other hand, impaired perfusion o f tumor tissue after P D T contributes to the antitumor effect based on ischemic necrosis (78).  I V . Influence of modifications in neutrophil activity on the response of tumors to P D T Neutrophils accumulate i n PDT-treated tumor tissue during and following treatment, and their contribution to the effectiveness o f this treatment has been demonstrated b y a number o f researchers.  DeVree et al demonstrated that the administration o f anti-granulocyte anti-serum  into tumor-bearing rats before and at least 5 days after P D T , did not affect tumor volume, and the tumors continue to grow at a normal rate. However, when the administration o f antiserum 22  was stopped, allowing neutrophil levels to increase, a delay i n tumor growth was recorded (118). Conversely, using granulocyte-colony stimulating factor ( G - C S F ) prior to and after P D T to increase circulating neutrophil numbers, P D T effectiveness i n delaying tumor regrowth was enhanced compared to saline controls (119). Similar results have also been observed in murine tumor models with a single intravenous dose o f a monoclonal antibody for the myeloid differentiation marker G R 1 , administered one hour prior to P D T light treatment.  In these  studies, the cure rate o f PDT-treated E M T 6 tumors was markedly decreased, while the recurrence o f treated S C C V I I tumors was accelerated (78). Based on this evidence it may be concluded that neutrophil-associated events i n P D T are indispensable for the efficacy o f this treatment modality. Other evidence supporting the above conclusion comes from studying the adjuvant effect o f mycobacterium cell wall extract on the curative effect o f P D T (76). The beneficial effect o f this agent was attributed to its enhancement o f neutrophil infiltration into PDT-treated E M T 6 tumors. However, depending on circumstances, the engagement o f activated neutrophils may both negatively and positively affect the tumor response to P D T . The cure rate o f PDT-treated S C C V I I murine tumors was augmented by the administration o f the anti-CD 18 monoclonal antibody before light treatment. B y inhibiting leukocyte-endothelium interactions, the occlusion o f blood vessels is presumably diminished allowing for an improved supply o f oxygen during light treatment.  23  HYPOTHESIS  The massive and rapid accumulation o f neutrophils from circulation into PDT-treated tumors is secured by a strong systemic response o f these cells, which play an indispensable role in the curative outcome o f P D T .  S P E C I F I C A I M S O F THIS P R O J E C T  1. T o examine changes i n peripheral blood leukocyte counts o f tumor-bearing mice following treatment with P D T .  2. T o determine whether i n mice P D T induces the enhanced production o f neutrophils from immature precursors i n the bone marrow, as well as the mobilization o f these cells from their storage and marginated pools.  3.  T o analyze L-selectin expression in neutrophils localized i n the bone marrow, peripheral blood, and PDT-treated tumor, to compare the activation status o f these cells before and after PDT.  24  MATERIALS AND METHODS  A l l plastics and glassware were obtained from V W R Canlab, Missisauga, Canada or G I B C O , Gaithersburg, U S A , unless otherwise specified. A l l chemicals and media were purchased from S I G M A Chemical C o . , St. Louis, U S A , unless otherwise specified.  I. T u m o r m o d e l s : 1. S C C V I I squamous cell carcinoma (120) and E M T 6 mammary sarcoma (121) tumors were grown in syngeneic, immunocompetent C 3 H / H e N and Balb/c mice, respectively. These cell lines were routinely maintained in vivo by biweekly intramuscular tumor brei inoculation. M i c e were sacrificed by CO2 inhalation and the tumors removed and minced using two #22 scalpel blades.  Subsequently the tumor tissue was repeatedly passed through two 18 gauge and 20  gauge needles, respectively, and diluted 5 times i n phosphate buffered saline (PBS). 0.1 m l o f tumor brei was inoculated into the thigh muscles o f anesthetized mice (122). For experiments, the tumor was removed using aseptic technique, chopped using two #22 scalpel blades, suspended i n 5 m l o f P B S and enzymatically digested with gentle rotation at 37°C for 30minutes ( S C C V I I ) or 15 minutes ( E M T 6 ) . The three enzyme cocktail used for disaggregation contained: DNase (type I) 0.6 mg/ml, collagenase (type IV) 0.24 mg/ml and Dispase (Boerhinger, Mannheim, Germany) 0.18mg/ml, diluted i n 5 mis o f cold P B S (122,123). The enzymes were added to the tumor just prior to incubation. The tumor cell suspension was then filtered through a 100pm nylon mesh filter using a 6cc syringe, and pelleted b y centrifugation at 600 rpm, and suspended i n P B S .  Cell concentration was determined b y hemacytometer count.  For  experiments, 1-2 x 10 S C C V I I and E M T 6 cells were inoculated subcutaneously on the sacral 6  region on the dorsal side o f animals, or 4 x 10 E M T 6 cells inoculated on the dorsal side o f the 6  25  hind leg footpad. During inoculation, the animals were anesthetized b y inhalation o f Metofane® (Associated Veterinary Purchasing Ltd., Abbotsford, Canada).  2. In vitro culture: E M T 6 tumor cells were cultured at 37°C, 5% CO2, and 9 5 % humidity, i n alpha-minimal  essential  medium  supplemented  with  10%  fetal  calf  serum  (HyClone  Laboratories Inc., Logan, U S A ) , 100 u,g/ml streptomycin, and 100 Units/ml penicillin, growing adherent to the bottom o f T75cm tissue culture flasks. T o harvest, a confluent monolayer o f cells was treated with T r y p s i n - E D T A (ethylenediaminetetraacetic  acid) solution ( G I B C O )  containing 0.25% trypsin and I m M E D T A 4 N a in H B S S (Hank's buffered salt solution), suspended i n P B S , washed once by centrifugation at 600 rpm, and resuspended i n P B S . A count o f cell viability was obtained using trypan blue dye exclusion. 2 x 10 cells were inoculated into 6  the footpad o f anesthetized animals.  A l l subcutaneous tumors were treated 7-8 days after inoculation and foot tumors 10-11 days after inoculation when the tumors reached an optimal P D T size o f 6-8 m m i n largest diameter.  The mice treated were 7-9 week old females, and kept i n the Joint A n i m a l Facility at the B . C . Cancer Research Centre where they were supplied with food and water ad libitum.  The A n i m a l  Ethics Committee o f the University o f British Columbia approved all experimental protocols.  II. PDT:  1 .Photosensitizers: 1.1  Photofrin®,  kindly provided by Q L T (Quadralogics Technologies Inc., Vancouver,  26  B.C.,Canada), was reconstituted i n 5% dextrose in H2O and used at a concentration o f 10 mg/kg. A volume o f 0.2 ml/20gram mouse was administered intravenously (i.v.) 24 hours prior to light treatment.  1.2  mTHPC  (metatetrahydroxyphenylchlorin, Scotia pharmaceuticals,  Great Britain) was  reconstituted in P E G - 4 0 0 solution containing ethanol, polyethylene glycol, and H2O i n a 2:3:5 v/v/v ratio, respectively. A volume o f 0.1ml/20gram mouse was administered i.v. 24 hours prior to light treatment.  2. Light Treatment: Light was administered by a tunable light source (Photon Technology International Inc., M o d e l A5000) equipped with a l k W Xenon bulb. This source was used to generate 6 3 0 ± 1 0 nm light for Photofrin-based P D T and 652±10 nm light for mTHPC-based P D T delivered through a 5mm-core diameter liquid light guide (2000A, Luminex, Munich, Germany). The power density at the illuminated spot encompassing the tumor and ~-lmm o f surrounding normal tissue was 100-120 m W / c m . 2  During light treatment each animal was restrained unanesthetized  in  specially designed lead holders exposing either the sacral region o f the back or the dorsal side o f the footpad.  3. Light dose: E M T 6 tumor was treated with a dose o f 60 J/cm , unless otherwise indicated, and the average 2  time per treatment was 8 minutes.  The S C C V I I tumor was treated with 150 J/cm , unless  otherwise indicated with an average time o f 20-30 minutes. Time duration was dependent on three parameters: (1) diameter o f the tumor, (2) power output o f the light source, and (3) light  27  dose.  III. G R 1  antibody  The hybridoma cell line, clone R B 6 - 8 C 5 producing rat IgG2b, was grown with the permission o f Dr. Gerald J. Spangrude (Department o f Medicine, University o f Utah) for the production o f the monoclonal  antibody reacting with mouse  G R - 1 antigen,  Ly-6G,  expressed  on  mouse  granulocytes. These cells were grown i n Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, U S A ) at 37°C, 5% CO2, and 95% humidity. The cells were transferred into serum- and protein-free hybridoma medium and incubated for two days at which time the supernatant was collected and the  antibody  concentrated to 1-1.5 mg/ml using 50,000 M W concentrating filters (Millipore Corporation, Bedford, U S A ) (124).  5 mg/kg o f the antibody solution was administered i.v. between 30  minutes and 1 hour prior to P D T light treatment.  IV. B C G vaccine: The Bacillus o f Calmette and Guerin ( B C G ) vaccine ( P A C I S  M D  , I A F Bio V a c  Inc., Laval  Quebec, Canada), was administered b y lifting the subcutaneous tumor and injected the solution slowly under the tumor using a 26-gauge needle. The dose used was 1 x 10 colony forming 7  units (CFU) per mouse.  V . G r o w t h delay:  S C C V I I subcutaneous (s.c.) tumors were grown on the sacral region o f the back on female 28  C 3 H / H e N mice, 6-8 weeks o f age. When the tumors reached optimal P D T size the mice were separated into two groups o f 8. One group was treated with Photofrin-based P D T alone and the second group was depleted o f circulating neutrophils with the i.v. adminisration o f 5 mg/kg monoclonal G R 1 antibody prior to P D T light treatment. Thereafter, the animals were monitored over a period o f 90 days, the time reference for tumor cures. Additional control groups consisted of tumor-bearing mice treated with a n t i - G R l antibody only or isotype control antibody, Rat IgG2b.  Rat IgG2b raised against the murine macrophage scavenger receptor, produced b y  hybridoma clone 2F8, was obtained purified i n azide-free form from Cedarlane Laboratories L t d . (Hornby, Canada) (124).  V I . Relative blood neutrophil levels: To obtain a white blood cell count, the tail vein o f an animal was dilated i n a warm water bath. Subsequently, using a sterile scalpel blade a small nick was made i n the tail and a sample o f blood collected by heparinized micropipet, smeared onto a clean glass slide, and left to air-dry. Samples were made i n duplicate. The air-dried specimens were stained using the Wright's stain (prepared according to manufacturer's instructions, Accustain, Sigma Chemical C o . , St. Louis, U S A ) for 30 seconds, and rinsed with water. Identifying morphological features, a differential white blood cell count was obtained using a Zeiss microscope at 2 0 X magnification. Cells were grouped as neutrophils, lymphocytes, monocytes, neutrophil band, and other, and counted until a total o f at least 200 cells were counted, in a non-specific number o f fields, on each slide. From that total, percent o f each cell type was calculated. The percentages were then converted into relative numbers, with 1 designating normal baseline levels.  29  VII. Obtaining total leukocyte numbers in circulation: To determine total blood leukocyte levels at various times after relative treatments, the tail vein o f the animal was dilated in a warm water bath, and a small nick in the tail was made using a sterile #22 scalpel blade. lOpl o f blood collected using a 20ul pipet-aid was placed into 90pl o f ice-cold lysing solution containing ammonium chloride 8.3 g/L, sodium bicarbonate 1 g / L , and 0. 1 m M ethylenediaminetetraacetic acid ( E D T A ) , to lyse erythrocytes, and kept on ice for twenty minutes. 900pJ o f lysing solution was added to each sample and a 10pJ aliquot placed on a hemacytometer to determine total leukocyte count per m l o f blood. The total number o f white blood cells was multiplied by the percent o f neutrophils (described i n part I V ) to determine the total number o f neutrophils in circulation at specific time points after treatment.  VIII. Control groups in blood neutrophil analysis Control groups o f tumor-free mice were either: (1) restrained for twenty minutes and released into their cages, (2) administered i.v. 0.2ml/20gram weight 5% dextrose i n H2O, (3) administered 10 mg/kg o f Photofrin i.v., (4) light dose o f 60 J/cm2 at 630 n m on the footpad, or (5) Photofrin-based P D T on the footpad. A single blood sample was collected from each mouse to determine total neutrophil numbers in circulation, and relative neutrophil levels.  IX. Systemic response of neutrophils to photodynamic therapy 1. T u m o r model a n d P D T : 2 x 10 E M T 6 tumor cells were inoculated onto the footpad o f anesthetized Balb/c mice. W h e n 6  the tumors reached a diameter o f 7 mm, 10 mg/kg o f Photofrin was administered i.v. to each mouse 24 hours prior to light treatment. The light dose given to each tumor was 60 J/cm .  30  2. Harvest of tumor, lung, blood, and bone marrow: 1. Tumor: Animals were sacrificed b y CO2 inhalation and tumors excised using a #22 scalpel blade and placed in a petri dish on ice. The tumors were chopped and made into a single cell suspension, as described i n section I, and suspended i n ice-cold Hank's buffered salt solution ( H B S S ) . 2. Lung: Lungs were removed using small scissors and placed i n a petri dish set on ice. The lungs were chopped with a #22 scalpel blade, then suspended i n 5 m l o f ice-cold P B S . The suspension was placed in a 6cc syringe. With the plunger, the lung tissue was pushed through a 100pm nylon mesh filter, attached on the end o f the syringe.  The filtrate was pelleted i n a  centrifuge at 600 rpm and resuspended i n erythrocyte lysing solution for 20 minutes on ice. The suspension was pelleted i n a centrifuge at 600 rpm and resuspended i n H B S S . 3 .Blood: 200-300pJ o f blood was collected b y cardiac puncture into an insulin syringe with attached 26 gauge (G) needle and immediately placed in a prepared 5cc polystyrene tube containing 3ml o f lysing solution, on ice for 20 minutes.  White blood cells were pelleted b y  centrifugation at 600 rpm and suspended i n H B S S . 4. Bone marrow: The femur o f the right hind leg was removed using sharp-tipped forceps and small surgical scissors.  Using a 2 6 G needle attached to a l c c syringe filled with P B S ,  marrow cells were flushed through the end o f the femur into a petri dish, and placed on ice. 3 m l o f P B S were added to the petri dish and the cells transferred into a 6cc syringe. U s i n g a plunger the cell suspension was pushed through a mesh filter, and the filtrate collected into a 12cc polystyrene tube.  The cells were pelleted i n a centrifuge at 600 rpm for 10 minutes, and  resuspended i n H B S S .  A 10pJ aliquot o f each suspension (tumor, lung, blood, and bone marrow) was placed on a  31  hemacytometer for the determination o f total cell count.  Each sample was concentrated or  diluted, as required, to a total cell count o f 0.5 - 1 x 10 i n H B S S prior to antibody staining for 6  flow cytometry.  3. Antibody staining and flow cytometry: All  antibodies were obtained from Pharmingen (San Diego, U S A ) .  Monoclonal  antibodies used for the detection o f cell type specific murine membrane antigens were antiC D 4 5 (pan-leukocyte marker), a n t i - G R l (myeloid differentiation surface marker), and anti-Lselectin. The antibodies G R 1 , L-selectin, and C D 4 5 , were directly conjugated to the fluorescent dyes phycoerythrin, fluorescein isothiocyanate, and Cy-chrome, respectively. A l l samples were centrifuged at 1000 rpm, and 100 p i o f supernatant prepared from hybridoma cells producing anti-mouse Fey receptor antibodies (to block the F c receptor mediated non-specific binding o f monoclonal antibodies), was added to each pellet and set on ice shielded from light for 10 minutes. Cells were washed once i n H B S S . lOOpi o f the antibody solution i n H B S S , diluted according to manufacturer's guidelines, was added to each pellet. H B S S used in this and following steps contained 0.02% sodium azide and O.lmg/ml bovine serum albumin. The cells were kept on ice for 25 minutes at which time they were washed once in H B S S and suspended i n 0.5ml ice-cold H B S S . The samples were kept on ice and shielded from direct light prior to analysis by flow cytometry using the Coulter Epics Elite E S P apparatus from Coulter Electronics. A 488nm laser was used to excite F I T C , P E and C Y - C H R O M , whose emissions were recorded through 530 ± 15, 580 ± 10, and 675 n m bandpass filters, respectively. 20 000 cells were analyzed for each sample.  32  4. GR1 and L-selectin analysis: Dead cells and debris were gated out using forward and side scatter light signals. In tumor and lung samples, white blood cells were separated from other cells, by their expression o f the C D 4 5 pan leukocyte surface marker. M y e l o i d cells were separated according to their expression o f GR1:  neutrophils/granulocytes as G R 1  expressing high levels o f GR1  + + +  , macrophages as G R (125). The population o f cells +  were then analyzed against their level o f L-selectin expression,  designated as high (++), and low (+).  X . Statistical analysis: In all experiments, data is presented as a mean ± S E M compared to non-treated controls. Analysis o f variance between the means o f control and treated sample groups was b y one-way A N O V A , unless otherwise indicated. The log-rank test was used when comparing tumor growth delay following P D T (Figure 1).  P-values less than 0.05 were considered statistically  significant.  33  RESULTS  SECTION 1 I. Growth delay following PDT is shortened with administration of G R 1 antibody:  Two groups o f eight S C C V I I tumor-bearing mice were treated with Photofrin-based P D T . One o f the two groups was injected intravenously with 5 mg/kg o f the monoclonal antibody G R 1 . These mice were then observed every 2-3 days for signs o f tumor regrowth. Results o f the experiment are shown i n Figure 1. A l l tumors responded to P D T , assessed as complete tumor ablation 24 hours after treatment. In the first group treated with P D T alone, all animals were tumor-free at day 16 after light treatment, and by day 23, 12.5 % o f the group was without tumor. The second group whose circulating neutrophils were depleted by the administration o f the a n t i - G R l antibody prior to light treatment, all animals were tumor-free at day 4 after P D T . Tumors recurred i n 50% o f the animals b y day 10, with no cures by day 14 after treatment. Treatment with G R 1 antibody alone showed no obvious effect on tumor growth, and the isotype control treatment with monoclonal antibody against mouse macrophage scavenger receptor (which belongs to the same class o f rat immunoglobulin, IgG2b, as G R 1 ) had no effect on the P D T response (124).  34  Time after PDT (days)  Figure 1: Neutrophil depletion reduces delay for tumor recurrence following PDT. T w o groups o f 8 s.c. S C C V I I back tumor bearing C 3 H / H e N mice were treated with P D T and monitored for tumor recurrence. The first group o f mice was treated with P D T alone as the second group received a combined treatment o f 5 mg/kg o f the a n t i - G R l antibody lhour prior to P D T light treatment. P D T : Photofrin 10 mg/kg injected i.v. 24 hours before 220 J / c m light delivery. Statistical significance was determined using the log-rank test. The delay for 50% tumor recurrence is significantly different (p<0.002). 2  35  II. Photofrin and mTHPC-based P D T stimulate an increase in circulating neutrophil levels in SCCVII-tumor-bearing mice  In order to investigate PDT-induced changes i n systemic levels o f neutrophils, a series o f experiments were undertaken i n which blood samples were collected from the tail vein o f mice at different time intervals before and/or after P D T or related treatments. In these experiments, one o f two protocols for blood sampling was followed: consecutive sampling or individual sampling. Consecutive sampling involved cutting nicks using a scalpel blade i n the tail o f a mouse to collect multiple blood samples over an observation period. Individual sampling meant that only one nick for a single sample o f blood was taken per mouse, thereby one group o f mice would have been used to collect blood at a particular time point.  Both m T H P C - and Photofrin-based P D T can cause a similar increase i n the level o f circulating neutrophils in S C C V I I tumor-bearing mice, as shown i n Figure 2. Neutrophil levels in treated animals rose 2.2 and 2.4 times above normal between 3 and 6 hours for Photofrin- and two different doses o f m T H P C - P D T , respectively, after the onset o f P D T light treatment.  A l l three doses o f P D T induced complete initial ablation o f treated  tumors, however, permanent cure rates for 0.6 mg/kg m T H P C . 0.3 mg/kg m T H P C , and Photofrin were 100%, 50%, and 0%, respectively (data not shown).  36  2.82.62.42.22.01.81.61.41.2-  •--10 mg/kg photofrin •#- 0.3 mg/kg mTHPC A- 0.6 mg/kg mTHPC  1.00.80.60  2  4  6  8  10  Time after the onset of light treatment (hours)  Figure 2: Photofrin- and mTHPC-based P D T of s.c. S C C V I I back tumors can induce as increase in circulating neutrophil levels, s.c. S C C V I I tumors on the backs o f three groups o f 8 C 3 H / H e N mice, were treated with either 10 mg/kg Photofrin and 150 J/cm , 0.3 mg/kg m T H P C and 110 J/cm , or 0.6 mg/kg m T H P C and 110 J/cm . A differential white blood cell count was determined from Wright stained blood smears, using the protocol o f consecutive sampling. Data is presented as mean ± se, * p<0.05, ** p<0.002, *** p<0.0001, compared to the control. 2  37  2  III. P D T induces a marked increase in circulating neutrophil levels:  Three groups o f S C C V I I tumor-bearing mice were used for this experiment, results o f which are shown i n Figure 3. The first group o f mice was treated with Photofrin-based P D T , the second was administered 5 mg/kg G R 1 antibody, and the third group received the antibody prior to light treatment o f Photofrin-based P D T .  B l o o d samples were  collected for each animal over a period o f 10 hours. M i c e treated with only the G R 1 antibody, were depleted o f neutrophils i n circulation over a time course o f the observation period. P D T induced a marked increase i n circulating neutrophil numbers with a peak 2.4 times that o f relative normal levels, at 3 hours after the onset o f light treatment. This increase persisted for a period o f at least 10 hours. A marked increase i n circulating neutrophil levels was also observed i n mice treated with the antibody prior to light treatment. In this group relative neutrophil levels rose to a peak o f 1.5 times that o f normal levels at 2.5 hours after the onset o f light treatment, however, this increase did not last more than 1 hour at which time levels fell down below normal.  38  —•—PDT — # — a n t i - G R 1 + PDT - A-  anti-GR1  Figure 3: P D T induces an increase in circulating neutrophil levels. A change in circulating neutrophil levels was compared in three groups o f 8 s.c. S C C V I I back tumor-bearing C 3 H / H e N mice. Tumors i n the first group were treated with P D T only, the second group received a combined treatment o f 5 mg/kg G R 1 antibody 1 hour prior to P D T light treatment, and the third group was administered the G R 1 antibody alone. P D T : Photofrin 10 mg/kg and 150 J/cm2. The light treatment lasted an average o f 20 minutes. Neutrophil levels were determined as described in Figure 2. Data are presented as mean ± se, ** p<0.005 compared to controls.  39  IV. P D T induces a rise in circulating neutrophil levels in EMT6-tumor bearing mice, but is suppressed in a combined treatment with B C G vaccine  Figure 4 illustrates the effect o f Photofrin-based P D T on neutrophil levels i n the blood o f Balb/c mice when either E M T 6 back tumors, or normal skin on tumor-free mice were treated.  In addition, the effect o f a peritumoral administration o f 1 x 10 C F U 7  B C G vaccine alone, which produces a different type o f inflammatory insult, or a combined treatment o f B C G and P D T on E M T 6 back tumors is demonstrated.  As  shown earlier, P D T induced a peak rise 2.5 times above normal at 2.5 hours after light treatment. P D T o f tumor-free, normal skin also caused a rise i n circulating neutrophil levels with a peak o f 2.2 times that o f normal at 1 hour after treatment. B C G did not stimulate a rise i n the level o f neutrophils i n blood, but induced a significant fall i n blood neutrophil levels at 10 and at 24 hours.  The combined treatment o f B C G and  P D T produced a modest increase i n neutrophil levels in circulation. This peak was reached at 1 hour after treatment, persisted for at least 10 hours, and fell back down to normal levels 24 hours later. The peak rise stimulated b y the combined treatment was well below the increase i n circulating neutrophil levels following either P D T on the E M T 6 tumor or that o f normal skin.  40  —•—PDT — • — P D T , tumor-free P D T and B C G  — A —  3.4  -  3.2  -  3.0  -  —•—BCG  2.8 2.6 2.4  -  2.2  -  2.0  -  1.8  -  1 .6 1 .4 1 .2 1 .0 0.8 - | 0.6 0.4 -4 0.2  T  T  6  2  0  T  8  - r 10 -  24  T i m e after the o n s e t of light t r e a t m e n t ( h o u r s )  Figure 4: P D T induces an increase in circulating neutrophil levels in both E M T 6 tumor and tumor-free Balb/c mice, whereas, B C G vaccine has a suppressive effect. Multiple blood samples were collected from each animal after treatment. Groups consisted o f 4-6 mice each. The first group o f s.c. back E M T 6 tumor-bearing female mice were treated with P D T , the second was treated with P D T on their tumor-free depilated backs, a third group received a combined treatment o f 10 C F U B C G vaccine and P D T , and the fourth group was administered B C G peritumoraly. P D T : 10 mg/kg Photofrin and 75 J / c m . Data is based on Wright stain analysis o f blood smears as explained i n Figure 2,and is presented as mean + se. * p<0.02, ** p< 0.0005 ompared to controls. 7  2  41  SECTION 2 I. Intravenous drug adminstration and tail bleeding capable of inducing an increase in circulating neutrophil levels:  Figure 5 shows the change i n blood neutrophil levels following an i.v. injection o f 10 mg/kg o f Photofrin. A n increase o f 2 times that o f the normal level was observed 1 hour after treatment.  Levels fell back down to normal 24 hours later. Consecutive sampling  from the tail vein o f otherwise non-treated animals also increased circulating neutrophil levels to a peak o f 2.1 that o f normal at 1 hour after the initial nick (See Figure 6). These levels remained slightly elevated 24 hours later.  42  T i m e (hours)  Figure 5: Increase in circulating neutrophil levels after i.v. administration of 10 mg/kg Photofrin. B l o o d was collected before, 1, 3, 6, and 24 hours after injection from 4 tumor-free Balb/c mice. Data was collected as explained i n Figure 2, and presented as mean ± se. * p<0.05, ** p< 0.005 compared to controls.  43  Figure 6: Relative changes i n circulating neutrophil levels over a 24 hour period of multiple blood sampling per animal. B l o o d was sampled from tail veins o f 3 tumor-free Balb/c mice. Data is based on Wright stain analysis, as described i n Figure 2 and presented as mean ± se. * p<0.05, ** p<0.005 compared to controls.  44  II. Changes in relative circulating neutrophil levels in non-PDT-treated mice: a.) i.v. injection of D±W, 20 minute restraint, or light alone of Balb/c tumor-free footpad:  Shown i n Figure 7, 5% dextrose i n H2O administered i.v. into a group o f mice can also induce an increase i n circulating neutrophil levels.  The peak was 1.4 times above  normal  • — — • —  restraint D ,W  — • — l i g h t alone on footpad (tumor-free) 1 .8 1 .7  1 .6 1 .5 CD  >  CD  CL  1 .4 1 .3 1 .2  O  i_  "5 CD C  T3  1 .1  1 .0  O O  0.9  X3  0.8  CD  as CD  DC  0.7 0.6 0.5 0.4 0.3  2  4  6  24  Tim e (hours) Figure 7: Changes in blood neutrophil levels after: i) i.v. injection of 0.2 ml/20 g mouse 5% dextrose in H 0 (D W), ii) 20 minute restraint, or iii) 60 J/cm , 630 nm of light on tumor-free footpad. Differential white blood cell counts were made as described in Figure 2, using the individual sampling protocol. 8 mice were used per time point. Data is presented as mean + se. *p<0.05 compared to control. 2  2  S  45  levels at 1 hour after injection, and dropped down to normal levels b y 6 hours after the injection. Twenty-minutes  o f restraint, as would be  experienced  during P D T light  treatment, also induced a peak increase i n blood neutrophil levels 1.6 times that o f normal at 1 hour after the mice were released from the restraint.  Neutrophil levels  dropped down to normal b y 6 hours after treatment. A l s o shown i n Figure 7 is the observed effect o f 630nm light delivered at a dose o f 60 J/cm , i n the absence o f photosensitizer. 2  When light was delivered to the footpad o f  Balb/c mice results suggest a suppressive effect o f light on circulating neutrophil levels, which persist below normal 24 hours later.  46  SECTION 3 I. Increase in circulating neutrophil levels is PDT-specific:  Figure 8 demonstrates the change in relative neutrophil levels i n circulation following Photofrin-based P D T . P D T on the footpad o f tumor-free mice induces a peak increase at 1 hour after light treatment 2 times that o f normal levels, an increase statistically higher than any other factor attributed to stress i n handling during treatment (discussed i n S E C T I O N 2), capable o f inducing a rise in neutrophil levels. These levels drop to normal 24 hours after treatment. P D T on the E M T 6 foot tumor also increased the neutrophil levels i n blood to a peak o f 2 times that o f normal, however, this increase occurred at 10 hours after treatment and remained statistically elevated 24 hours after treatment. Figure 9 indicates that P D T had the strongest effect on the s.c. E M T 6 back tumor, with a peak o f 2.3 times that o f normal levels persisting for at least 10 hours. Levels drop at 24 hours but remain statistically above normal. Light delivered to s.c. E M T 6 tumors grown on the sacral region on the back o f mice, induced an increase i n blood neutrophil levels with several peaks over a 24 hour time course. A t 1 hour after light exposure, levels increased 1.8 times that o f normal, dropped to 1.3 at 3 hours, rise at 6 hours to 1.5 times that o f normal levels and fall down to normal at 10 hours only to rise again 24 hours later. In Figure 9 a third curve represents the normalized P D T effect, corrected for the oscillations i n neutrophil levels following light treatment alone.  47  Figure 8: Changes in circulating neutrophil levels following PDT on the foot. P D T (10 mg/kg Photofrin and 60 J/cm ) was applied to: i.) footpad, and ii.) E M T 6 foot tumor, o f Balb/c mice. Blood was sampled from 6 mice at various times after light treatment. Data was collected as described i n Figure 7 using individual sampling, and presented as mean ± se. * p< 0.05, ** p< 0.005 compared to non-treated controls. 2  48  E M T 6 s.c. back tumor  3.0  — • — PDT minus light only --O--PDT --A-- light only n  2.82.62.42.22.01.81.61.41.21.00.80.6-•  ~r o  2  4  6  8  -W/-T10 24  Time after onset of light treatment (hours)  Figure 9: Normalized changes in circulating neutrophil levels following P D T on E M T 6 back tumor. Shown i n this figure are the data for P D T (10 mg/kg Photofrin and 60 J/cm ), light alone, and the corrected P D T effect minus light alone on the s.c. E M T 6 back tumor o f Balb/c mice. B l o o d was sampled from 6 mice at various times after light treatment. Data was collected as described i n Figure 7 using individual sampling, and presented as mean ± se. * p< 0.05, ** p<0.005 compared to non-treated controls. 2  49  SECTION 4 I. Analysis of total circulating leukocyte numbers following PDT treatment of tumors and normal skin: Changes in total blood leukocyte counts and total neutrophil counts in PDT-treated mice are presented in Tables 1 and 2.  Total numbers of white blood cells increased  dramatically from 11 ± 2.0 x 10 per ml of blood to 24 ± 2.8 x 10 and 18 ± 1.9 x 10 at 6  6  6  3 and 6 hours post treatment, respectively, for mice treated with PDT on their tumor-free footpad (Table 1). A marked increase also occurred in mice whose back EMT6 tumors were treated with PDT, with the greatest difference observed at 3 hours post light treatment when the total leukocyte numbers per ml of blood rose to 29.9 ± 2.6 x 10 . 6  Neutrophil numbers (Table 2) also increased significantly at these corresponding time points, with a peak of 2.3 times and 6 times that of numbers normally found in circulation for the treatment on footpad and back EMT6 tumors, respectively, 3 hours after treatment.  Although, there does not seem to be a significant change in total  leukocyte numbers following PDT on EMT6 foot tumors, an increase in neutrophil numbers was observed at 10, and 16 hours, followed by a peak increase of 1.9 times that of normal numbers, at 24 hours after treatment. Although oscillations did occur in total numbers of lymphocytes (Table 3), changes observed were not consistent. No obvious changes in other leukocytes, such as monocytes, were detected in the blood of treated mice.  50  Table 1: Total circulating leukocyte numbers per ml of blood following Photofrin based-PDT of: i) tumor-free footpad, ii) s.c. EMT6 back tumor, and iii) EMT6 foot tumor, in Balb/c mice.  x 10 leukocytes/ml of blood 6  Time after PDT light (hours)  Footpad  back EMT6 tumor  non-treated  11 ± 2 . 0  13.8 ± 1.1  0.5  15 ± 1.3  18.8 ± 3 . 3  -/-  1  16 + 1.3 *  12 ± 1.4  -/-  3  24 ± 2 . 8 **  29.9 ± 2 . 6 *  11 ± 2 . 4 7  6  18 ± 1.9 *  17.4 ± 2 . 3  11 ± 1.64  10  10 ±2.25  25 ± 7.16 *  9.3 ± 1.52  16  16 + 2.1 *  24  14 ± 1.91  -/14.2 ± 1.0  Foot EMT6 tumor  13 ± 1.37  13.3 ± 0 . 8 8 15.5 ± 2 . 0  Blood samples for each time point following light treatment were taken from 6-8 mice, by individual sampling protocol. PDT dose was as described in Figure 9. Mean values based on hemacytometer counts are presented ± se. * p< 0.05, ** p<0.005 compared to non-treated controls.  51  Table 2: Total circulating neutrophil numbers following Photofrin-based P D T of: i) Balb/c footpad, ii) s.c. EMT6 back tumor, and iii) EMT6 foot tumor. x 10 neutrophils/ml of blood 6  Footpad  back EMT6 tumor  non-treated  2.9 ± 1.1  2.95 ±0.38  0.5  4.5 ±0.87  8.1 ±2.3  -/-  1  5.2 ±0.64 *  7.0± 1.04  -/-  3  6.8 ±1.9 **  18.3 ± 5 *  4.2 ± 1.5  6  4.2 ±0.64 *  9.8 ± 1.4 *  5.2 ±0.51 *  10  2.9 ±0.98  10±2.9 *  4.9 ±0.63 *  16  3.3 ±0.47  24  2.6 ± 0.46  Time after PDT light (hours)  -/-  5.6 ±0.58  Foot EMT6 tumor  3.4 ±0.58  5.4 ±0.87 * 6.5 ±0.46 *  Blood samples for each measurement following light treatment were collected from 6-8 mice, using the individual sampling protocol. Data is based on hemacytometer counts and Wright stain analysis of blood smears. PDT dose was as described in Figure 9. Data is presented as mean ± se. * p<0.05, ** p<0.005 compared to non-treated controls.  52  Table 3: Total circulating lymphocyte numbers per ml of blood following Photofrinbased P D T of: I) tumor-free footpad, ii) s.c. EMT6 back tumor, iii) EMT6 foot tumor, in Balb/c mice.  x 10 Ivmphocvtes/ml of blood 6  Time after P D T light (hours)  back E M T 6 tumor  Footpad  Foot E M T 6 tumor  9 ± 0.96  9.2 ± 1.9  11 ± 0.73  0.5  10.4± 0.72  11± 1.2  -/-  1  10.1± 0.57  5.1± 0.9 *  -/-  3  17 ± 2 . 1 *  12 ±1.01  7.0+1.0  6  13± 1.5*  7.5 ± 1.1 *  5.6± 0.94 *  10  7.2± 1.6  8.9± 2.6  4.3 ± 0 . 9 *  16  13 ± 2 . 4  -/-  7.9 ± 0.1  24  11± 1.5  non-treated  8.5± 0.96  8.9±  1.5  B l o o d samples for each time point following light treatment were taken from 6-8 mice, by individual sampling protocol. P D T dose was as described i n Figure 9. Mean values based on hemacytometer counts and Wright stain analysis, are presented ± se. * p< 0.05, ** p< 0.005 compared to non-treated controls.  53  II. Control treatments also induced a change in total circulating neutrophil numbers  leukocyte and  Table 4 shows a stress-related increase i n total white blood cells i n Balb/c mice. The increase was from normal levels o f 13.8 ± 1.1 x 10 /ml to a peak level o f 21.5 ± 3 x 10 /ml 6  6  and 16.6 ± 2.2<x 10 /ml at 3 hours after the i.v. administration o f D W , or b y restraint alone, 6  5  respectively.  The total number o f neutrophils can also be elevated i n both cases as  summarized i n Table 4. The peak in mice that were administered D5W i.v. occurred 1 hour after injection, and rose to 8.54 ± 1.3 x 10 per m l o f blood from 2.3 ± 0.38 x 10 . The peak 6  6  for restrained mice, 5.4 ± 0 . 8 1 x 10 /ml, occurred 3 hours after release. Total numbers o f 6  lymphocyte and other leukocytes, such as monocytes, and band neutrophils did not change significantly.  Table 4: Total leukocyte and neutrophil numbers i n mice following either i.v. injection of D5W or 20 minute restraint. x 10 leukocytes/ml of blood  x 10 neutrophils/ml of blood  6  Time after Treatment (hours)  6  D W  Restraint  D W  Restraint  13.8 ± 1.1  13.8 ± 1.1  2.3 ± 0 . 3 8  2.3 ± 0 . 3 8  1  16.3 ± 2 . 1  9.5 ± 3 . 9  8.4 ± 1 . 3 *  3.9 ± 0 . 9 8  3  21.5 ± 3 *  16.6 ± 2 . 2 *  6.5 ± 1 . 6 *  5.4 ± 0 . 8 1 *  16.2 ± 1  13.4 ± 1.9  3.7 ± 0 . 7 1  4.0 ± 0 . 5 8  23.2 ± 4 . 6  12.5 ± 1.2  3.6 ± 0 . 7 5  3.1 ± 0 . 5 4  non-treated  6 24  5  5  B l o o d samples for each timepoint following light treatment were taken from 6-8 mice, b y individual sampling. Mean values based on hemacytometer counts and Wright stain analysis o f blood smears are presented ± se. * p<0.05 compared to nontreatedcontrols  54  Table 5: Total circulating leukocyte and neutrophil numbers in mice following a light dose of 60 J/cm , in the absence of photosensitizer. 2  x 10 leukocytes/ml of blood  xlO neutrophils/ml  6  Time after Light (hours)  Footpad  6  back E M T 6 tumor  Footpad  of blood  back E M T 6 tumor  10.7 ± 1.0  13.8 ± 1.1  2.9 ± 0 . 5 6  2.9 ± 0 . 3 8  15.9 ± 0 . 7 6  9.8 ± 2 . 0  4.4 ± 0.24  4.8 ± 0 . 8 3  3  22.4 ± 4 . 7 *  29.1 ± 5 . 8 *  5.8 ± 1.8 *  9.5 ± 2 . 0 *  6  17.2 ± 1.8 *  13.4 ± 3.1  2.8 ± 0 . 8 8  5.9 ± 1 . 8 *  treated 1  '  10  -/-  16.9 ± 2 . 2  24  16.6 ± 2 . 7  14.3 ± 2 . 2  -/2.2 ± 0 . 2 1  4.0 ± 0.68 5.5 + 1.1 *  B l o o d samples for each timepoint were collected from Balb/c mice whose footpad or back E M T 6 tumor was illuminated with 60 J / c m o f 630 nm wavelength o f red light. B l o o d samples for each time point following light treatment were taken from 6-8 mice. Mean values are presented ± se, based on hemacytometer counts and Wright stain analysis. * p<0.05 compared to non-treated controls. 2  Table 5 summarizes the increase i n total leukocyte and neutrophil numbers i n circulation following light treatment in the absence o f photosensitizer, on the footpad o f mice or on the E M T 6 tumor grown on the sacral region o f their backs. 60 J/cm o f light delivered on the footpad o f mice induced a peak increase i n total white blood cell numbers o f 22.4 ± 4.7 x 10  6  at 3 hours after treatment, 2.1 times greater than normal levels o f 10.7 ± 1.0 x 10 per m l o f 6  blood.  The same light dose induced a very similar increase i n total circulating leukocyte  numbers when illuminating back E M T 6 tumors.  55  The neutrophil levels increased two-fold at 3 hours, i n mice whose tumor-free footpad was illuminated with 60 J/cm o f 630nm light. The levels o f these cells increased 3.1 times i n mice whose back s.c. E M T 6 tumors were treated with light alone.  56  S E C T I O N 5: Systemic response of GRl-positive cells to P D T  Four groups o f 3-6 E M T 6 tumor-bearing Balb/c mice were treated with Photofrin-based P D T , and a fifth group was not treated. F l o w cytometry analysis was the method used to decipher the G R l - p o s i t i v e cell content o f tumors, lungs, blood and bone marrow i n these mice, at 1.5, 6, 10, 24 hours following P D T light treatment (Figure 10). changes  are  commented  in  detail  57  in  the  presentation  of  The observed  figures  11-14.  Lung Turn or  6.0 **  o o  -  vi  o Q.  <+—  o  4.5  —  Bone marrow  4.0 -  >  "•+—•  |  —  -  "CD  rr o  5.0  Blood  I  5.5 -  3.5 3.0 2.5  — _  2.0  —  1 .5  —  1 .0  —  0.5  -  >  CD CD >  Re  CO  0.0 -0.5 -2  ~r  "T 4  T 6  T 8  /h-T-  10 12 14 24 2 T i m e after P D T light t r e a t m e n t ( h o u r s ) 0  F i g u r e 10: Systemic response of G R l - p o s i t i v e cells to P D T . Four groups o f 3-6 E M T 6 foot tumor-bearing Balb/c mice were treated with P D T and a fifth group was untreated. Groups o f mice had their lungs, tumor, blood, and bone marrow cells collected at different time points following P D T (10 mg/kg Photofrin and 60 J/cm ). Data is based on flow cytometry analysis, and is presented as mean ± se. ** p<0.005, * p<0.05 compared to non-treated controls. 2  58  L  Tumors: The cell composition o f E M T 6 rumors prior to P D T treatment generally  consisted o f 70% tumor associated macrophages ( T A M ) , 10% neutrophils and the remaining 20% were malignant  Figure 11: Changes in cell content of GRl-positive cell population in E M T 6 tumors following P D T . E M T 6 foot tumors from 3-6 Balb/c mice were excised and analyzed b y flow cytometry for GR1 expression 1.5, 6, 10, and 24 hours after P D T light treatment. P D T : 10 mg/kg Photofrin and 60 J/cm2. Data is presented as mean ± se. ** p<0.005, and p<0.05 compared to non-treated controls.  59  cells. Following P D T treatment a change i n the overall cell content occurs with a decrease i n malignant and T A M cells which die during treatment, and an increase i n infiltrating neutrophil and macrophage populations at 10 and 24 hours post treatment.  These changes were also  observed b y Wright stain analysis o f prepared tumor cell suspensions. M y e l o i d cells express various levels o f G R 1 , where neutrophils express the highest levels, monocytes and less mature neutrophils express medium levels, and macrophages express the lowest. A s shown in Figure 11, P D T induced a continuous influx o f G R l - p o s i t i v e cells in E M T 6 tumors over a 24-hour time interval. The majority o f these cells expressed high levels o f G R 1 , and forward and side scatter signals indicate that these are most likely neutrophils. A significant increase 1.97 ± 0.58 times over normal levels was detected 1.5 hours post P D T .  A peak rise was at 10 hours following  treatment, 3.12 + 0.41 times over normal levels. A t 24 hours, levels were still elevated at 2.7 ± 0.38 times above normal.  II. L u n g s : Figure 12 shows that the accumulation o f neutrophils, designated as G R l - p o s i t i v e cells, i n lungs peaked at 6 hours after P D T treatment o f E M T 6 tumors, 4.79 ± 0.83 times that o f normal levels i n pulmonary vessels. The levels o f these cells decreased to normal at 10 hours and then rose again at 24 hours to 1.69 ± 0.18 times above normal. The cell content o f resident macrophage and other cell populations, do not change i n the 24 hour interval following P D T treatment.  60  T i m e a f t e r P D T light t r e a t m e n t ( h o u r s )  Figure 12: Change in the levels of GRl-positive cells in lungs following P D T . Lungs o f 3-6 E M T 6 foot tumor-bearing mice were excised and analyzed b y flow cytometry for GR1 expression at 1.5, 6, 10, and 24 hours following P D T treatment, and compared to lungs excised from non-treated mice. P D T : 10 mg/kg Photofrin and 60 J / c m . Data are presented as mean ± se. ** p< 0.005, * p< 0.05 compared to non-treated controls. 2  61  III. Blood: Figure 13 illustrates the change i n levels o f neutrophils in blood o f treated mice. A rise to 2.35 ± 0.24 times that o f normal circulating levels o f G R l - p o s i t i v e cells was observed 10 hours after treatment. Levels returned to normal at 24 hours after P D T .  T i m e after P D T light t r e a t m e n t ( h o u r s )  Figure 13: Changes in the GRl-positive cell content of blood following treatment with P D T . B l o o d collected from 3-6 E M T 6 foot tumor-bearing mice 1.5, 6, 10, and 24 hours following P D T light treatment. P D T : 10 mg/kg Photofrin and 60 J/cm . Data is based on flow cytometry analysis and presented as mean ± se. ** p<0.005 compared to non-treated controls. 2  62  I V . Bone marrow: The change i n G R l - p o s i t i v e cell content in bone marrow is shown i n Figure 14. There was a marked drop o f 20 % from normal levels in the cell population expressing m i d high levels o f G R 1 , at 6 hours and a 55% drop at 10 hours after treatment.  Levels return to  normal at 24 hours after P D T .  Time after P D T light treatment (hours) Figure 14: Changes in GRl-positive cell content of bone marrow following P D T . Bone marrow cells were harvested from 3-6 E M T 6 foot tumor-bearing mice and analyzed b y flow cytometry for G R 1 expression, 1.5, 6, 10, and 24 hours following P D T light treatment. P D T : 10 mg/kg Photofrin and 60 J/cm . Data is presented as mean ± se. * p<0.05 compared to non-treated controls. 2  63  Section 6: L-selectin expression on neutrophils following PDT In an attempt to uncover their activation state, flow cytometry analysis was used to detect changes in L-selectin ( C D 6 2 L ) expression in granulocyte populations o f the bone marrow and lungs o f E M T 6 foot tumor-bearing mice following P D T (Photofrin 10 mg/kg and 60 J/cm ), over a 24-hour interval.  Lungs  Bone marrow  F i g u r e 1 5 : C h a n g e s i n the c o n t e n t o f g r a n u l o c y t e s e x p r e s s i n g h i g h levels o f L -  Bone marrow cells and lungs were excised from 3-4 E M T 6 foot tumor-bearing Balb/c mice, 1.5, 6, 10, and 24 hours following P D T . F l o w cytometry analysis was used to detect changes in the percent o f granulocytes expressing high levels o f L-selectin. P D T : Photofrin 10 mg/kg and 60 J/cm . Data is presented as mean ± se. * p< 0.005 compared to nontreated controls. selectin i n b o n e m a r r o w a n d lungs.  64  Cells expressing high levels o f L-selectin increased from 2.5% to 36% as shown i n Figure 15, with a subsequent decrease from ~97% to 64% o f cells expressing l o w levels o f L-selectin, i n the total granulocyte population o f bone marrow 24 hours following P D T . N o significant change was detected i n the lungs.  W e also examined a change i n L-selectin expression i n G R l - p o s i t i v e  cells i n tumors. Figure 16 illustrates a change from 8% o f all G R l - p o s i t i v e cells expressing high levels o f L-selectin to 53%, with a corresponding decrease from 9 1 % to 46% o f all G R l - p o s i t i v e cells expressing l o w levels o f L-selectin over a 24-hour time interval following P D T treatment.  Figure 16: Changes in L-selectin expression of GRl-positive cells in tumors following PDT. E M T 6 foot tumors were excised 1.5, 6 and 24 hours from host Balb/c mice, following treatment with P D T . Using flow cytometry techniques, a change i n the G R l - p o s i t i v e cell content expressing high levels o f L-selectin was detected. P D T : Photofrin 10 mg/kg and 60 J/cm . Data is presented as mean ± se. * p< 0.005 compared to non-treated controls. 2  65  Changes i n the percent o f circulating neutrophils expressing low, medium, and high levels o f L selectin i n blood are shown i n Figure 17. In a non-treated control, the majority o f circulating neutrophils i n E M T 6 tumor-bearing mice expressed medium levels o f L-selectin, while high levels were expressed by a very small content o f the neutrophil population. Ten hours following Photofrin-based P D T , there was a decrease from 20% to 5% o f all neutrophils expressing low levels o f L-selectin, and a corresponding increase from 77% to 92% expressing medium levels. A t 24 hours however, 1/3 o f all neutrophils expressed high levels o f L-selectin, an increase from 4% (1/25) i n control levels, which caused a significant decrease i n the cell content expressing medium concentrations o f L-selectin compared to normal levels. Dot-plots representing changes in L-selectin expression i n blood, tumor, and bone marrow, are shown i n figures 18 and 19.  66  li^fel L-selectin  low  —  100-1  L-selectin  medium  I  I L-selectin  high  908070-  CO  Q.  o  •*-> ~i CD c  6050-1  40-| 30 20100  T m  tin  JL  fa  nontreated  1.5  10  24  Time after P D T light treatment (hours)  Figure 17: Changes in L-selectin expression of neutrophils in circulation following PDT. E M T 6 foot tumors o n Balb/c mice were treated with P D T (Photofrin 10 mg/kg and 60 J/cm ) and their circulating neutrophils analysed b y flow cytometry for a change i n L-selectin expression: low, medium, or high. Data is presented as mean ± se. * p< 0.005 compared to non-treated controls. 2  67  Blood  10 hr post PDT  Control  24 hr post P D T  1000 o I  DO O U H  I_a_ H c n  r  I I  i  Forward scatter  Figure 18: Flow cytometry analysis dot-plots of PDT-induced changes i n L-selectin expression on G R 1 cells in peripheral blood. Representative examples o f flow cytometry-generated dot-plots, with the ordinate showing fluorescence o f F I T C conjugated to anti-mouse C D 6 2 L (L-selectin) in arbitrary units per cell, and light forward scatter on the abscissa. (PDT: Photofrin 10 mg/kg, 60 J/cm ). 2  68  r  Control  24 hr post PDT  1000 i  Tumor  i  "5  1  1  r  n  i  1  1  r  t/3  00  .v..  JO U H  .  * ' .  = 1  i  1  1  1  r  1  marrow  B  ..... .....  1  F o r w a r d scatter Figure 19: Flow cytometry analysis dot-plots of PDT-induced changes in L-selectin expression on G R 1 cells in tumor and bone marrow. Representative examples o f flow-cytometry-generated dot-plots, with the ordinate showing fluorescence o f F I T C conjugated to anti-mouse C D 6 2 L (L-selectin) i n arbitrary units per cell, and light forward scatter on the abscissa. ( P D T : Photofrin 10 mg/kg, 60 J/cm ). 2  69  DISCUSSION  A s the first wave in the host response to PDT-elicited inflammation, neutrophils are attracted to a site o f P D T damage (76,77,114). Inflammatory chemotactic signals released into immediate surroundings and circulation from treated lesions, attract neutrophils which can contribute to the management o f tumor cell death in a number o f ways. A s large number o f neutrophils accumulate i n PDT-treated tumors, their adherence to the endothelium may promote the formation o f a neutrophil aggregate. In this way, neutrophils may enhance blood flow stasis associated with P D T , contributing to tumor hypoxia, and consequent ischemic tumor cell death. Activated neutrophils promote platelet aggregation through the release o f thromboxane, again amplifying reduction i n blood flow, which leads to tumor regression (13,78).  In addition,  activated neutrophils release proteolytic substances and components produced v i a the N A D P H oxidase membrane system, which i n a synergistic manner inflict localized tumor damage. Subsequently, both tumor vasculature and parenchyma become susceptible to cytotoxic capabilities o f neutrophils.  In particular, endothelial cells are targeted resulting i n the  breakdown o f basement membrane,  creating leaky vasculature,  and increased  vascular  permeability with consequent edema formation and hemorrhage (75). I f neutrophils are capable o f migrating from the vessel lumen, they may come into direct contact and inflict death to tumor cells. Whether or not neutrophils are able to extravasate into PDT-treated tissue may depend on a number o f factors. Adherence to endothelium is crucial and dependent on the expression o f adhesion molecules (81,82). L-selectin on polymorphonuclear leukocytes and P/E-selectin on endothelial cells, are crucial for initial contact, and subsequent neutrophil diapedesis between endothelial cells is mediated by members o f the integrin family o f adhesion molecules (86).  70  The level o f endogenous nitric oxide (NO) production i n tumors, which can vary significantly i n human tumors and among different experimental tumor models (13), may play an important role in regulating adhesion, since N O is a strong inhibitor o f adhesion molecule expression (126,127).  This suggests an indirect effect o f N O i n the control o f neutrophil extravasation,  implying a possible hindrance i n the rate o f tumor cures by P D T . These issues are currently under active investigation in our laboratory. Chemotactic signals, presumably produced and released by activated neutrophils following P D T , initiate and attract an influx o f inflammatory cells to the lesion. Neutrophils are followed by macrophages/monocytes, and mast cells, which arrive i n waves following P D T treatment (77).  A l l o f the above mentioned functions o f activated neutrophils support the  hypothesis that neutrophils play an indispensable role for effective P D T . The rapid, marked sequestration o f neutrophils into P D T treated tissue has been welldocumented (77,78,114).  However, it was not determined whether  such a protracted  mobilization o f neutrophils is associated with a systemic response o f these cells to P D T . Does accumulation o f neutrophils into tumors following P D T result i n a change i n the level o f these cells i n circulation, or their storage and/or marginated pools i n organs such as bone marrow and lungs? This issue has received little attention thus far, aside from a group i n the Netherlands. D e Vree and co-workers observed a rapid rise i n circulating neutrophil levels i n the blood o f rhabdomyosarcoma tumor-bearing rats following Photofrin-based P D T (119). This finding and related revelations in earlier work o f our laboratory, prompted the first phase o f this thesis project to investigate a change i n the circulating level o f neutrophils following Photofrin-based P D T i n murine tumor models. The results presented in this thesis show that P D T did induce a marked rise in blood neutrophil levels.  This was demonstrated with two tumor models ( S C C V I I squamous cell  71  carcinoma, and E M T 6 mammary sarcoma) growing i n two different strains o f mice ( C 3 H / H e N and Balb/c, respectively). A similar, although perhaps somewhat less pronounced effect was observed with P D T treatment o f normal skin. PDT-induced changes i n circulating leukocytes were almost exclusively limited to alterations i n neutrophil levels. Changes i n the lymphocyte content o f blood were noticed on several isolated occasions, but this was not consistent and may be attributed to experimental fluctuations. There were no indications o f changes in the levels o f circulating monocytes or other types o f white blood cells, aside from the occasional appearance o f immature neutrophils (band cells). The presence o f band cells hints to enhanced neutrophil production and differentiation from cell precursors i n the bone marrow as a response to P D T induced inflammation. The leukocyte content in mouse blood is characterized by a marked predominance o f lymphoid populations, comprising over 70% o f all nucleated cells i n circulation. Neutrophils normally make up 25-30% o f the total, whereas monocytes and other white blood cells usually do not exceed 1% (78). A marked rise in the level o f circulating neutrophils to about 60% o f all leukocytes, was observed i n PDT-treated S C C V I I tumor bearing mice lasting for at least 10 hours following light treatment.  The magnitude o f the PDT-induced neutrophilia, is further  illustrated by neutrophil total counts which rose over six times above normal levels i n mice at peak time intervals, i.e. from below 3 x 10 /ml (normal levels i n mice) to over 18 x 10 /ml, 6  respectively.  This caused the lymphocyte levels to drop below 40%.  6  The total count o f  circulating leukocytes i n these situations increased more than two-fold which can all be attributed to changes i n neutrophil levels. These changes are demonstrating that P D T induces a pronounced leukocytosis caused by neutrophils, i.e. neutrophilia. A n increased level o f neutrophils in circulation commonly accompanies a strong acute inflammation  caused by bacterial infections, tissue injury or acute traumatic  72  disorders  (128,129). Neutrophilia is in some cases caused by neoplasia, a phenomenon not observed with our mouse tumor models. Rather, infarction and hemorrhage that occurs i n P D T treated tumor tissue are likely responsible for the induction o f neutrophilia, generally caused b y the release o f interleukin 1 (IL-1) and tumor necrosis factor alpha ( T N F - a ) from macrophages and other cells, following bacterial infections or tissue injury (129). These cytokines, known to be induced by P D T (119,130), directly promote the accelerated release o f neutrophils from bone marrow, with subsequent generation o f colony-stimulating factors b y macrophages  and T-lymphocytes,  inducing the proliferation o f bone marrow hemopoietic precursor cells (129). In addition, a pool o f neutrophils temporarily marginated along vascular walls can rapidly be mobilized b y specific molecular signals (best known are epinephrine, or corticosteroids) (129), some o f which may also be released from PDT-treated sites. Interestingly, the G R 1 antibody-mediated depletion o f the circulating neutrophil pool prior to P D T light treatment, reduces the delay for tumor recurrence following Photofrin-based P D T o f the S C C V I I murine tumor (Figure 1).  This suggests the relevance o f a systemic  response o f neutrophils for successful P D T . Surprisingly, it was also observed that the P D T treatment following  anti-GRl-mediated neutrophil depletion, induced a marked rise i n  circulating neutrophil levels, although this did not persist for more than one hour (Figure 3). Therefore, it seems that tumor control b y P D T requires a prolonged mobilization o f neutrophils, reflected b y a rise i n neutrophil levels i n blood following treatment.  A pool o f neutrophils  unscathed b y the antibody presumably supplied a surge o f neutrophils into circulation observed in Figure 3. In addition to the FDA-approved photosensitizer Photofrin, there are several new photosensitizers showing promising results in clinical testing (12). A t least some o f them can be expected  to  share with  Photofrin the  ability  73  to  induce  systemic  neutrophilia  when  photodynamically activated by light energy, as evidenced by our results with the photosensitizer mTHPC.  Interestingly, very similar changes i n blood neutrophil counts were incited b y two  different doses o f mTHPC-based P D T and Photofrin-based P D T (Figure 2). Another form o f inflammatory insult initiated by the peritumoral application o f the live B C G vaccine (used to induce inflammation i n the clinical treatment o f bladder cancer (131)), however, was not found to provoke a rise i n neutrophil levels in circulation (Figure 4), although it does induce neutrophil accumulation in E M T 6 tumors (Korbelik, unpublished). Instead, B C G alone seems to suppress blood neutrophil levels and also abrogates the rise induced by P D T . Since the mice undergo little stress in handling during a peritumoral application o f B C G , the possibility that neutrophilia associated with P D T may originate i n other stress-related factors experienced  i n handling o f the  animals during P D T treatment, required  examination.  Neutrophils are known to be the first inflammatory cell to respond and be on alert in any trauma. A neutrophilic state can, for instance, be induced in sheep b y a loud noise (132), and as seen in our mouse models, when these animals were subjected to blood collection b y tail bleeding. Indeed, the method o f consecutive blood sampling by putting a small nick i n the tail vein and, i n this way, collecting multiple blood samples from each mouse over a period o f 24 hours, stimulated an inflammatory response, characterized by neutrophilia (Figure 6).  A  neutrophilic state is also observed with a single bolus injection o f Photofrin administered intravenously, followed b y a 24-hour period o f multiple, consecutive blood collections (Figure 5). These results prompted us to repeat experiments involving P D T , and follow through on a number o f control studies to determine the possibility o f a masked neutrophilic state. Instead o f multiple blood collections per animal over a time course o f 24 hours after treatment, i n subsequent experiments, we chose to collect a single sample o f blood from a group o f mice at each time point before and following P D T light treatment.  74  In this manner, a  neutrophilic response due to repeated tail bleeding was avoided. In the absence o f P D T , mice were also subjected to stress factors such as, intravenous administration o f 5% dextrose in H2O (the solution i n which we reconstitute Photofrin prior to use), or the restraint i n which mice are immobilized during light treatment. These stress factors involved i n the methodology o f P D T i n mice, can individually induce an increase in circulating neutrophil levels (Figure 7). Initial experiments collecting multiple blood specimens from individual mice after P D T , were undertaken using mice bearing E M T 6 and S C C V I I tumors on their depilated backs. This created the possibility for light exposure to proximal organs, such as the liver or spleen. This may be a concern since photosensitizer is taken-up b y virtually all cells i n the body but especially b y phagocytic cells o f the reticuloendothelial system (35,133).  T w o scenarios  comparing the effect o f light, i n the absence o f photosensitizer, on (1) the s.c. E M T 6 back tumor and .(2) the tumor-free footpad o f mice, produced very different outcomes. P D T light only treatment on the tumor-free foot, as the rest o f the body was shielded from light during treatment, seemed to have a suppressive effect on circulating neutrophil levels, whereas oscillations i n neutrophil levels above normal occur when light was directed on a s.c. E M T 6 back tumor (Figure 8).  A complementary, or alternate explanation for these findings may  originate from the fact that the blood flow and vascular network through the foot differs from that on the skin o f the back. These factors are also likely responsible for the fact that the level o f increase i n circulating neutrophils following P D T seems to depend on the position o f the tumor during treatment. These considerations prompted us to compare the effect o f Photofrin-based P D T on the tumor-free footpad, E M T 6 foot tumor, and s.c. E M T 6 back tumor. In all three cases, P D T had an independent effect o f increasing circulating neutrophil levels significantly higher than any other stress-related factor experienced by the animal during treatment, although time  75  kinetics differ slightly in each case (Figure 9). The delayed peak i n neutrophil levels 10 hours following P D T on the E M T 6 tumor grown on the foot, compared to the initial rise and peak i n the two alternate groups at 1 hour, could be due to a combination o f reasons. A hallmark trait o f the P D T effect is edema formation (12,75). A t the time o f light treatment, i n the small area as is a mouse footpad a tumor already has limited capacity to swell. Therefore, the release o f signals from the P D T lesion into circulation may be hampered and delayed by severe vasoconstriction. P D T treatment o f the s.c. E M T 6 back tumor induced the most enhanced rise i n circulating neutrophil levels. Reasons for this may be an amplified response to PDT-elicited inflammatory signals released from both the treated tumor site, and also organs i n close proximity to the tumor affected during light treatment. From the data described here, it has been deduced that P D T results i n a massive sequestration o f G R l - p o s i t i v e cells, determined to be neutrophils by Wright stain analysis and due to their high expression o f the G R 1 surface marker compared to other myeloid cells (125), into circulation. In response to inflammatory signals released b y P D T , neutrophils are released into circulation presumably from their storage/marginated pools located at different sites i n the body (134,135,136).  The bone marrow for instance, has been shown to increase the rate o f  polymorphonuclear leukocyte ( P M N s ) production, shorten their maturation time, and release both mature and immature neutrophils into circulation i n response to inflammation and stress (128,137).  Such a response was shown to be induced b y bacterial challenge i n rabbit lungs  (138,139).  This phenomenon had up to date not been described for inflammation associated  with P D T . To decipher possible sources o f neutrophils released into circulation following P D T , the blood, bone marrow, tumor and lungs (representing tissue containing marginated pools o f neutrophils) from PDT-treated E M T 6 foot tumor bearing mice were examined (Figure 10).  76  F l o w cytometry analysis revealed a 50% drop in the neutrophil cell content o f bone marrow in response to PDT-elicited inflammation.  This release o f neutrophils from bone marrow  following P D T presumably occurs i n response to inflammatory chemotactic signals released into circulation from the treated site.  Neutrophils are released into circulation but seem to  undergo a bottleneck effect in the pulmonary vasculature. Perhaps blood pressure is lower, or the vessels i n the microvasculature o f lungs are smaller i n diameter than that encountered i n other parts o f the body, reducing the speed at which neutrophils can pass. The accumulation o f neutrophils i n tumors and blood at ten hours after treatment appears to occur due to a cumulative effect o f neutrophils released into circulation as they manage to travel through the lungs, and also from bone marrow i n a continuous wave o f release.  Although the level o f  neutrophils i n both blood and bone marrow returns to normal 24 hours after treatment, levels in tumors remain elevated. Perhaps this can be attributed to a second wave o f neutrophil release from the bone marrow between 10 and 24 hour intervals following treatment, suggested by a second rise i n neutrophil levels i n the lungs, observed 24 hours after treatment. Recent reports have suggested that P M N s i n bone marrow have different characteristics compared to their older counterparts i n circulation. Older neutrophils are less deformable, and have a decreased ability to respond to chemotactic stimuli. Younger neutrophils may have an enhanced ability to attach and extravasate in a site o f inflammation, whereas mature neutrophils have a greater ability to degranulate and produce cytotoxic, oxygenated products (140). These traits are not particularly useful when trying to separate the two populations.  However,  granulocytes from the bone marrow express higher concentrations o f L-selectin than do older, mature cells (141). This adhesion molecule that mediates rolling o f leukocytes along vascular endothelium, may aid the migration o f immature neutrophils into circulation (81).  B y flow  cytometry analysis, L-selectin expression can therefore be indicative o f the maturation stage o f a  77  neutrophil and also o f its activation state, since the expression o f L-selectin is downregulated once neutrophils reach a site o f inflammation (86).  In our E M T 6 tumor model, neutrophils  normally had low levels o f L-selectin with a small percentage expressing high levels. F o l l o w i n g P D T however, G R l - p o s i t i v e cells infiltrating treated tumors express high and l o w levels o f L selectin i n a 1:1 ratio. In accordance, the concentration o f L-selectin on circulating neutrophils, normally at a medium range, increases, as does that o f the granulocyte population harvested from bone marrow (Figures 15,17). The majority o f granulocytes i n the lungs expressed l o w concentrations o f L-selectin, with no significant changes following P D T (Figure 15). From these results one could postulate that PDT-elicited inflammation induced a systemic response o f neutrophils i n our murine tumor models.  The massive sequestration o f  neutrophils into PDT-treated tumors was accompanied by a marked increase o f these cells i n circulation. This PDT-induced neutrophilia may be attributed to the release o f both mature and immature granulocytes from bone marrow i n response to inflammatory signals released from the treated lesion.  Compiled, our findings seem to suggest that the presumed importance o f  neutrophils for effective P D T as an anti-tumor modality, relies on the synergistic actions o f older and less mature neutrophils. Neutrophils invading PDT-treated tumors inevitably die (mostly b y apoptosis) at the site within a short time interval after their arrival (Korbelik, unpublished results). These dying cells appear to be replaced constantly b y new waves o f neutrophils sequestered from circulation, a process which seems to last throughout the first day following P D T treatment.  It seems  therefore, that huge numbers o f neutrophils are required during this period, a process secured b y their mobilization from storage pools and accelerated generation o f young neutrophils from myeloid precursors.  78  FUTURE DIRECTIONS  W e have only begun to uncover the knowledge on the activity o f neutrophils that contributes to the destruction o f PDT-treated solid cancers. The extent o f its relevance for clinical P D T practice remains to be elucidated by further research. Several avenues o f investigation have opened-up based on the findings o f this thesis. One o f the main questions is whether or not the variable sensitivity o f tumors to P D T stems from the engagement o f neutrophils in treated lesions. In this respect it would be important to identify biological characteristics o f tumors critical in determining neutrophil activity following P D T treatment. Factors such as tumor perfusion/vascularization, stromal structure including types o f tumor associated host immune cells, profiles o f locally released cytokines, nitric oxide and other mediators, seem to be obvious targets i n such an investigation. The evidence o f superoxide generation, and o f decreased then subsequent increased tumor blood flow, following P D T , strongly suggests the manifestation o f ischemiareperfusion insult in this treatment.  The dominant role o f neutrophils in the infliction o f  damage mediated by this form o f insult, has to be taken into account and investigated. Can the curative effect o f P D T be improved by modulating the sequestration and activity o f neutrophils i n treated tumors?  Ongoing studies i n our laboratory suggest that a  positive answer to this question appears highly likely.  For instance, agents modulating the  engagement o f integrins or selectins, or the levels o f nitric oxide can enhance the curative effect o f P D T . Further research for supporting this approach should be very productive. Further studies to supplement the results collected i n this project could be aimed at verifying that inflammatory signals specific for neutrophils are indeed being released into circulation from a PDT-treated site. Collecting the serum o f treated tumor-bearing mice at  79  various time intervals following PDT, and subsequently introducing it intravenously to naive recipient mice, may confirm the presence of chemotactic signals in circulation, by the induction of neutrophilia in those recipient animals. If this does turn out to be the case, using molecular biology techniques, the next step would be to identify these inflammatory signals. Although IL-1 and TNF-oc are primary candidates in the specific mediation of neutrophilia, other well-established neutrophil attractants such as complement factor 5 a, and the cytokine interleukin-8 (IL-8), or even novel previously undefined molecules cannot be ruled out. The accumulation of activated neutrophils in PDT-treated tumors is well known, however, their localization is not well documented.  It would be revealing to find out the  extent of extravasation of these sequestered neutrophils from the vasculature in the PDTtreated lesion, and whether this pattern varies between tumors of different sensitivity to PDT. This may clarify which cell type(s) in a tumor are being affected by the cytotoxic capabilities of neutrophils.  Such details may possibly be elucidated using fluorescence microscopy  methods to study PDT-treated tumor sections. One issue to be taken into consideration when interpreting the results of this thesis is the relative size and location of treated murine tumors.  Obviously, the tumor size (and  consequently the PDT light-exposed area) related to the body size in the mouse is much larger than that encountered in the human situation. A marked rise in circulating neutrophil levels may not be seen in humans for the treatment of smaller lesions, but may be observed when rather large areas of skin, in psoriasis for example, are treated. We hope to be able to perform a preliminary investigation of patient's blood samples in the near future.  80  REFERENCES  (1) Pathak M A , Kramer D M , Fitzpatrick T B . (1974) Photobiology and photochemistry o f furocoumarins (psoralens) In: Fitzpatrick T B , Pathak M A , Harber L C , Seiji M , Kukita A , editors. Sunlight and man. Tokyo: University o f Tokyo Press, 335-368. (2) Daniel M D , H i l l JS. (1991) A history o f photodvnamic therapy. Aust N Z J Surg, 61, 340-348. (3) Jesionek A , con Tappenier H . (1903) Zur behandlung der Hautkarcinome fluorescierenden stoffen. Munchen M e d Wochenscher, 47, 2042-2051.  mit  (4) Hausman W . (1911) D i e sensiboliserende Wirkung das Hematoporphyrins. Biochem Z 30, 276-316. (5) Auler H , Banzer G . (1942) Untersuchungen uberdie Rolle geschwulstkranken Menschen und Tieren. Z krebsforsch, 53, 65-68.  der  Porphyrine  bei  (6) Gregorie H G Jr, Horger E O , Ward J L . (1968) Hematoporphyrin-derivative fluorescence i n malignant neoplasms. A n n Surg 167, 820-828. (7) Dougherty T J . (1974) Activated dyes as antitumor agents. J Natl Cancer Inst, 51, 1333-1336. (8) Dougherty T J , Grindey G E , Fiel R. (1975) Photoradiation therapy. II. Cure o f animal tumors with hematoporphyrin and light. J Natl Cancer Inst, 55, 115-121. (9) Dougherty T J , Kaufman J E , Goldfarb A . (1978) Photoradiationtherapv for the treatment o f malignant tumors. Cancer Res., 38, 2628-2635. (10) Dougherty T J . (1984) Photoradiation therapy. U r o l Suppl 23, 61. (11) Dougherty T J . (1987) Photosensitizers: Therapy and detection o f malignant Photochem Photobiol, 45, 879-889.  tumors.  (12) Dougherty T J , Gomer, C J , Henderson B W , Jori Giulio, Kessel D , Korbelik M , M o a n J, Peng Q. (1998) Photodvnamic Therapy. J Natl Cancer Inst, 90 (12), 889-905. (13) Korbelik M , Cecic I, Shibuya H . (1997) The role o f nitric oxide i n the response o f cancerous lesions to photodvnamic therapy. Recent Res Devel i n Photochem & Photobiol, 1, 267-276. (14) Pass H I . (1993) Photodvnamic therapy i n oncology: Mechanisms and clinical use. J Natl Cancer Inst, 85, 443-456. (15) Fisher A M R , Murphree A L , Gomer C J . (1995) Clinical and preclinical photodvnamic therapy. Lasers Surg M e d , 17, 2. (16) Dougherty T J . (1986) Photosensitization o f malignant tumors. Semin Surg Oncol, 2, 24-27. 81  (17) Santoro O, Bandieramonte G , M e l l o n i E . (1990) Photodynamic therapy b y topical mesotetraphenylporphinesulfonate tetrasodium salt administration i n superficial basal cell carcinomas. Cancer Res, 50, 4501-4503. (18) Waldow S M , Lobraico R V , Kohler I K . (1987) Photodynamic therapy for treatment o f malignant cutaneous lesions. Lasers Surg M e d , 7, 451-456. (19) Gluckman J L , Waner M , Shumrick K . (1986) Photodynamic therapy. A viable alternative to conventional therapy for early lesions o f the upper aerodigestive tract? Reflections on a 5-vear experience. Laryngoscope, 191, 36-42. (20) Shikowitz M J , Steinberg B M , Abramson A L . (1986) Hematoporphyrin derivative therapy o f papillomas: Experimental study. A r c h Otolaryngol Head Neck Surg, 118, 25-29. (21) Lightdale C J , Heier S K , Marcon N E . (1995) Photodynamic therapy with porfimer sodium versus thermal ablation therapy with N d : Y A G laser for palliation o f esophageal cancer: a multicenter randomized trial. Gastrointest Endosc, 42, 507-512. (22) Pass H I , Pogrebniak H W . (1992) Photodynamic therapy for thoracic malignancies. Semin Surg Oncol, 8, 217-225. (23) L a m S, Palcic B , M c L e a n D . (1990) Detection o f early lung cancer using low dose Photofrin II. Chest, 97, 333-337. (24) L a m S, Crofton C , Cory P. (1991) Combined photodynamic therapy ( P D T ) using photofrin and radiotherapy ( X R T ) versus radiotherapy alone in patients with inoperable distribution nonsmall cell bronchogenic cancer. Proc SPIE Proc , 20-28. (25) Hayata Y , Kato H , Okitsu T. (1985) Photodynamic therapy with hematoporphyrin derivative i n cancer o f the upper gastrointestinal tract. Semin Surg Oncol, 1, 1-11. (26) Barr H , Krasner N , Boulos P B . (1990) Photodynamic therapy for colorectal cancer: A quantitative pilot study. B r J Surg, 77, 93-96. (27) Rettenmaier M A , Berman M L , Disaia P J . (1984) Gynecologic uses o f photoradiation therapy. In: Porphyrin Localization and Treatment o f Tumors. Doiron D T , Gomer C J , editors. A l a n R. Liss, N e w York. (28) Sindelar W F , DeLaney T F , Tochner Z . (1991) Technique o f photodynamic therapy for disseminated intraperitoneal malignancies: Phase I study. A r c h Surg, 126, 318-324. (29) Hisazumi H , M i s a k i T, M i y o s h i N . (1983) Photoradiation therapy o f bladder tumors. J U r o l , 130, 685-687. (30) Benson R C Jr, Farrow G M , Kinsey J H . (1982) Detection and localization o f in situ carcinoma o f the bladder with hematoporphyrin derivative. M a y o C l i n Proc, 57, 548-555.  82  (31) Razum N , Snyder A , Doiron D . (1996) SnET2: Clinical update. Proc SPIE, 2675, 43-46. (32) Leung J . (1994) Photosensitizers i n photodvnamic therapy. Semin Oncol, 21,4-10. (33) Gragoudas E , Schmidt-Erfurth U , Sickenkey M . (1997) Results and preliminary dosimetry o f photodvnamic therapy for choroidal neovascularization i n age-related molecular degeneration in a phase I/II study. Assoc Res vision Opthamology, 38, 73 (Abstract). (34) Grosjean P, Savary J, Wagnieres G . (1993) Tetra(m-hydroxyphenyl) Chlorin Clinical photodvnamic therapy o f early bronchial and oseophageal cancers. Laser M e d S c i , 8, 235-243. (35) Henderson B W , Dougherty T J . (1992) H o w does photodvnamic therapy work? Photochem Photobiol, 55, 145-157. (36) Hasan T, Parrish J A . (1997) Photodvnamic therapy o f cancer. Cancer Medicine, V o l . 1, Holland JF, Bast R C Jr, Morton D L , Frei E III, Kufe D W , Weichselbaum R R , editors, W i l l i a m s & W i l k i n s , Baltimore, 739-751. (37) Marcus S L . (1992) Clinical photodvnamic therapy: The continuing evolution. Photodvnamic therapy. Henderson B W , Dougherty T J , editors, Marcel Dekker Inc, N e w Y o r k , 219-268. (38) R i s H B , Altermatt H J , Nachkur B , Stewart C M , Wang Q, L i m C K , Bonnett R , Altkaus U . (1993) Effect of drug-light interval on photodvnamic therapy with metatetrahydroxyphenylchlorin in malignant mesothelioma. Int J Cancer, 53, 141-146/ (39) Svaasand L O . (1985) Photodvnamic therapy and photothermic response o f malignant tumors. M e d P h y s , 12,455-461. (40) Henderson B W , W a l d o w S M , Potter W R , Dougherty T J . (1985) Interaction o f photodvnamic therapy and hyperthermia: tumor response and cell survival studies after treatment o f mice in vivo. Cancer Res, 45, 6071-6077. (41) M o a n J. (1986) Porphyrin photosensitization and phototherapy. Photochem Photobiol. 43, 681-690. (42) Dougherty T J . (1996) A brief history o f clinical photodvnamic therapy development at Roswell Park Cancer Institute. J C l i n Laser M e d , 14, 219-221. (43) Henderson B W , Fingar V H . (1994) The role o f vascular photodamage in photodvnamic therapy. Photochem & Photobiol, 59 (suppl.) 1S-2S. (44) Korbelik M , K r o s l G . (1994) Cellular levels o f photosensitizers i n tumors: the role o f proximity to the blood supply. B r J Cancer, 70, 604-610. (45) Soncin M , Polo L , Reddi E , Jori G , Kenney M E , Cheng G , Rodgers M A J . (1995) Effect o f axial ligation and delivery system on the tumor-localizing and photosensitizing properties o f Ge(IV)-octabutoxv-phthalocyanines. B r J Cancer, 71, 727-732. 83  (46) A l l i s o n B A , Haydn-Pritchard P, Richter A M , L e v y J G . (1990) The plasma distribution o f benzoporphyrin derivative and the effects o f plasma lipoproteins on its biodistribution. Photochem Photobiol, 52, 501-507. (47) Korbelik M , K r o s l G . (1996) Photofrin accumulation i n malignant and host cell populations o f various tumors. B r J Cancer, 73, 506-513. (48) Korbelik M , K r o s l G , Chaplin D J (1991) Photofrin uptake by murine macrophages. Cancer Res, 51,2251-2255. (49) Korbelik M , K r o s l G . Olive P L , Chaplin D J . (1991) Distribution o f photofrin between tumor cells and tumor associated macrophages. B r J Cancer, 64, 508-512. (50) M i l a s L , W i k e J, Hinter N , Volpe J, Basic I. (1987) Macrophage content o f murine sarcomas and carcinomas: association with tumor growth parameters and tumor radiocurabilitv. Cancer Res, 47, 1069-1075. (51) Davies C L , Ranheim T, Rofstad E K , M o a n J, Lindmo T. (1988) Relationship between changes i n antigen expression and protein sunthesis i n human melanoma cells after hyperthermia and photodynamic treatment. B r J Cancer, 58, 306-313. (52) L i n G S , A l - D a k a n A A , Gibson D P . (1986) Inhibition o f D N A and protein synthesis and cell division by photoactivated haematoporphyrin derivative i n hamster ovary cells. B r J Cancer, 53, 265-269. (53) Christensen T, M o a n J , Smedshammer L . (1985) Influence o f hematoporphyrin derivative ( H P D ) and light on the attachment o f cells to the substratum. Photochem Photobiophys, 10, 53. (54) Kessel D . (1986) Sites o f photosensitization by derivatives o f hematoporphyrin. Photochem Photobiol, 44, 489-493. (55) M o a n J, Pettersen E O , Christensen T. (1979) The mechanism o f photodynamic inactivtion o f human cells in vitro i n the presence o f hematoporphyrin. B r J Cancer, 39, 398-407. (56) V o l d e n G , Christensen T , M o a n J. (1981) Photodynamic membrane damage o f hematoporphyrin derivative-treated N H I K 3025 cells in vitro. Photobiochem Photobiophys, 3, 105-111. (57) M o a n J, M c G h i e J, Jacobsen P B . (1983) Photodynamic effects on cells in vitro exposed to hematoporphyrin derivative and light. Photochem Photobiol, 37, 599-604. (58) Specht K G , Rodgers M A J . (1990) Depolarization o f mouse myeloma cell membrane during photodynamic action. Photochem Photobiol, 51, 319-324. (59) M o a n J, Christensen T. (1981) Cellular uptake hematoporphyrin. Photochem Photobiophys, 2, 291-299.  84  and  photodynamic  effect  of  (60) Henderson B W , Donovan J M . (1989) Release o f prostaglandin photodvnamic treatment in vitro. Cancer Res, 49, 6896-6900.  E? from cells b y  (61) Ochsner M . (1997) Photophysical and photobiological processes i n the photodvnamic therapy o f tumors. Photochem Photobiol B : B i o l , 39, 1-18. (62) Kessel D , Woodburn K . (1995) Selective photodvnamic inactivation o f a multidrug transporter b y a cationic photosensitising agent. B r J Cancer, 71, 306-310. (63) Joshi P G , Joshi K J , Mishra S, Joshi N K . (1994) C a influx induced by photodvnamic action i n human cerebral plioma ( U - 8 7 M G ) cells: Possible involvement o f a calcium channel. Photochem Photobiol, 60, 244-248. 2 +  (64) Davies C L , Western A , Lindmo T, M o a n J. (1986) Changes i n antigen expression on human F M E melanoma cells after exposure to photoactivated hematoporphyrin derivatives. Cancer res, 46, 6068-6072. (65) Thomas JP, Girotti A W . (1989) Role o f lipid peroxidation i n hematoporphyrin derivativesensitized photokilling o f tumor cells: protective effects o f glutathione peroxidase. Cancer Res, 49, 1682-1686. (66) Berg K , M o a n J. (1994) Lysosomes as photochemical targets. Int J Cancer, 59, 814-822. (67) H i l f R, Smail D B , Murant R S . (1984) Hematoporphyrin derivative-induced photosensitivity of mitochondrial succinate dehydrogenase and selected cytosolic enzymes o f R 3 2 3 0 A C mammary adenocarcinomas o f rats. Cancer Res, 44, 1483-1488. (68) H i l f R , Murant R S , Narayanan U . (1986) Relationship o f mitochondrial function and cellular adenosine triphosphate levels to hematoporphyrin derivative-induced photosensitization in R 3 2 3 0 A C mammary tumors. Cancer Res 46, 211-217. (69) Oleinick N . (1998) Apoptosis i n response to photodvnamic therapy. Photodvnamic News, Vol l,Num2. (70) Luo Y , Kessel D . (1997) Initiation o f apoptosis versus necrosis by photodvnamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol, 66, 479-483. (71) Kessel D , Luo Y , Deng Y , Chang D K . (1997) The role o f subcellular localization i n initiation o f apoptosis by photodynamic therapy. Photochem Photobiol, 65, 422-426. (72) Kessel D , Luo Y . (1998) Mitochondrial photodamage Photochem Photobiol B : B i o l , 42, 89-95.  and PDT-induced apoptosis. J  (73) H i c k m a n J A , Potten C S , Merritt A J , Fisher T C . (1994) Apoptosis and cancer chemotherapy. Phil Trans R Soc Lond B , 345, 319-325. (74) Korbelik, M . (1996) Induction o f tumor immunity by photodvnamic therapy. J C l i n Laser M e d Surg, 14, 329-334. 85  (75) Fingar V H . (1996) Vascular effects o f photodynamic therapy. J C l i n Laser M e d Surg, 14, 323-328. (76) Korbelik M , Cecic I. (1998) Enhancement o f tumor response to photodynamic therapy b y adjuvant mycobacterium cell-wall treatment. J Photochem Photobiol, B : B i o l , 44, 151-158. (77) K r o s l G , Korbelik M , Dougherty G J . (1995) Induction o f immune cell infiltration into murine S C C V I I tumor by photofrin-based photodynamic therapy. B r J Cancer, 71, 549-555. (78) Cecic I, Korbelik M . (1999) Neutrophil-associated events i n the inflammation o f cancerous tissue following treatment with photodynamic therapy. Trends Photochem Photobiol, i n press. (79) Korbelik M , Krosl G , Krosl J, Dougherty G J . (1996) Potentiation o f ph'otodvanmic therapy elicited anti-tumor response b y localized treatment with granulocyte-macrophage colony stimulating factor. Cancer Res, 56, 3281-3286. (80) Korbelik M , Dougherty G J . (1999) Photodynamic therapy mediated immune response against subcutaneous mouse tumors. Cancer Res, 59, i n press. (81) Janeway C A , Travers, P. (1996) Immunobiology. Current B i o l o g y Ltd./Garland Publishing Inc, London. (82) Tonnesen M G , Anderson D C , Springer T A , Knedler A , A v d i N , Henson P M . (1989) Adherence o f neutrophils to cultured human microvascular endothelial cells. J C l i n Invest, 83, 637-645. (83) Warren JS, W a r d P A , Johnson K J . (1990) Hematology, M c G r a w - H i l l , N e w Work. (84) Sekiya S, Gotoh S, Yamashita T, Watanabe T, Saito S, Sendo F. (1989) Selective depletion o f rat neutrophils by in vivo administration o f a monoclonal antibody. Leukocyte B i o l , 46, 96102. (85) Haslett C . (1997) Granulocyte apoptosis and inflammatory disease. B r M e d B u l l , 53, 669683. (86) Kishimoto T, Jutila M A , Berg E L , Butcher E . (1989) Neutrophil Mac-1 and M E L - 1 4 adhesion proteins inversely regulated b y chemotactic factors. Science, 245, 1238-1241. (87) Smith W C . (1993) Endothelial adhesion molecules and their role i n inflammation. C a n J Physiol Pharmacol, 71, 76-87. (88) L i u Q, Kishimoto T K , Mainolfi E , Deleon R P , Myers C , Moretz R C . (1998) Dynamic expression o f L-selectin in cell-to-cell interactions between neutrophils and endothelial cells in vitro. Exptl C e l l Res, 243, 87-93. (89) Malech H L , G a l l i n JI (1988) Current concepts: immunology. Neutrophils i n human disease. N Engl J M e d , 317, 687-694. 86  (90) Henson P M , Johnston R B Jr. (1987) Tissue injury i n inflammation. Oxidants, proteinases, and cationic proteins. J C l i n Invest, 79, 669-674. (91) Weiss SJ. (1989) Tissue destruction by neutrophils. N Engl J M e d , 320, 365-376. (92) Test ST, Weiss SJ. (1986) The generation o f utilization o f chlorinated oxidants by human neutrophils. A d v Free Radical B i o l M e d , 2, 91-116. (93) Klebanoff SJ. (1988) Inflammation: Basic principles and clinical correlations. G a l l i n JI, Goldstein I M , editors, Raven Press, N e w York. (94) Savill J, Haslett C . (1995) Granulocyte clearance by apoptosis i n the resolution o f inflammation. C e l l B i o l , 6, 385-393. (95) M o u l d i n g D A , Quayle J A , Hart C A , Edwards S W . (1998) M c l - 1 expression i n human neutrophils: regulation by cytokines and correlation with cell survival. Blood, 92, 2495-2502. (96) Savill JS, W y l l i e A H , Henson J E , Walport M J , Henson P M , Haslett C . (1989) Macrophage phagocytosis o f aging neutrophils i n inflammation. J C l i n Invest, 83, 865-875. (97) Bellingan G J , Caldwell H , H o w i e S E M , Dransfield I, Haslett C . (1996) In vivo fate o f inflammatory macrophage during the resolution o f inflammation. J Immun, 157, 2577-2585. (98) H e v i n M , Friguet B , Fauve R M . (1990) Inflammation and antitumor resistance.V. Production o f a cytostatic factor following cooperation o f elicited polymorphonuclear leukocytes and macrophages. Int J Cancer, 46, 533-538. (99) Renard N , Lienard D , Lespagnard L , Eggermont A , Hermann R, Lejeune F. (1994) Early endothelium activation and polymorphonuclear cell invasion precede specific necrosis o f human melanoma and sarcoma treated b y intravascular high-dose tumor necrosis factor alpha ( T N F alpha). Int J Cancer, 57, 656-663. (100) Midorikawa Y , Yamashita T, Sendo F . (1990) Modulation o f the immune response to transplanted tumors in rats b y selective depletion o f neutrophils in vivo using a monoclonal antibody: abrogation o f specific transplantation resistance to chemical carcinogen-induced syngeneic tumors b y selective depletion o f neutrophils in vivo. Cancer Res, 50, 6243-6247. (101) Lichtenstein A , Kahle J. (1985) Anti-tumor effect o f inflammatory neutrophils: characteristics o f in vivo generation and in vitro tumor cell lysis. Int J Cancer, 35, 121-127. (102) Vadas M A , N i c o l a N A , Metcalf D . (1983) Activation o f antibody-dependent cell mediated cytotoxicity o f human neutrophils and eosinophils by separate colony-stimulating factors. J Immunol, 130, 795-799. (103) MacPherson G G , North R J . (1986) Endotoxin-mediated necrosis and regression o f established tumors in the mouse. A correlative study o f quantitative changes i n blood flow and ultrastructural morphology. Cancer Immun Immunother, 21, 209-216. 87  (104) Colombo M P , Lombardi L , Stoppacciaro A , M i l a n i C , Parenza M , Bottazi B , Parmiani G . (1992) Granulocyte colony-stimulating factor ( G - C S F ) gene transduction i n murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo. J Immunol, 149, 113-119. (105) Katano M , Torisu M . (1982) Neutrophil-mediated tumor cell destruction i n cancer ascites. Cancer, 50, 62-68. (106) Lindermann A , Riedel D , Oater W , Meuer S C , Blohm D , Merstelsmann R H , Herrmann F. (1988) Granulocyte/macrophage colony-stimulating factor induces interleukin I production b y human polymorphonuclear neutrophils. J Immunol, 140, 837-839. (107) Pekarek L A , Starr B A , Toledano A Y , Schreiber H . (1995) Inhibition o f tumor growth b y elimination o f granulocytes. J E x p M e d , 181, 435-440. (108) Hernandez L A , Grisham M B , T w o h i g B , Arfors K E , Harlan J M , Granger D N . (1987) R o l e o f neutrophils i n ischemia-Zreperfusion-induced microvascular injury. A m J Physiol, 253, H699703. (109) Y o s h i k a w a T, Kokura S, Oyamada H , Iinuma S, Nishimura S, Kaneko T, Naito Y , K o n d o M . (1994) Anti-tumor effect o f ischemia/reperfusion injury induced b y transient embolization. Cancer Res, 54, 5033-5055. (110) Y o s h i k a w a T, Kokura S, Tainaka K , Naito Y , Kondo M . (1995) A novel cancer therapy based on oxygen radicals. Cancer Res, 55, 1617-1620. (111) Parkins C S , Dennis M F , Stratford M R L , H i l l S A , Chaplin D J . (1995) Ischemia reperfusion injury i n tumors: the role o f oxygen radicals and nitric oxide. Cancer Res, 55, 6026-6029. (112) Fingar V H , Wieman T J , Wiehle S A , Cerrito P B . (1992) The role o f microvascular damage in photodynamic therapy: the effect o f treatment on vessel constriction, permeability, and leukocyte adhesion. Cancer Res, 52,4914-4921. (113) Bellnier D A , Henderson B W . (1992) Photodynamic therapy: Basic principles and clinical applications. Henderson B W , Dougherty T J , editors. Marcel Dekker, N e w Y o r k . (114) Gollnick S O , L i u X , Owczarczak B , Musser D A , Henderson B W . (1997) Altered expression o f Interleukin 6 and Interleukin 10 as a result o f photodynamic therapy in vivo. Cancer Res, 57, 3904-3909. (115) van Geel IPJ, Oppelar H , Rijken P F J W , Bernsen H J J A , Hagemeier N E M , van der K o g e l A J , Hodgkiss R J , Stewart F A . (1996) Vascular perfusion and hypoxic areas i n RIF-1 tumors after photodynamic therapy. B r J Cancer, 73, 288-293. (116) Sluiter W , deVree W J A , Fontijne-Dorsman A N R D , Koster JF. (1996) Photodynamic treatment o f human endothelial cells promotes the adherence o f neutrophils in vitro. B r J Cancer, 73, 1335-1340.  88  (117) M a X , Weyrich A S , Lefer D J , Lefer A M . (1993) Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. C i r c Res, 72, 403-412. (118) de Vree W J A , Essers M C , de Brujin H S , Starr W M , Koster JF, Sluiter W . (1996) Evidence for an important role o f neutrophils i n the efficacy o f photodynamic therapy in vivo. Cancer Res, 56, 2908-2911. (119) de Vree W J A , Essers M C , Koster JF, Sluiter W . (1997) Role o f interleukin 1 and granulocyte colony-stimulating factor i n photofrin-based photodynamic therapy o f rat rhabdomyosarcoma tumors. Cancer Res, 57, 2555-2558. (120) Olive P L , Chaplin D J , Durand R E . (1985) Pharmakokinetics. binding and distribution o f Hoechst 33342 i n spheroids and murine tumors. B r J Cancer, 52, 739-746. (121) R o c k w e l l S. (1986) Absence o f contact effects i n irradiated E M T 6 - R w tumors. R a d Res, 107, 375-381. (122) Korbelik M . (1993) Distribution o f disulfonated and tetrasulfonated aluminum phthalocyanine between malignant and host cell populations o f a murine fibrosarcoma. Photochem Photobiol, 20, 173-181. (123) M c B r i d e W H , Thacker J D , Comora S, Economou J, K e l l e y D , Dubinett S M , Hogge D , Dougherty G J . (1992) Genetic modification o f a murine fibrosarcoma to produce IL7 stimulates host cell infiltration and tumor immunity. Cancer Res, 52, 3931-3937. (124) Korbelik M , Cecic I. (1999) Contribution o f myeloid and lymphoid cells to the curative outcome o f mouse sarcoma treatment b y photodynamic therapy. Cancer Letters, 137,91-98. (125) Lagasse E , Weissman I L . (1996) F l o w cytometric identification o f murine neutrophils and monocytes. J Immun Methods, 197, 139-150. (126) Kubes P, Suzuki M , Granger D N . (1991) Nitric oxide: A n endogenous modulator o f leukocyte adhesion. Proc Natl A c a d Sci U S A , 88, 4651-4655. (127) Gaboury J, Woodman R C , Granger D N , Reinhardt P, Kubes P. (1993) Nitric oxide prevents leukocyte adherence: role o f superoxide. A m J Physiol, 265, H862-H867. (128) Kubo H , Graham L , D o y l e N A , Quinlan W M , H o g g J C , Doerschuk C M . (1998) Complement fragment-induced release o f neutrophils from bone marrow and sequestration within pulmonary capillaries i n rabbits. Blood, 92, 283-290. (129) Rubin E , Farber J L , editors. (1999) Pathology. Lippincott-Raven, N e w Y o r k . (130) Anderson C , Hrabovsky S, M c K i n l e y S, Tubesing Y , Tang K , Dunbar R, Mukhtar H , Elmets C A . (1997) Phthalocyanine photodynamic therapy: disparate effects o f pharmacologic inhibitors on cutaneous photosensitivity and on tumor regression. Photochem Photobiol, 65, 895901. 89  (131) L a m m D L , van der Margolese R G , Weiss D W . (1992) Incidence and treatment o f complications o f Bacillus Calmette-Guerin intravesical therapy i n superficial bladder cancer. J U r o l , 147, 596-600. (132) Staub N C , Schultz E L , Albertine K H . (1982) Leucocytes and pulmonary microvascular injury. Annals N e w Y o r k Academy o f Sciences. (133) Hamblin M R , Newman E L . (1004) O n the mechanism o f the tumor-localising effect i n photodvnamic therapy. J Photochem Photobiol B : B i o l , 23, 3-8. (134) L i e n D C , Wagner W W Jr, Capen R L , Haslett C , Hanson W L , Hofmeister S E , Henson P M , Worthern G S . (1987) Physiological neutrophil sequestration in the lung: visual evidence for localization i n capillaries. J A p p l Physiol, 62, 1236-1243. (135) Downey G P , Worthern G S , Henson P M , Hyde D M . (1993) Neutrophil sequestration and migration i n localized pulmonary inflammation. A m Rev Respir Dis, 147, 168-176. (136) Doerschuk C M , A l l a r d M F , Martin B A , MacKenzie A , Autor A P , H o g g J C . (1987) Marginated pool o f neutrophils in rabbit lungs. J A p p l Physiol, 63, 1806-1815. (137) Bainton D F . (1988) Phagocytic cells: developmental biology o f neutrophils and eosinophils. In: Inflammation: Basic principles and clinical correlations. G a l l i n JI, Goldstein I M , editors. Raven, N e w Y o r k . (138) Terashima T, Wiggs B , English D , H o g g J C , V a n Eeden SF. (1996) Polymorphonuclear leukocyte transit times in bone marrow during streptococcal pneumonia. A m J Physiol, 271, L587-L592. (139) Sato Y , V a n Eeden SF, English D , H o g g J C . (1998) Bacteremic pneumococcal pneumonia: Bone marrow release and pulmonary sequestration o f neutrophils. Crit Care M e d , 26, 501-509. (140) Tanji-Matsuba K , V a n Eeden S F , Saito Y , Okazawa M , K l u t M E , Hayashi S, H o g g J C . (1997) Functional changes i n aging polymorphonuclear leukocytes. Circulation, 97, 91-98. (141) V a n Eeden S, Miyagashima R, Haley L , Hogg H C . (1995) L-selectin expression increases on peripheral blood polymorphonuclear leukocytes during acute marrow release. A m J Repir Crit Care M e d , 151,500-507.  90  

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