"Medicine, Faculty of"@en . "Pathology and Laboratory Medicine, Department of"@en . "DSpace"@en . "UBCV"@en . "Saulnier, Ronald Betnoit"@en . "2010-12-07T22:19:10Z"@en . "1991"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Little is known about the mechanisms which cause tumor cells to become invasive. For this thesis an in vitro tumor cell invasion assay was developed and used to investigate the role of a family of cell surface receptors, called integrins, in the invasion of tumor cells across basement membranes. It was also used to isolate an invasive cell line in order to study some of its properties.\r\nTwo osteosarcoma cell lines, HOS and MNNG-HOS, with known in vivo metastatic potentials were assayed in the in vitro invasion assay. Invariably, the highly tumorigenic and metastatic MNNG-HOS cells demonstrated greater invasive ability than the non-tumorigenic HOS cells. The chemical transformation of HOS into tumorigenic MNNG-HOS cells resulted in an increase in the expression of \u00CE\u00B1\u00E2\u0082\u0081\u00CE\u00B2\u00E2\u0082\u0081, \u00CE\u00B1\u00E2\u0082\u0082\u00CE\u00B2\u00E2\u0082\u0081 and \u00CE\u00B1\u00E2\u0082\u0086\u00CE\u00B2\u00E2\u0082\u0081 integrins which are laminin and collagen receptors. The expression of \u00CE\u00B1\u00E2\u0082\u0083\u00CE\u00B2\u00E2\u0082\u0081 and \u00CE\u00B1\u00E2\u0082\u0085\u00CE\u00B2\u00E2\u0082\u0081, were unchanged on MNNG-HOS cells and the expression of \u00CE\u00B1v\u00CE\u00B2\u00E2\u0082\u0083 was strongly downregulated on the more invasive cells. The invasion of HOS and MNNG-HOS cells through matrigel could be significantly inhibited when anti-fibronectin receptor or anti-\u00CE\u00B1\u00E2\u0082\u0086 subunit antibodies were present in the invasion assay, demonstrating the important role of integrins in tumor cell invasion.\r\nThe in vitro invasion assay described in this thesis was used to isolate a more invasive cell line from the prostate carcinoma cell line, PC-3, and called IPC-3. The morphology of these cells was distinct from the parent population, showing a spherical morphology as opposed to the triangular or spindle shaped morphology of PC-3 cells. These cells were also several times more invasive than the PC-3 cells and proliferated at a faster rate than the parent PC-3 cells. IPC-3 cells gradually lost their invasive potential after several months in tissue culture but retained their morphology and the characteristic expression of integrins. Adhesion of PC-3 and IPC-3 cells to purified extracellular matrix components revealed that IPC-3 attached well to laminin and to vitronectin. In adhesion kinetic experiments to purified extracellular matrix proteins, IPC-3 cells attached more quickly than PC-3 cells to larninin and vitronectin. Although the IPC-3 cells attached to the extracellular matrix proteins, fibronectin, vitronectin, laminin and collagen type IV, they were only able to spread on laminin and required several hours to do so. PC-3 cells also attached well to the extracellular matrix proteins but required only several minutes to spread on the matrix proteins including laminin. When plated on stock matrigel PC-3 cells organized themselves in tube-like structures while IPC-3 cells aggregated in clusters.\r\nAnalysis of the integrins on PC-3 and IPC-3 cells demonstrated that IPC-3 cells downregulated the expression of the \u00CE\u00B1\u00E2\u0082\u0081\u00CE\u00B2\u00E2\u0082\u0081,\u00CE\u00B1\u00E2\u0082\u0082\u00CE\u00B2\u00E2\u0082\u0081 and an almost completely downregulated the \u00CE\u00B1\u00E2\u0082\u0083\u00CE\u00B2\u00E2\u0082\u0081 integrin while the expression of the fibronectin receptor, \u00CE\u00B1\u00E2\u0082\u0085\u00CE\u00B2\u00E2\u0082\u0081,, and the vitronectin receptor, \u00CE\u00B1\u00E2\u0085\u00B4\u00CE\u00B2\u00E2\u0082\u0081, were unchanged. The expression of \u00CE\u00B1\u00E2\u0082\u0086\u00CE\u00B2\u00E2\u0082\u0081, in both PC-3 and IPC-3 cells was not prominent. However the \u00CE\u00B1\u00E2\u0082\u0086\u00CE\u00B2\u00E2\u0082\u0084 receptor was present in large amounts and was upregulated in IPC-3 cells, particularly the 200 kDa subunit of \u00CE\u00B2\u00E2\u0082\u0084.\r\nImmunofluorescence staining of PC-3 and IPC-3 cells demonstrated that PC-3 cells distributed their \u00CE\u00B1\u00E2\u0082\u0083\u00CE\u00B2\u00E2\u0082\u0081 and \u00CE\u00B1\u00E2\u0082\u0086\u00CE\u00B2\u00E2\u0082\u0084 integrin receptors mainly along the cell periphery and their \u00CE\u00B1\u00E2\u0085\u00B4\u00CE\u00B2\u00E2\u0082\u0083 receptor in focal adhesion plaques, while the invasive IPC-3 cells concentrated their integrin receptors in circular adhesion structures.\r\nAlthough much remains to be learned about integrins, they have an instrumental role in the invasion of tumor cells across basement membranes during the metastatic cascade of malignant cells."@en . "https://circle.library.ubc.ca/rest/handle/2429/30317?expand=metadata"@en . "ANALYSIS OF INTEGRINS AND CELL ADHESION ON INVASIVE TUMOR CELL LINES USING AN IN VITRO INVASION ASSAY by Ronald Benoit Saulnier B.Sc, Acadia University, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1991 \u00C2\u00A9 Ronald Benoit Saulnier, 1991 V In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT ii Little is known about the mechanisms which cause tumor cells to become invasive. For this thesis an in vitro tumor cell invasion assay was developed and used to investigate the role of a family of cell surface receptors, called integrins, in the invasion of tumor cells across basement membranes. It was also used to isolate an invasive cell line in order to study some of its properties. Two osteosarcoma cell lines, HOS and MNNG-HOS, with known in vivo metastatic potentials were assayed in the in vitro invasion assay. Invariably, the highly tumorigenic and metastatic MNNG-HOS cells demonstrated greater invasive ability than the non-tumorigenic HOS cells. The chemical transformation of HOS into tumorigenic MNNG-HOS cells resulted in an increase in the expression of oc-jB,, o^Bj and o^Bj integrins which are laminin and collagen receptors. The expression of c^Bi and o^B, were unchanged on MNNG-HOS cells and the expression of ocvB3 was strongly downregulated on the more invasive cells. The invasion of HOS and MNNG-HOS cells through matrigel could be significantly inhibited when anti-fibronectin receptor or anti-ot^ subunit antibodies were present in the invasion assay, demonstrating the important role of integrins in tumor cell invasion. The in vitro invasion assay described in this thesis was used to isolate a more invasive cell line from the prostate carcinoma cell line, PC-3, and called IPC-3. The morphology of these cells was distinct from the parent population, showing a spherical morphology as opposed to the triangular or spindle shaped morphology of PC-3 cells. These cells were also several times more invasive than the PC-3 cells and proliferated at a faster rate than the parent PC-3 cells. IPC-3 cells gradually lost their invasive potential after several months in tissue culture but retained their morphology and the characteristic expression of integrins. Adhesion of PC-3 and IPC-3 cells to iii purified extracellular matrix components revealed that IPC-3 attached well to laminin and to vitronectin. In adhesion kinetic experiments to purified extracellular matrix proteins, IPC-3 cells attached more quickly than PC-3 cells to larninin and vitronectin. Although the IPC-3 cells attached to the extracellular matrix proteins, fibronectin, vitronectin, laminin and collagen type IV, they were only able to spread on laminin and required several hours to do so. PC-3 cells also attached well to the extracellular matrix proteins but required only several minutes to spread on the matrix proteins including laminin. When plated on stock matrigel PC-3 cells organized themselves in tube-like structures while IPC-3 cells aggregated in clusters. Analysis of the integrins on PC-3 and IPC-3 cells demonstrated that IPC-3 cells downregulated the expression of the a,B,, and an almost completely downregulated the OjBi integrin while the expression of the fibronectin receptor, otsB,, and the vitronectin receptor, 0 ,^63, were unchanged. The expression of o^ B, in both PC-3 and IPC-3 cells was not prominent. However the 0^64 receptor was present in large amounts and was upregulated in IPC-3 cells, particularly the 200 kDa subunit of B4. Immunofluorescence staining of PC-3 and IPC-3 cells demonstrated that PC-3 cells distributed their (X3B1 and cc6B4 integrin receptors mainly along the cell periphery and their o^ Bj receptor in focal adhesion plaques, while the invasive IPC-3 cells concentrated their integrin receptors in circular adhesion structures. Although much remains to be learned about integrins, they have an instrumental role in the invasion of tumor cells across basement membranes during the metastatic cascade of malignant cells. iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS xi INTRODUCTION 1 1. Overview 1 2. Pathogenesis of Metastasis 2 3. Metastatic Phenotype 6 a. Proteases 6 b. MHC antigens 8 c. Oncogenes 8 d. Extracellular matrix 10 4. Basement Membranes 12 a. Structure and Function 12 b. Type IV collagen 14 c. Laminin 14 d. Fibronectin 16 e. Entactin and Heparan Sulfate Proteoglycan 17 d. Vitronectin 18 V Table of Contents 5. Extracellular matrix receptors 23 6. Invasion assays 28 MATERIALS AND METHODS 30 1. Cells 30 2. Antibodies 30 3. Matrix proteins 31 4. Surface labelling and immunoprecipitation 31 5. In vitro invasion assay 32 6. Cell adhesion assay 33 7. Morphology 34 8. Immunofluorescence 34 9. Proliferation rate 35 10. Adhesion kinetics 35 RESULTS 37 1. In vitro invasion assay 37 2. Invasion of PC-3 and IMR90 cells 43 3. Invasion of HOS and MNNG-HOS cells 43 4. Expression of integrins on HOS and MNNG-HOS cells 46 5. Isolation of an invasive cell line (IPC-3) 49 6. Growth rate of PC-3 and IPC-3 51 vi Table of Contents 7. Adhesion of PC-3 and BPC-3 cells on extracellular matrix proteins 57 8. Adhesion kinetics of PC-3 and IPC-3 cells 66 9. Morphology of PC-3 and IPC-3 cells on extracellular matrix proteins 66 10. Expression of integrins on PC-3 and IPC-3 cells 73 11. Immunofluorescence of PC-3 and IPC-3 cells using anti-integrin antibodies . . 78 DISCUSSION AND CONCLUSIONS 86 BIBLIOGRAPHY 95 LIST OF TABLES vii Table I Integrin superfamily 25 Table II Invasion of PC-3 and IMR90 in vitro 44 Table III Invasion of MNNG-HOS and HOS cells in vitro 45 Table IV Invasion of MNNG-HOS and HOS cells in the presence of anti-integrin antibodies 50 Table V Loss of invasion of IPC-3 cells in culture 54 viii LIST OF FIGURES Figure 1. Three step invasion hypothesis across the basement membrane 5 Figure 2. Schematic diagram of extracellular matrix proteins 20 Figure 3. Association of integrin a and B subunits within the integrin family of adhesion receptors 26 Figure 4. Photograph of transwells 38 Figure 5. Schematic diagram of the in vitro invasion assay 39 Figure 6. Relationship between matrigel concentration and invasion of PC-3 cells 42 Figure 7. Immunoprecipitation of 125I-surface labeled HOS and MNNG-HOS using anti-integrin antibodies 48 Figure 8. Photograph of PC-3 and IPC-3 cells in tissue culture 53 Figure 9. Proliferation rate of PC-3 and IPC-3 cells cultured in DMEM containing 10% FCS 56 Figure 10. Adhesion of PC-3 and IPC-3 cells to fibronectin 59 Figure 11. Adhesion of PC-3 and IPC-3 cells to vitronectin 59 Figure 12. Adhesion of PC-3 and IPC-3 cells to laminin 61 Figure 13. Adhesion of PC-3 and IPC-3 cells to type I collagen 61 Figure 14. Adhesion of PC-3 and IPC-3 cells to type IV collagen 63 Figure 15. Adhesion of PC-3 cells to fibronectin, vitronectin, laminin and collagen type I and rv 65 Figure 16. Adhesion of IPC-3 cells to fibronectin, vitronectin, laminin and collagen type I and rv 65 Figure 17. Adhesion kinetics of PC-3 cells on fibronectin, laminin, vitronectin and type IV collagen 68 Figure 18. Adhesion kinetics of IPC-3 cells on fibronectin, laminin, vitronectin and type IV collagen 68 ix Figure 19. Morphology of PC-3 and IPC-3 cells on fibronectin, vitronectin, laminin, and collagen I and IV 70 Figure 20. Morphology of PC-3 and IPC-3 cells cultured on matrigel 72 Figure 21. Immunoprecipitation of 125I-surface labelled PC-3 and IPC-3 cells with anti-integrin antibodies 75 Figure 22. Immunoprecipitation of PC-3 and IPC-3 cells using anti-vitronectin receptor and anti-otg 77 Figure 23. Immunofluorescence staining of PC-3 and IPC-3 cells using the monoclonal anti-integrin antibody P1B5 (anti-ot,) 81 Figure 24. Immunofluorescence staining of PC-3 and IPC-3 cells using the polyclonal anti-vitronectin antibody 83 Figure 25. Immunofluorescence staining of PC-3 and IPC-3 cells with the monoclonal anti-integrin antibody J1B5 (anti-Og) 85 LIST OF ABBREVIATIONS BSA bovine serun albumin CPM counts per minute DMEM Dulbecco's Modified Eagle Medium DPM disintigrations per minute EDTA Ethylenediaminetetraacetic acid FCS fetal calf serum MHC major histocompatibility complex PBS phosphate buffer saline PMSF phenylmethylsulfonyl fluoride RIPA radioimmunoprecipitation assay SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electroph TIMP tissue inhibitor of metalloproteinases amino acids R arginine G glycine D aspartic acid Y tyrosine I isoleucine S serine L leucine E glutamic acid xi Acknowledgments I would like to express my sincerest gratitude: To my supervisor, Shoki Dedhar (Department of Advance Therapeutics, Cancer Research Centre), for his guidance and help throughout my project. To the members of my committee, Gerry Krystal, Ross MacGillivary, Nelly Auersperg and Haden Pritchard for their critical evaluation, insightful comments and questions during the preparation of my thesis. To the other members of our lab, Mumtaz Rojiani and Kathy Robertson for their assistance with several experiments. Special thanks go to Virginia Gray for her patience and technical assistance, especially during the first several months. To Sarah Maines-Bandiera for her help the photography of the immunofluorescence data. To Spencer Kong for his assistance with the preparation of the computer graphics. Finally, to the staff in the departments of Advance Therapeutics and Cancer Endocrinology for providing a pleasant working environment. 1 INTRODUCTION Overview The management and treatment of cancer patients is a growing concern with the increasing number of cancer patients being diagnosed each year. Many patients have benign neoplasms which are not life-threatening and can be treated with surgery. However, of greater importance are the individuals who have malignant tumors capable of metastasizing. Metastasis is defined as the spread of malignant tumor cells from their origin to a distant site by one of three possible routes:(i) the bloodstream, (ii) the lymphatic system or, (iii) across body cavities. It is these patients having malignant tumors which are of concern. For many of these patients the treatments currently available are not sufficient to eradicate the entire tumor. The few cells that survive treatment will reestablish new lesions and eventually lead to the patient's death (Fidler et al., 1978). One of the more serious problems in treating cancer is that malignant tumors have often metastasized before they are clinically detectable. Often, at the time of diagnosis, a very large number of cells have established themselves at distant sites making surgery a futile attempt at removing the lesion. Chemotherapy and radiation therapy are sometimes beneficial but often make the patient very ill. More advanced and sophisticated methods for treating cancer are therefore required. In order to develop better methods, we must first understand more about the mechanisms involved in tumor cell invasion and metastasis and more about the properties of malignant cells. 2 PATHOGENESIS OF METASTASIS Tumor cell metastasis is a very complex multi-step process. In order for metastatic lesions to be established, the tumor cell must successfully complete several steps during the metastatic cascade (Poste and Fidler, 1980). A tumor arises from a single transformed cell which grows slowly as a small avascular lesion called an \"in situ\" carcinoma. Once the tumor has attained a sufficient size it becomes vascularized and grows much more rapidly giving rise to a large heterogenous population of cells, some of which have acquired the genetic and biochemical, characteristics that enable them to become metastatic. The cells which have acquired these metastatic characteristics must first detach from their neighbouring cells and invade any surrounding matrix, then penetrate the basement membrane of the nearby blood or lymphatic vessels (Fidler and Hart, 1982). Once in the circulatory or lymphatic system, the tumor cells must evade the host immune defences such as the lymphocytes and macrophages. The invasive cells must cross the basement membrane of the blood vessel a second time and establish a colony in a new location (Fidler and Hart, 1982). If the tumor is incapable of completing any of these steps because it is lacking an important biochemical or phenotypic characteristic such as proteases, extracellular matrix receptors or growth factors, it will be eliminated. The metastatic process is a very inefficient process (Weiss, 1983). Few cells which leave the primary lesion actually survive long enough to establish metastases. Poste and Fidler, (1980) injected radiolabelled murine B16 melanoma cells into mice and measured the radioactivity from the cells having established metastases in the various organs. From their experiments they concluded that far less than 0.1% of the cells were able to colonize and form new lesions. The invasion of tumor cells through the extracellular matrix , especially the basement membrane, is often the first step in the metastatic process. The basement membrane is considered 3 a significant barrier for the tumor cell and is therefore an important area to investigate. Liotta et al., (1986) have described a 3 step mechanism for the invasion of tumor cells across a basement membrane (Fig. 1). The first step involves the attachment of tumor cells to the components of the basement membrane such as laminin or type IV collagen via cell surface receptors, many of which belong to a superfamily of adhesion receptors called integrins. The second step involves the release of proteolytic enzymes such as collagenases, stromelysin and plasminogen activator by the tumor cell which degrade the components of the basement membrane. The third and final step is the active migration of the tumor cell through the broken down matrix. The cell surface receptors for the extracellular matrix play an important role in all three steps of the invasion process. Attachment of the tumor cell to the basement membrane, mediated by receptors for extracellular matrix molecules, is crucial for invasion. If attachment is inhibited with antibodies directed against cell surface receptors or peptides containing the cell binding domain, the cells are unable to invade (Kramer et al., 1989, De Luca et al., 1990, Dedhar and Saulnier, 1990). There is now evidence that integrins may be involved in signal transduction from the extracellular matrix to the nucleus. Werb et al.,(1989) have shown that 4 Figure 1 Three step invasion hypothesis Step 1: The tumor cell attaches to the basement membrane via cell surface receptors called integrins. Step 2: The tumor cell releases proteolytic enzymes which degrade the components of the basement membrane. Step 3: The tumor cell actively migrates through the weakened area of the basement membrane. Figure from Liotta et al., 1986 INVASION THROUGH THE BASEMENT MEMBRANE 5 ^ LAMININ RECEPTOR \u00E2\u0080\u00A2jC, LAMININ STEP 1: ATTACHMENT TYPE IV COLLAGENASE STEP 2: DISSOLUTION / * i \ \ STEP 3: LOCOMOTION 6 antibodies to the integrin oc5 subunit and RGD containing peptides from fibronectin stimulate the release of proteolytic enzymes from rabbit synovial fibroblasts when coated to plastic. Turpeenniemi-Hujanen et al.(1986) have shown that the attachment of human and murine melanoma cells to laminin promotes the release of type IV collagenase. Kanemoto et al., (1990) have shown that a segment in the E8 segment of the A chain, called PA22-2, may be responsible for the release of type IV collagenase. As well, the integrins a4Bx and o^ B, have been found to be important in facilitating CD3 mediated T cell proliferation (Shimizu et al., 1989, Davis et al., 1990). Lastly, during the migration of tumor cells through the degraded basement membrane, there is successive attachment and release of integrins to extracellular matrix proteins which enables the tumor cells to migrate through the basement membrane and metastasize to other organs. METASTATIC PHENOTYPE Proteases Although the mechanisms which cause cells to become metastatic are not known, malignant cells have several characteristic features which are crucial to the cell's ability to metastasize through the host tissues and establish new lesions. One important characteristic of invasive cells is their ability to release proteolytic enzymes which degrade the extracellular matrix (Mignatti et al., 1986, Liotta et al., 1979). The three major classes of degradative enzymes found in tumor cells are the serine proteases (tissue-type and urokinase-type plasminogen activators), the matrix metalloproteinases (interstitial collagenases, type IV collagenase, gelatinase and stromelysin), and the cysteine proteases (cathepsin B,L and H)(Nicolson, 1989). Although metalloproteinases are important in the invasion of tumor cells and the degradation of basement 7 membrane components, not all tumor cells with an invasive phenotype express elevated levels of metalloproteinases. Some cells use the serine protease, plasminogen activator, almost exclusively to degrade the extracellular matrix (Mackay et al., 1990). The tissue inhibitor of metalloproteinases (TIMP) is also an important factor in tumor cell invasion. The regulation of protease activity is partially regulated by the expression of protease inhibitors (Khokha and Denhardt, 1989). In some cases, the cell's increased ability to degrade its surrounding matrix may not be caused by increased protease secretion but by decreased levels of protease inhibitors. Khokha et al. (1989) have shown an inverse correlation between the expression of TIMP and the invasive potential of tumor cells. By transfecting the plasmid pNMH-aT, designed to produce antisense TIMP RNA into Swiss 3T3 cells, they were able to show a decrease in TIMP expression and an increase in metastatic potential. They also observed that cells with TIMP antisense mRNA in the cytoplasm were able to produce as much TIMP as the controls and therefore concluded that the antisense mRNA exerted its inhibitory effect in the nucleus and not in the cytoplasm. TIMP may also affect tumor cell invasion indirectly by influencing the microenvironment. Reduction of proteases permits the tumor cells to lay down and adhere to extracellular matrix (Khokha and Denhardt, 1989). Edwards et al. (1987) have shown that the expression of TIMP can be regulated by TGF-B and that TGF-B alone did not have an effect on the expression of TIMP. However, in the presence of other growth factors such as FGF or EGF, TGF-B was shown to upregulate the expression of TIMP in tumor cells. 8 MHC antigens Another important characteristic of metastatic cells is their ability to evade the host immune system. A key component in immunogenic recognition of tumor cells are the class I major histocompatibility (MHC) proteins. In the murine system the surface expression of the H-2K/H-2D gene products have been correlated to metastatic capacity in vivo (Eisenbach et al., 1986). It was found that the ratio of H-2K/H-2D expressed on the cell surface was more important than the overall expression of the two proteins. Eisenbach et al., (1986) concluded from their experiments that cells with a high H-2K/H-2D ratio did not form many metastatic lesions when injected into mice while cells with a low H-2K/H-2D ratio were able to form numerous metastatic lesions. Increasing the expression of H-2K with interferon a or B or retinoic acid or by gene transfection of the H-2K gene in the highly metastatic murine 3LL tumor cell line reduced the cell's ability to form metastases (Gelber et al., 1989, Wallich et al., 1985). Thus the increase or decrease in metastatic ability of the tumor cells in vivo was attributed to the increased or decreased immunogenicity of the cells. Oncogenes Oncogenes are a group of genes, whose products, when altered or ectopically expressed, are capable of causing cell immortalization and transformation (Weiss, 1986). They normally control a wide variety of cellular functions such as proliferation, differentiation, morphology, intercellular communication and motility (Greenberg et al., 1989). Although the expression of cellular or viral oncogenes is associated with the transformed phenotype, there is no conclusive evidence that any particular oncogene(s) is/are consistently associated with the malignant phenotype; however, certain oncogenes such as the ras and myc oncogenes have been associated with the metastatic phenotype in some tumor cells. 9 There are three varieties of the ras oncogene, the Harvey-ras, Kirsten-ras, and N-ras genes. All three encode 21 kDa proteins that display homology to G proteins which act as second messengers in the transduction of signals in the cell. The 21 kd protein encoded by the ras oncogene is a guanine nucleotide binding protein associated with the inner surface of the plasma membrane and binds to GTP or GDP with high affinity (Greenberg et al., 1989). This protein was also found to have an intrinsic GTPase activity that catalyzed the hydrolysis of GTP to GDP, and the coincident inactivation of ras. NIH/3T3 cells transfected with DNA containing the activated H-ras or K-ras oncogene were able to form metastases in nude mice while the parent or spontaneously transformed NIH/3T3 cells could not form metastases (Thorgeirsson et al., 1985). Other cell lines such as rat embryo cells and mouse lymphoma cells transfected with the ras oncogene were also able to form metastases in nude mice (Muschel and Liotta, 1988, McKenna et al., 1990). Although the ras oncogene is able to induce the metastatic phenotype in some cells, not all metastatic cells express the ras oncogene. This suggests that other oncogenes may be required to induce the metastatic phenotype or that the ras oncogene is only able to induce a phenotypic change in some cells (Greenberg et al., 1989). The myc family of oncogenes are nuclear oncogenes, originally isolated from the avian myelocytomatosis virus. Although little is known about the biochemistry of its 65 kDa protein, the myc oncogene has been associated with the metastatic phenotype in some tumors (Nicolson, 1986). Human signet ring gastric carcinoma cells capable of forming metastasis in nude mice were found to have an amplified myc oncogene (Yanagihara, et al., 1991). Ras and myc are the oncogenes commonly associated with a metastatic phenotype. However, occasionally other oncogenes such as raf, src and fms have been correlated with metastatic progression (Greenberg 10 Extracellular matrix The extracellular matrix plays a substantial role in cell differentiation, embryogenesis, wound repair and tumor cell invasion (McDonald, 1989, Ekblom et al., 1986). The major components of the extracellular matrix include collagens, elastins, proteoglycans, and glycoproteins (Labat-Robert et al., 1990). The more common extracellular matrix proteins include the glycoproteins fibronectin, laminin, vitronectin, and collagens type I-VII. Collagens I-III are found primarily in bone, cartilage and the extracellular space. Type IV collagen and laminin are found exclusively in basement membranes (Laurie et al., 1982). Types V and VII collagen are associated with the attachment of the basement membrane to the underlying connective tissue (Bachinger et al., 1990). Fibronectin is a very abundant extracellular matrix protein which is found in the extracellular space of numerous cell types while vitronectin is predominantly found in the serum. There are three important molecular interactions associated with extracellular matrix proteins: (1) self-aggregation to form ordered structures, (2) interactions with other matrix proteins forming large complexes and (3) interactions with the cell surface to promote cell adhesion (Ruoslahti et al., 1985). The intrinsic interconnecting network of extracellular matrix proteins in basement membranes includes all three types of intermolecular interactions (Yurchenco and Schittny, 1990, Yurchenco et al., 1986). Several of the extracellular matrix proteins, such as laminin and fibronectin have more than one cell binding domain. The most studied is the cell binding domain mediated by the tripeptide RGD, found in fibronectin. The RGD sequence was determined by progressively trying smaller and smaller synthetic peptides from the fibronectin molecule until binding affinity was lost. It was found that only the three amino acids, Arginine-Glycine-Aspartic acid (RGD) were required for cell attachment (Pierschbacher and Ruoslahti, 1984, Yamada and Kennedy, 1985). 11 Since its role in cell adhesion was described in fibronectin, the RGD sequence was found to have similar properties in several other extracellular matrix molecules including, vitronectin (Suzuki et al., 1985), von Willebrand factor (Cheresh and Spiro, 1987, Dejana et al., 1989), tenascin (Bourdon and Ruoslahti, 1989), laminin (Sasaki et al., 1988), thrombospondin (Lawler et al., 1988) and fibrinogen (Ruoslahti and Pierschbacher, 1986). The RGD peptide in many of these extracellular matrix proteins is recognized by integrin receptors (D'Souza et al., 1988, Smith and Cheresh, 1988). RGD peptides in solution can inhibit cell attachment and tumor cell invasion by interfering with the interaction of the cell surface receptors with the extracellular matrix proteins (Gehlsen et al., 1988a, Bretti et al., 1989, Saiki et al., 1989). Hence the extracellular matrix-receptor interactions are important in cell adhesion and tumor cell invasion. 12 BASEMENT MEMBRANES Structure and Function Basement membranes are specialized extracellular matrix structures which are composed of three layers. Directly underlying the cells is the electron-lucid lamina rara, then a more electron-dense layer called the lamina densa followed by a second electron-lucid layer which interfaces with the underlying extracellular matrix (Abrahamson,1986). Basement membranes are found underlying epithelial cells and endothelial cells and surrounding muscle cells, adipocytes, and Schwann cells. These membranes are almost exclusively produced by the cells apposing them. The basement membrane is anchored to its underlying matrix by fibrils composed of type V and VII collagens (Keene et al., 1987). Basement membrane components are the first extracellular matrix products produced during development and are required for the adhesion, migration, growth and differentiation of cells (Timpl and Dziadek, 1986). Several functions have been described for basement membranes including maintenance of tissue architecture by providing a sheet-like support to which cells attach, serving as a physical boundary for cells, and functioning as a molecular filter preventing passage of proteins (Martin et al., 1988). There is some variability in the composition and thickness (30-300nm) of basement membranes depending on their location (Timpl and Dziadek, 1986). However a typical basement membrane is composed of several ubiquitous components; type IV collagen, laminin, Nidogen/Entactin and various sulfated proteoglycans, the predominant one being Heparan sulfate proteoglycan (Martin et al., 1988). Fibronectin is an important and abundant extracellular matrix molecule but is only occasionally found in basement membranes (Laurie et al., 1982). It is probable that basement membranes contain a number of less abundant components which are yet to be isolated and characterized. Components such as BM 40/osteonectin/SPARC, amyloid P component, Bullous pemphigoid antigen, AE26 antigen and EBA antigen are present in low amounts and are not 13 found in all basement membranes (Kolega and Manabe, 1990). Changes in basement membranes are hallmarks of several diseases such as a thickened renal glomerular basement membrane in diabetes, autoimmune diseases such as Goodpasture's syndrome (Spargo and Taylor, 1988) and neoplastic lesions. The basement membrane surrounding a neoplastic lesion may appear structurally normal. However, in many cases the basement membrane components are either reduced in number or simply not properly assembled, weakening the rigid structure of the basement membrane and facilitating the passage of tumor cells (Ingber et al., 1981). Occasionally, the most important histological distinguishing feature between malignant and benign tumors is the presence of a basement membrane surrounding the lesion, benign tumors being encapsulated by a basement membrane and malignant tumors having degraded and invaded through some areas of the basement membrane (Liotta et al., 1986). A murine tumor which produces large amounts of basement membrane is the EHS (Engelbreth Holm-Swarm) tumor. It has provided valuable information in the isolation and characterization of basement membrane proteins (Inoue and Leblond, 1985). The matrix produced by the EHS tumor can be reconstituted and used in vitro as a basement membrane substitute. Reconstituted basement membrane, sold commercially as Matrigel, has been shown to be structurally and functionally similar to the basement membranes found in vivo and is now commonly used in in vitro invasion assays.(Kleinman et al., 1982, Kleinman et al., 1983). 14 Type IV collagen Type IV collagen, found exclusively in basement membranes, is a heterotrimer composed of two al(IV) chains and an a2(IV) chain. Each heterotrimer (monomer) is approximately 400 nm in length and contains a protease resistant NCI (noncollagenous) domain at the carboxyl end followed by a large helical region interrupted by several non-helical regions. Type IV collagen self-assembles in a time and temperature dependant manner. The monomers bind end-to-end via the NCI domain and laterally via the 7S domain located at the amino terminal end to form large chicken-wire-like networks (Fig 2a,p.20), providing a structural framework to the basement membrane (Babel and Glanville, 1984). Type IV collagen can also bind to other extracellular matrix proteins such as heparin sulfate proteoglycans, heparin, laminin and fibronectin (Laurie et al., 1986, Koliakos et al., 1989). Herbst et al., (1988) have shown that type IV collagen and a pepsin-generated triple-helical fragment of type IV collagen were much more effective in mediating cell attachment and migration of aortic endothelial cells than were laminin and the NCI domain of type IV collagen. Type IV collagen not only provides a framework for the basement membrane but is also important in cell adhesion. Laminin Laminin is a large glycoprotein (Mr 850,000) ubiquitous to basement membranes. It was originally isolated from neutral extracts of the mouse Engelbreth-Holm-Swarm (EHS) tumor cell line (Timpl et al., 1979). Laminin is a cross-shaped molecule composed of three different polypeptide chains, an A chain (400,000), a B l chain (210,000) and a B2 chain (200,000), linked together by inter and intrachain disulfide bonds (Pikkarainen et al., 1988, Sasaki et al., 1987, Vuolteenaho et al., 1990)(Fig. 2b). The Bl and B2 chains of laminin show considerable 15 homology suggesting that they were derived from the same ancestral gene (Sasaki and Yamada, 1987). Laminin is a multifunctional molecule which promotes cellular adhesion, growth, migration (Wewer et al., 1987), tumor cell invasion and differentiation of cells (Ekblom et al., 1980, Kleinman et al., 1985, Vukicevic et al., 1990, Ocalan et al., 1988). At the 2 cell stage of embryogenesis only the B1 chain of laminin is synthesized. Laminin does not appear in intact form until the morula stage (Ekblom et al. 1986). The expression of the laminin A chain was found to correspond with the development of cell polarity during embryonic development of murine kidney tubules (Klein et al., 1988). Laminin can also bind to a number of matrix proteins found in the basement membrane such as entactin, type IV collagen via the globular domains at the end of the long and short arms, and to heparan sulfate proteoglycans. Laminin can also bind to other laminin molecules, via the E4 and E l domains, forming large aggregates (Martin and Timpl, 1987). Laminin is a multidomain molecule possessing several functional binding sites. The 20 amino acid F9 site, located on the internal globular domain of the B l chain, and the YIGSR sequence, located in a cysteine rich region of the Bl chain are both involved in cell attachment (Kleinman and Weeks, 1989) (Fig. 2c). The YIGSR peptide also promotes cell migration and inhibits the formation of lung metastases when injected with B16F10 melanoma cells in vivo (Iwamoto et al., 1987, Kanemoto et al., 1990). The RGD sequence on the A chain is also involved in cell attachment (Kleinman et al., 1990). Also found on the A chain is the 19 amino acid PA22-2 segment which promotes cell adhesion, neurite outgrowth and induces collagenase IV activity (Kanemoto et al., 1990). Laminin also has three known binding sites for heparin, the F9, E8 and AC15 domains (Kouzi-Koliakos et al., 1989) (Fig. 2b). The laminin-heparin interaction is a calcium dependent interaction which modulates the polymerization of laminin 16 molecules (Yurchenco et al., 1990). Recently, other molecules which are homologous to laminin have been found in basement membranes. Merosin, which is homologous to the carboxy terminal end of the laminin A (Fig. 2d) chain appears in the basal lamina of Schwann cells, striated muscle and trophoblasts (Leivo and Engvall, 1988, Ehrig et al., 1990). In mice, merosin only appears later in development suggesting that it may have a role in the differentiation or maturation of tissues (Hunter et al., 1989a). Another laminin-like molecule, called S-laminin (Fig. 2e), shares a 40% sequence homology with the B l chain of laminin and is concentrated at synaptic sites in muscles and is also present at other locations such as peripheral nerve and glomerular basement membranes (Hunter et al., 1989b). Neurons from embryonic chick ciliary ganglia were able to adhere to plates coated with S-laminin. The use of successively smaller peptides revealed the cell binding domain of S-laminin as a tripeptide sequence called LRE (Hunter et al., 1989b). Fibronectin Fibronectin is an important multifunctional extracellular matrix glycoprotein (Mr 400,000) which has many biological functions similar to laminin such as cell migration (Lacovara, et al., 1984), adhesion, cell invasion (Ruoslahti, 1984), morphogenesis and development (Ruoslahti, 1988). Fibronectin can be present in both soluble forms in plasma and other body fluids or in insoluble forms in the extracellular matrix (Rocco et al., 1987). It is present in some basement membranes but has not been isolated in the EHS tumor extracellular matrix or the glomerular basement membrane (Laurie et al., 1982, Kleinman et al., 1986). Fibronectin is composed of two similar chains made of three different type repeats which are linked near the carboxyl end by disulfide bonds (Ruoslahti, 1988)(Fig. 2f). The molecule possesses several binding domains including two fibrin and heparin binding domains, a collagen and gelatin binding domain, and two cell binding 17 domains, one found in the central region of all the fibronectin molecules and containing the RGD sequence (Ruoslahti, 1988). The other cell binding site is found in an alternatively spliced region of the fibronectin molecule and is called the IIICS domain (Guan and Hynes, 1990, Schwarzbauer et al., 1989). Entactin/Nidogen and heparan sulfate proteoglycans Entactin (nidogen) is a 150 kDa sulfated glycoprotein found only in basement membranes. It is dumbbell shaped having a globular domain at each end (Carlin et al., 1981, Timpl et al., 1983)(Fig. 2g). Entactin also contains an RGD sequence which is partially responsible for cell adhesion (Chakravarti et al., 1990). Entactin binds most strongly to the B2 chain of laminin near the centre of the cross in a 1:1 ratio but can also bind to the triple helix of the type IV collagen (Mann et al., 1989). The complexes that laminin forms with entactin in equimolar proportions are very stable and divalent cation dependent (Paulsson, 1988). Heparan sulfate proteoglycans are the most predominant proteoglycan found in basement membranes and in the extracellular matrix. Other proteoglycans found in basement membranes are chondroitin and dermatin sulfate proteoglycans (Fujiwara et al., 1984). Proteoglycans range in size from 75-350 kDa, however, the majority of those found in basement membranes are 130 kDa (Martin et al., 1988). Heparan sulfate proteoglycans function as selective filtration barriers and may form large aggregates which bind to other basement membrane components helping to maintain the structural integrity of the matrix (Yurchenco et al., 1987). 18 Vitronectin Vitronectin/serum spreading factor/S protein is a 75 kDa glycoprotein found predominantly in the serum and occasionally in the extracellular space (Hayman et al., 1983). Vitronectin functions as a complement regulatory protein in plasma (Preissner, 1989), plays a role in the aggregation of platelets (Asch and Podack, 1990) and promotes cell adhesion and spreading. Vitronectin also contains an active RGD sequence which is recognized by integrin receptors (Thiagarajan and Kelly, 1988, Pytela et al., 1985b). Schematic diagrams of extracellular matrix proteins (a) Type IV collagen (b,c) laminin. Figure from Anderson, 1990 (d) S-laminin. Figure from Anderson, 1990 (e) Merosin Figure from Anderson, 1990 (f) fibronectin. Figure from Ruoslahti, 1988 (g) Entactin. Figure from Mann et al., 1989 20 21 22 Fibronectin heparin gelatin fibrin collagen c t U heparin I I I C S (Ibrln RGD S S NM, ^ }{]{}omx>{}{}{}{>aaDL^ ^ Entactin g 23 EXTRACELLULAR MATRIX RECEPTORS The interactions between cells and the extracellular matrix is mediated by cell surface receptors many of which belong to a superfamily of adhesion receptors called integrins. Integrins are a family of transmembrane glycoprotein heterodimers composed of an a and B subunit noncovalently bound together which mediate cell-cell and cell-matrix interactions (Hynes, 1987, Buck, 1987, Albelda and Buck, 1990) and play a key role in embryogenesis, wound healing, cell differentiation, cell migration, bacterial cell invasion (Isberg and Leong, 1990) and tumor cell invasion (Dedhar, 1990, Ruoslahti and Pierschbacher, 1987). Both the a and B subunits possess large extracellular domains, a transmembrane domain and short cytoplasmic domains, with the exception of the B4 subunit which has a 118 kDa cytoplasmic domain (Hogervorst et al., 1990, Suzuki and Naitoh, 1990). The superfamily of integrins was initially classified according to their B subunits and formed three major groups. The integrins sharing the B, subunit are the largest group having six different a subunits and bind fibronectin, collagens and laminin (table 1). The leucocyte adhesion molecules, LFA-1, MAC-1, and PI50,95 are only found on lymphoid and myeloid cells and share a common B2 subunit (Larson and Springer, 1990). The third group of integrins, the cytoadhesion molecules, consists of the vitronectin receptor (0 ,^63) and the platelet gpIIb/flTa receptor which share a common B3 subunit. The isolation and characterization of several new B subunits, Bp (Holzmann and Weissman, 1989), Bs (Freed et al., 1989), B5 (Ramaswamy and Hemler, 1990) and B6 (Sheppard et al., 1990) and the finding that a subunits could associate with multiple B subunits has forced a new classification for integrins (Fig. 3). The members of the Bt family possess mainly extracellular matrix molecules as ligands, many of which are components of basement membranes. Integrin 0 , 6 , was initially isolated from 24 the surface of T lymphocytes two weeks after in vitro activation (Hemler et al., 1984) but it is also expressed on a number of other cell types (Dejana and Lauri, 1990). It is composed of an a subunit of Mr 200,000 and a Mr 110,000 ^ subunit and functions as a collagen and laminin receptor (Tawil et al., 1990). The rat analogue of the a t subunit contains an I domain, which is an additional 200 amino acids inserted in the a subunits of the leukocyte adhesion molecules and the Oj subunit of the integrin o^ Bj. The a t subunit also contains three divalent cation binding sites within its extracellular domain (Ignatius et al., 1990). The integrin Oj subunit, Mr 150,000, was initially isolated on activated T cells but is also expressed on platelets, fibroblasts, and endothelial and epithelial cells from many different tissues (Zutter and Santoro, 1990, Hemler et al., 1984).The otj subunit also binds to collagen and laminin (Elices and Hemler, 1989). However its ligand specificity varies with different cell lines, binding to collagen on some cell lines and to laminin on others (Languino et al., 1989, Kirchhfer et al., 1990). The oc^ ! complex also possesses an I-domain and three divalent cation binding sites (Takada and Hemler, 1989). The o^ B, complex is a promiscuous receptor capable of binding to laminin, fibronectin and collagen (Sanchez-Madrid et al., 1986). It is located on a large number of cell types and functions in cell-matrix adhesion (Carter et al., 1990b). It is also located at intercellular contact sites and therefore may also function in cell-cell interactions (Kaufmann et al., 1989). Gehlsen et al.(1989) have shown that the OjBi complex binds to the Bl chain of laminin in an RGD independent manner. The integrin o^ B, differs from the other integrins of the B, family in that it is only weakly associated to its B subunit and easily undergoes partial cleavage into 80 and 70 kDa fragments (Hemler et al., 1987). It is expressed most abundantly on thymocytes, peripheral blood lymphocytes, monocytes, T and B cell lines and 25 Table 1 Integrin superfamily of cell adhesion receptors integrin subunits molecular mass ligands M i 200/110 laminin (El), collagen M i 160/110 laminin, collagen M i 150/110 laminin, collagen (I,IV,VI), fibronectin M i 140/110 cell-cell, fibronectin (CS-1) M P 140/100 M i 155/110 fibronectin M , 140/110 laminin (E8) M 4 140/210 laminin ? aLB2 170/90 ICAM-1, ICAM-2 165/90 C3bi, fibrinogen aiso02 145/90 M i 160/110 fibronectin, vitronectin M\u00E2\u0080\u009E 160/110 fibronectin, collagen avBs 160/100 fibronectin, vitronectin M s 160/105 avB3 160/105 laminin, Von Willebrand factor vitronectin, fibrinogen, osteopontin,thrombospondin aiibB3 140/105 fibrinogen, fibronectin,vitronectin, Von Willebrand factor, thrombospondin 26 27 myelomonocytic cell lines (Takada et al., 1989) but is expressed on most adhesive cells in low amounts. The integrin ct4Bi functions in both cell-cell and cell-matrix adhesion (Hemler et al., 1990). Its ligands are the VCAM-1 (vascular cell adhesion molecule) and the UICS region of the fibronectin molecule which is an alternatively spliced region (Elices et al., 1990, Mould et al., 1990). The ct4 subunit is also able to associate with the B p subunit and functions as a lymphocyte homing receptor (Holzmann and Weissman, 1989). The classical fibronectin receptor, has a 155 kDa cc-subunit and binds to the RGD sequence of fibronectin (Pytela et al., 1985a). During keratinocyte differentiation the loss of adhesiveness precedes the loss of from the surface of the cells (Adams and Watt, 1990). Giancotti and Ruoslahti (1990) transfected the oc5 and B, cDNA into transformed Chinese hamster ovary cells and found that the cells which overexpressed oCsBj also secreted more fibronectin and were nontumorigenic as compared to the control cells, associating the increase in the expression of a 5 B! with a higher degree of cell differentiation (Dedhar et al., 1987). The integrin has been found on platelets and a large number of other cell types (Sonnenberg et al., 1988, Gehlsen et al., 1988a). This is the classical larninin receptor and binds to the E8 fragment of laminin (Sonnenberg et al., 1990) as opposed to which binds to the E l domain (Hall et al., 1990). The Og subunit is also able to bind the B 4 subunit (Hemler et al., 1989, Kennel et al., 1989). The B 4 subunit, primarily expressed in epithelial cells, can be expressed in three forms: 200 kDa, 180 kDa and 125 kDa. The difference in size is a result of alternative splicing of the cytoplasmic domain (Suzuki and Naitoh, 1990). The 0^64 complex also called TSP-180 is found in epithelial carcinoma cell lines but its ligand still remains uncertain (Hogervorst et al., 1990). The oty subunit is associated with multiple B subunits. It was first described as associating with the B3 subunit which behaves as a vitronectin receptor. This heterodimer also binds to fibrinogen, von Willebrand factor, osteopontin and thrombospondin in an RGD-dependent manner 28 (Smith and Cheresh, 1990). The a,, subunit has since been shown to associate with the Bs (Freed et al., 1989), B5 (Ramaswamy and Hemler, 1990), Bn and Bt (Dedhar and Gray, 1990) subunits. INVASION ASSAYS In vitro invasion assays are now commonly used to determine the invasive potential of tumor cell lines instead of in vivo invasion assays (Albini et al., 1987, Kramer et al., 1986, Terranova et al., 1986). They have advantages over the in vivo assays in that the experimental conditions can be more closely regulated and the assay can be performed in several hours as opposed to the 15-30 days required for in vivo experiments. Although simpler and quicker, in vitro invasion assays are not an adequate substitute for in vivo experiments. In vitro invasion assays are designed to look only at the invasive potential of cells, i.e. only one step in the complex metastatic process (Yagel et al., 1989). Several different in vitro invasion assays are in use today including an amnion invasion assay, a transwell (Repesh, 1989) and Boyden chamber or a modified Boyden chamber assay (Albini et al., 1987, Terranova et al., 1986). The introduction of matrigel in in vitro invasion assays is now widely used by researchers looking at tumor cell invasion and metastatic potential of tumor cells (Kramer et al., 1986, Repesh, 1989). A comparison between the amnion and the reconstituted basement membrane revealed that fewer cells were able to invade amnion membranes and that the results were more consistent when using matrigel. Cells invading through the reconstituted basement membrane can be isolated and recultured to select a more invasive cell line. This method of isolation is more feasible compared to using the human amnion assay because the cells invading through the amnion are difficult to isolate (Hendrix et al., 1989). 29 To understand the role of integrins in tumor cell invasion better, an in vitro invasion assay was developed and used to determine the invasive potential of two osteosarcoma cell lines and the effect of anti-integrin antibodies on their invasion. An invasive cell population was isolated using the in vitro invasion assay and its adhesive properties to extracellular matrices and expression of integrins investigated using cell attachment assays and immunofluorescence. Materials and Methods 30 Cells Prostate carcinoma cell lines, PC-3 and DU145, human lung fibroblasts, IMR90, and human osteosarcoma cell lines, HOS and MNNG-HOS were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (with 1% glutamine, penicillin 100 ug/ml and streptomycin 100 Ug/ml) supplemented with 10% heat inactivated fetal calf serum. The cells were passaged every five days. To detach the cells, the medium was aspirated and replaced with 5 mM EDTA in PBS for 2-5 min. The EDTA was then aspirated and the cells removed from the flask by pipetting media directly onto the cells. Antibodies Anti-fibronectin receptor and anti-vitronectin receptor polyclonal antibodies as well as anti-Oj (P1E6) and anti-a, (P1B5) monoclonal antibodies were purchased from Telios. Anti-a5 (BIIG2), anti-ctg (GoH3) and anti-B, (A2BI1) monoclonal antibodies were a gift from C. Damsky (U. of California, San Francisco). Normal preimmune serum was obtained from our own New Zealand white rabbits. Rhodamine conjugated antibodies (Goat anti-rat, Goat anti-rabbit and Rabbit anti-mouse) were purchased from Jackson ImmunoResearch. 31 Matrix proteins Collagen types I and IV were obtained from Sigma Chemical Company. Human fibronectin and vitronectin were purchased from Telios. Mouse Laminin was purchased from Gibco. BSA was obtained from Sigma. Matrigel and ITS+ were purchased from Collaborative Research (Boston Mass.) Surface labelling and immunoprecipitation Cells were detached from tissue culture flasks with 5 mM EDTA in PBS, washed twice in PBS, and surface labelled with 0.05 mCi 1 2 5I in the presence of Iodogen (Pierce) for 30 min at room temperature. The cells were then washed three times in PBS to remove free label and lysed in 500 ul Radioimmunoprecipitation assay (RJPA) lysis buffer containing 2.0 mM phenylmethylsulfonyl fluoride (PMSF) for at least 30 min on ice with agitation. The lysate was centrifuged in a microcentrifuge (12,000 rpm) and the pellet discarded. The number of cpm in the supernatant was determined by counting a 5 ul aliquot in a Beckman Gamma 7000 gamma counter. The supernatant (lysate) was then aliquoted such that 6xl06 cpm were used in each reaction. Anti-integrin antibodies were added to the lysate aliquots and incubated overnight at 4\u00C2\u00B0C on a tube rotator. The primary antibodies of mouse origin required the addition of a rabbit antimouse secondary antibody which was added several hours after the primary antibody. The following day, 50 pi protein A Sepharose in PBS was added to the lysate and incubated at 4\u00C2\u00B0C on a tube rotator for at least 2 h. Samples were then washed twice with RIPA containing 0.5 M NaCl and twice with RIPA (no NaCl) both containing 1.0 mM PMSF. The protein A Sepharose beads containing the antibody-integrin complexes were resuspended in 60 ul sample buffer 32 (62.5mM Tris, 10% glycerol, 2.3% SDS and bromphenol blue) and boiled for 3 min. The samples were then loaded on a 7.5% SDS-polyacrylamide gel and electrophoresed under nonreducing conditions. Once the dye front had migrated off the gel, the gel was fixed in gel fixative (10% glacial acetic acid, 37.5% methanol in dH20, and bromophenol blue) for at least 3 h with agitation. The gel was then rinsed in gel fixative without bromophenol blue for several minutes, then rinsed with dH 20 and placed on Whatman filter backing paper. The gel was then dried in a Bio-Rad model 583 Gel Dryer for 1.5 h and subjected to autoradiography using Kodak Diagnostic X-OMAT AR Film. In vitro Invasion assay Transwell membranes (Costar) were coated with type I collagen (100 (Xl/ml) for 24 h prior to the assay and kept at 4\u00C2\u00B0C . The matrigel was thawed several hours prior to the assay at 4\u00C2\u00B0C. Once thawed the matrigel was diluted 1:5 in cold PBS and mixed thoroughly by pipetting and kept on ice until ready to use. Excess type I collagen solution was pipetted off the membrane and 6.0 mm diameter, alcohol sterilized polycarbonate membranes with 12 |im pores were placed on the collagen coated membrane. The diluted matrigel (40 ul) was carefully pipetted into each well and then placed in a 37\u00C2\u00B0C, 5.0% CO z incubator for 30 min while cells were harvested. Cells, prelabelled with 0.05 mCi 3H-thymidine (20 Ci/mmol) for 24 h, were harvested with 5 mM EDTA, then washed twice in DMEM containing 0.5ml ITS+\100 ml DMEM and diluted to 5x10s cells/ml in the same medium. Medium containing cells (200 |il) was placed in the top chamber and 1 ml DMEM containing 0.5 ml ITS+M00 ml medium in the bottom chamber. Two aliquots of cells (200 p:l) were placed in 10 ml scintillation fluid and counted in a Beckman LS 6800 scintillation counter for initial counts. The transwells were incubated at 37\u00C2\u00B0C, 5.0% C0 2 33 for 24 h. Once the assay was completed media from the top and bottom chambers were pipetted off and the cells fixed in methanol for 5-10 min. The methanol was pipetted off and the top membrane carefully removed with forceps. Once the methanol had dried, the transwell membrane was cut with a sharp scalpel. The membranes were then placed in 10 ml scintillation fluid and the radioactivity measured in a scintillation counter and the percent invasion calculated using the following equation. average dpm of cells having invaded x 100 = percent cells average dpm of cells placed in top chamber Cell adhesion assay Cell attachment assays to extracellular matrix molecules were performed in 96-well, non-tissue culture, flat bottom plates (Libra). The plates were coated overnight at 4\u00C2\u00B0C with 0.156-20 Ug/ml laminin, fibronectin, vitronectin, type I collagen, type IV collagen and Bovine Serum Albumin (BSA) as a control. Two hours prior to the assay all coated wells were blocked with DMEM containing 2.5 mg/ml BSA. Cells prelabelled for 24 h with 3H-thymidine were harvested with 5 mM EDTA and washed in DMEM containing 2.5 mg/ml BSA. PC-3 cells were plated at a concentration of 4x10* cells/ well and IPC-3 at a concentration of 8xl04 cells/ well in a total of 100 pi DMEM containing 2.5 mg/ml BSA. After 1 h at 37\u00C2\u00B0C, 5.0% C 0 2 all wells were gently rinsed with PBS to remove unattached cells. Attached cells were removed with 50 ul lOmM 34 EDTA, 0.1% Triton-XlOO and the radioactivity counted in a scintillation counter. Morphology The morphology of PC-3 and D?C-3 cells on extracellular matrix proteins were studied in 96-well, flat bottom, non-tissue culture titer plates (Linbro). The plates were coated with 10 (ig/ml BSA, fibronectin, vitronectin, laminin, and collagen types I and rv overnight at 4\u00C2\u00B0C. 2 h prior to the assay, the wells were blocked with 100 ul DMEM containing 2.5 mg/ml BSA. PC-3 cells were plated at a concentration of 30,000 cells/well and IPC-3 cells at a concentration of 50,000 cells/well and then incubated at 37\u00C2\u00B0C. The cells were photographed on a Wild M40 inverted biological microscope after 24 h. Immunofluorescence PC-3 and IPC-3 cells grown in DMEM containing 10% FCS were harvested with 5 mM EDTA, washed in DMEM and then diluted to 2x10s and 4x10s cells/ml respectively in DMEM containing 10% FCS. Alcohol rinsed and heat sterilized 12 mm circular coverslips (Fisher) were placed in the bottom of 24 well plates. Medium containing cells (0.5 ml) was placed in each well and incubated at 37\u00C2\u00B0C for 48 h. The cells were gendy washed twice in PBS and fixed in 2.0% paraformaldehyde in PBS (pH 7.2) for 1 h at 4\u00C2\u00B0C. The cells were then washed twice with PBS and incubated for 5-10 min in 0.1% Triton-XlOO in PBS to permeabilize the cells. The coverslips and cells were blocked with 1% BSA in PBS for 30 min at room temperature and then washed once in PBS. The cells were covered with a 1:200 dilution of anti-integrin antibody in PBS containing 1% BSA for 1 h at room temperature. The cells were washed extensively (1 h) 35 with PBS, then incubated with a 1:100 dilution of rhodamine conjugated antibody for 1 h at room temperature, in darkness. The cells were then washed 3 times and mounted on slides (Micromaster) and sealed with clear nail polish and kept at 4\u00C2\u00B0C in the dark. Control slides were incubated for the same time periods as the treated slides with rabbit or mouse preimmune serum. Slides were photographed on a Zeiss Axiophot epifluorescence microscope. Photographs of the controls were printed such that the cells were barely visible and those of the treated cells were printed under the same light and aperture conditions as the controls. Determination of Proliferation rate PC-3 and IPC-3 cells grown in DMEM containing 10 % FCS were harvested with 5 mM EDTA and washed in DMEM containing 10% FCS. The cells were diluted to 1x10s cells/ml in DMEM containing 10% FCS. Medium containing cells (1 ml) was added to each T25 and incubated at 37\u00C2\u00B0C. Every 24 h, 3 T25 flasks of each cell line were harvested using 5 mM EDTA, resuspended in PBS and counted in a Coulter counter. Adhesion Kinetics Adhesion kinetic assays were performed in 96-well, non-tissue culture, flat bottom plates (Libro). The plates were coated with 10 ul/ml BSA, fibronectin, laminin, type IV collagen and 5 ul/ml vitronectin for 2 h at 37 \u00C2\u00B0C. Two hours prior to the assay all wells were blocked with 100 pi DMEM containing 2.5 mg/ml BSA to prevent nonspecific binding. PC-3 cells were plated at a concentration of 30,000 cells/well and IPC-3 cells at a concentration of 50,000 cells/well. Plates were then centrifuged at 1200 rpm for 1 min. At designated time points duplicate wells were 36 gently rinsed with PBS to remove unattached cells and then fixed with 3.7% paraformaldehyde in PBS. Plates were kept at 37\u00C2\u00B0C between time points. After completion of the assay the fixative was replaced with 3.7% paraformaldehyde in PBS containing 0.25% toluidine blue and allowed to stain overnight. The plates were then thoroughly rinsed with distilled water and the absorbance measured in an ELISA plate reader at 492 OD. 37 RESULTS In vitro invasion assay In order to investigate the invasive potential of tumor cells and the role of integrins in tumor cell invasion, an in vitro invasion assay using transwells (Costar) (Fig. 4) and reconstituted basement membrane, matrigel (Collaborative Research), was developed. The first problem encountered during the development of the assay was the removal of the matrigel from the transwell membrane. The matrigel remaining on the membrane with the invasive cells after the invasion assay retained the Toluidine blue, causing high background staining and making it difficult to count the cells. Another possible method to quantitate the number of cells having invaded was to prelabel them with 3H-thymidine. In this case, some cells which had not invaded or partially invaded and remained in the matrigel which could not be removed from the membrane and were counted as part of the invasive cells. A third method investigated was digesting the matrigel with Dispase (Collaborative Research). The enzyme would also digest the extracellular matrix to which the invasive cells were attached, causing them to detach during washings. In order to rectify these problems, another membrane with 12 |im pores was cut to the size of the transwell and placed over the transwell membrane (8.0 |im pores) which was previously covered with 100 ul/ml type I collagen to permit the invasive cells to attach. The in vitro invasion assay that we finally settled on is illustrated in figure 5. In the final procedure, the matrigel was diluted in cold PBS, placed in the transwell and allowed to gel at 37 \u00C2\u00B0C. Cells prelabeled for 24 h with 3H-thymidine were suspended in DMEM containing 1% ITS+ (Collaborative Research) and placed in the top chamber. The cells were allowed to invade for 24 h at 37\u00C2\u00B0C, 5.0% C0 2 . 39 Figure 5 Schematic diagram of the in vitro invasion assay using transwells and Matrigel 40 During this time, the invasive cells penetrated through the matrigel and the 12 p:m pores of the upper membrane to attach to the bottom collagen coated membrane. After fixation with methanol, the top membrane and matrigel could be easily removed with curved forceps leaving behind the bottom membrane with the invasive cells. The bottom membrane was then removed and the radioactivity counted in a scintillation counter. Aliquots of the cells placed in the top chamber were taken prior to the assay and the percent invasion calculated. The first objective after having developed the assay was to optimize the conditions for invasion. The parameters examined were (i) the number of cells, (ii) the concentration of matrigel, and (iii) the time required for the cells to invade. For these experiments, a prostate carcinoma cell line PC-3 was used. PC-3 cells are an undifferentiated cells which are known to be invasive in vitro (Albini et al., 1987).An adequate number of cells was required in the assay such that a quatitiative number of cells would invade through the matrigel and the top membrane. By varying the number of cells, an optimal cell number of lxlO5 cells/well allowed a sufficient number of cells to invade and did not result in overcrowding during invasion. Several dilutions of matrigel in PBS were also tried to determine which concentration would permit only the invasive cells to invade in a reasonable period of time. An optimal concentration of matrigel would discriminate between the invasive and non-invasive cells. The relationship between the number of cells having invaded through the matrigel and the dilution of matrigel is shown in figure 6. Stock matrigel (8.8 mg/ml) and a 1:2 dilution did not permit any cells to invade, even after 48 h, while concentrations of less than 1 mg/ml did not gel properly and permitted a large number of cells to invade. A dilution of 1:5 (1.75 mg/ml) was optimal, allowing a reasonable number of cells to invade during a 24 h incubation period. 41 Figure 6 Relationship between matrigel concentration and invasion of PC-3 cells 1x10s cells were placed in each well with varying concentrations of matrigel and allowed to invade for 24 h at 37\u00C2\u00B0C, 5.0% C0 2 . The cells were then fixed and stained with methanol containing 0.5% toluidine blue. The cells were counted visually using a Zeiss inverted microscope. Matrigel Concentration vs invasion No. of cells 1000 i \u00E2\u0080\u0094 Matrigel cone mg/ml 43 Invasion of PC-3 and IMR90 Cells Once the conditions of the invasion assay were optimized it was necessary to compare different cell lines and verify the ability of the invasion assay to discriminate between invasive and non-invasive cells. An undifferentiated malignant prostate carcinoma cell line, PC-3 and a normal human lung fibroblast cell line, IMR90, were assayed simultaneously as described in the Materials and Methods. Although the actual percent of invasive cells varied between experiments, a higher percentage of PC-3 cells invaded through the matrigel in every case (Table 2). Since normal fibroblasts are able to migrate, the small degree of invasion observed with IMR90 was expected. Invasion of HOS and MNNG-HOS cells After having developed and optimized the conditions of the invasion assay, two osteosarcoma cell lines with known in vivo potential were assayed. The human osteosarcoma cell line (HOS) is a non-tumorigenic tumor cell line which is unable to form tumors in nude mice (Rhim et al.,1975). Its chemically transformed counterpart, MNNG-HOS, is very tumorigenic and is able to form tumors in nude mice. The two cell lines were assayed simultaneously under the same conditions described for PC-3 and IMR90 cells. Although there was some interassay variability MNNG-HOS cells were consistantly and considerably more invasive than HOS cells (Table 3). 44 Table 2 Invasion of PC-3 and IMR90 cell lines in vitro percent invasion cells experiment 1 experiment 2 PC-3 3.70 \u00C2\u00B1 0.80 1.54 \u00C2\u00B10.21 IMR90 0.38 + 0.14 0.58 + 0.12 Values are stated as percent invasion \u00C2\u00B1 standard error. Difference in invasiveness between PC-3 and IMR90 cells are statistically significant (p<0.05) as determined by the students T-test In all experiments n=4 Table 3 Invasion of osteosarcoma cell lines, HOS and MNNGHOS Percent cells invaded Experiment HOS MNNG-HOS IMR90 1 1.71 \u00C2\u00B1 0.84 4.65 \u00C2\u00B1 1.2 0.70 \u00C2\u00B10 .16 2 4.97 \u00C2\u00B1 1.71 8.90 \u00C2\u00B1 1.62 0.82 \u00C2\u00B1 0.20 3 1.81 \u00C2\u00B1 1.31 6.79 \u00C2\u00B1 1.05 0.59 \u00C2\u00B10 .13 4 1.87 \u00C2\u00B1 0.43 2.94 \u00C2\u00B1 0.85 \u00E2\u0080\u0094 Values are given in percent invasion \u00C2\u00B1 standard error Difference in invasion between HOS and MNNG-HOS cells are statistically significant (p<0.05) as determined by the Mann-Whitney U test. In all experiments n=4 46 Expression of integrins on HOS and MNNG-HOS Cells Having observed a difference in the invasive potential between HOS and MNNG-HOS, the expression of integrins was investigated on the two cell lines. The integrins were immunoprecipitated using anti-integrin antibodies. Immunoprecipitations with the anti-6, monoclonal antibody generated two bands, a 110 kDa band (B( subunit) and a 150 kDa band containing the Oj, ct,, 0%, and a 6 subunits. Both bands were present in both cell lines but were upregulated in MNNG-HOS cells (Fig. 7a, lanes 1 and 2). Anti-ct, monoclonal antibody generated similar amounts of the 110 kDa Bx and 150 kDa fx, subunits in both cell lines (lanes 3 and 4). Neither HOS nor MNNG-HOS cells expressed high levels of the a 5 subunit in immunoprecipitations using anti-oc5 monoclonal antibodies (lanes 5 and 6). Immunoprecipitations using anti-o^ monoclonal antibodies demonstrated that MNNG-HOS cells upregulated the 140 kDa Og subunit compared to the HOS cells (lanes 7 and 8). The 180 kDa a, subunit was also upregulated in MNNG-HOS cells when immunoprecipitated with anti-oc! monoclonal antibodies (lanes 9 and 10). MNNG-HOS cells also upregulated the 150 kDa subunit in immunoprecipitations using anti-Oj monoclonal antibodies (lanes 11 and 12). Immunoprecipitations with anti-vitronectin receptor polyclonal antibody generated 3 bands. A 160 kDa ov subunit, its 97 kDa associated B3 subunit and the Bt subunit which cross reacts with the antibody. MNNG-HOS cells strongly downregulated the vitronectin receptor, especially the B3 subunit (Fig. 7b, lane 2). To investigate the role of the integrins, in particular the integrins of the Bj family in the invasion process through matrigel, antibodies directed against the integrin subunits were incubated with the cells prior to and during the invasion 47 Figure 7a,b Autoradiograph of a 7.5% sodium dodecyl sulfate polyacrylamide gel. Integrins from 1 2 5I surfaced labeled HOS (odd numbered lanes) and MNNG-HOS (even numbered lanes) cells were immunoprecipitated under non-reducing conditions with anti-B^ lanes 1 and 2; anti-o ,^ lanes 3 and 4; anti-a5, lanes 5 and 6; anti-o ,^ lanes 7 and 8; anti-al5 lanes 9 and 10; Oj, lanes 11 and 12 and anti-VNR (Fig. 7b, lanes 1 and 2). After electrophoresis the gel was fixed and stained in gel fixative containing Coomassie blue then dried and exposed to Kodak diagnostic film. 48 quK 1 2 3 4 5 6 7 8 9 10 11 12 2\u00C2\u00B0\u00C2\u00B0\" 41 \u00E2\u0080\u0094a, :g,2.3>5 K 200 -49 assay. A polyclonal antibody to the fibronectin receptor (directed to the Bj subunit) and a monoclonal antibody directed to the 0% subunit (GoH3) of were preincubated for 20 min with the cells prior to the assay and were present during the assay. Table 4 shows the invasion of HOS and MNNG-HOS cells in the presence of anti-integrin antibodies. The invasion of HOS cells was inhibited approximately 46% and MNNG-HOS cells 36% in the presence of a polyclonal anti-fibronectin receptor (1:50 dilution) as compared to controls using rabbit preimmune serum. The integrin laminin receptor oCgBj was of particular interest because it was strongly upregulated on the more invasive MNNG-HOS cells. Preincubation of HOS and MNNG-HOS cells with the monoclonal antibody GoH3 (1:2 dilution) inhibited invasion of HOS cells by approximately 70% but had no inhibitory effect on the invasion of MNNG-HOS cells. Suspecting that there was not sufficient antibody present to affect receptor-ligand interactions, we concentrated the antibody five fold and repeated the experiment. After concentration of the GoH3 antibody the invasion of MNNG-HOS was reduced by 41% (Table 4). Isolation of an invasive cell line (IPC-3) In order to study the properties of invasive cells, it is important to obtain a homogenous population of invasive cells. The in vitro invasion assay serves a dual purpose in that the cells having invaded through the matrigel can be easily isolated from the non- invasive cells and recultured. A more invasive cell line from the prostate carcinoma cell line, PC-3, was isolated using the in vitro invasion assay. After a 36 h incubation period, the membrane containing the matrigel and non-invasive cells was removed leaving the membrane containing the invasive cells. 50 Table 4 Inhibition of invasion of HOS and MNNGHOS using ocFNR and anti-ac antibodies Cells % cells invaded % inhibition HOS Contol 5.0 \u00C2\u00B1 1.2 \u00E2\u0080\u0094 Anti-FNR antibody (1:50) 2.7 \u00C2\u00B1 0 . 8 46 Anti-a6 antibody (1:2) 1.5 \u00C2\u00B1 0.4 70 MNNG-HOS Control 8.1 \u00C2\u00B1 1.5 --Anti-FNR antibody (1:50) 5.2 \u00C2\u00B1 1.1 36 Anti-a6 antibody (1:2) 8.3 \u00C2\u00B1 1.7 ~ Anti-Og antibody (5 fold more concentrated) 4.8 \u00C2\u00B11 .2 41 Values are given as percent invasion \u00C2\u00B1 standard error The difference in invasive potential between control and treatment groups were statistically significant (p<0.05) as determined by the Mann-Whitney U test. In all experiments n=4 51 The membranes were cut from the transwell and placed in DMEM containing 10% FCS and allowed to grow at 37\u00C2\u00B0C, 5% C0 2 . Once a sufficient number of cells was obtained they were assayed again and those having invaded recultured as before. After three successive passages through the matrigel, a more invasive cell line called IPC-3 was obtained. The difference in the morphology and size of the parent PC-3 and more invasive IPC-3 cell lines in tissue culture was studied (Fig. 8). The more invasive cells were smaller in size and remain spherical in shape (Fig. 8b) as compared to the PC-3 cells which were flatter and spindle or triangular shaped (Fig. 8a). When both cell lines were assayed simultaneously, the invasive IPC-3 cells were several times more invasive than the parent PC-3 cells. However, after several weeks in tissue culture the IPC-3 cells gradually lost their invasive potential and became similar to the PC-3 cells (Table 5). Because the IPC-3 cells initially behaved different from the PC-3 cells therefore the possibility of cell contamination was investigated. A fresh freezer stock of PC-3 cells (passage 8) was cultured and assayed under the same conditions. A cell line similar in morphology, growth rate, and integrin expression was isolated a second time (Fig. 8c). Growth rate of PC-3 and IPC-3 cells Typical growth curves for PC-3 and IPC-3 cells are shown in Figure 9. On subculturing, both PC-3 and IPC-3 cells started in a lag phase followed by an exponential phase. IPC-3 cells began the exponential phase sooner than PC-3 and remained in log phase for the 7 days recorded. PC-3 cells had a smaller log phase and started to level off after as they became confluent. The doubling time for PC-3 cells during log phase was 20 h and 28 h in prelog phase. IPC-3 cells had a slightly shorter doubling time of 22 h during log phase and 24 h during prelog phase. 52 Figure 8 Photograph of PC-3(a), the first IPC-3 isolate(b) and the second isolate (c). The cells were cultured in DMEM containing 10% FCS. magnification 40x 54 Table V Difference in invasive potential between PC-3 and IPC-3 Weeks after isolation % invasion PC-3 % invasion IPC-3 ratio 2 0.64 + 0.12 5.4+1.29 8.4 4 0.32 \u00C2\u00B1 0.06 1.1 \u00C2\u00B10 .12 3.4 10 1.98 \u00C2\u00B1 0.73 3.55 \u00C2\u00B1 0.91 1.8 14 1.51 \u00C2\u00B1 0.21 2.35 \u00C2\u00B1 0.60 1.6 Values are given in percent invasion \u00C2\u00B1 standard error In all experiments n=4 55 Figure 9 Growth curves for PC-3 and IPC-3 cells PC-3 and IPC-3 cells were plated at a concentation of 1x10s cells/T25 in DMEM containing 10% FCS and incubated at 37\u00C2\u00B0C, 5.0% CO z . Three flasks of each cell line were harvested every 24 h and counted in a Coulter counter. 56 PROLIFERATION RATE OF PC3 & IPC3 57 Adhesion of PC-3 and IPC-3 cells to extracellular matrix proteins The adhesion of tumor cells to the extracellular matrix is the first step in the invasion process. Therefore the adhesion of PC-3 and IPC-3 cells to fibronectin, vitronectin, laminin, and collagens type I and IV was investigated (Figs. 10-14). PC-3 and IPC-3 did not adhere well to concentrations of fibronectin below 1.25 fig/ml However, at concentrations greater than 2.5 |ig/ml the adhesion of PC-3 cells to fibronectin was greater than that seen with IPC-3 cells (Fig. 10). The adhesion of IPC-3 cells to vitronectin was greater than PC-3 at concentrations below 2.5 (ig/ml but both cell lines had similar adhesion profiles at protein concentrations between 5 (ig/ml and 20 |ig/ml (Fig. 11). There was very little difference in the adhesion of PC-3 and IPC-3 cells to laminin at all concentrations assayed (0.156-20 ug/ml) with the exception of 1.25 (ig/ml where a greater percentage of PC-3 cells attached to the substrate (Fig. 12). Both PC-3 and IPC-3 cells attached well to collagens type I and IV. For both sustrates a greater percentage of PC-3 cells adhered than IPC-3 cells at all protein concentrations studied (Figs. 13 and 14). Both PC-3 and IPC-3 reached maximal binding between 5-10 |ig/ml for fibronectin, laminin and collagen types I and IV while on vitronectin they bound maximally at approximately 2.5-5.0 ug/ml. Comparing the adhesion of PC-3 to the five extracellular matrix components assayed (Fig. 15), a greater percentage of PC-3 cells attached to type IV collagen at very low concentrations. At concentrations above 2.5 ug/ml, PC-3 cells had similar adhesion curves for all five substrates. A greater percentage of IPC-3 cells attached to vitronectin at low concentrations and to laminin at substrate concentrations above 5.0 |ig/ml (Fig. 16). Fewer IPC-3 cells attached to fibronectin at both high and low protein concentrations. 58 Figure 10 Adhesion of PC-3 and IPC-3 to fibronectin 96 well, flat bottom, microtiter plates were coated with two fold serial dilutions of fibronectin (20-0.156 ug/ml). Cells, prelabeled with 3H-thymidime were plated at a concentration of 4xl04 (PC-3) and 8X104 (IPC-3) cells/well, then incubated at 37\u00C2\u00B0C, 5.0% CO z for 1 h. Attached cells were removed with 50 pi 10 mM EDTA, 0.1% Triton-XlOO and the radioactivity counted in a B-scintillation counter. Figure 11 Adhesion of PC-3 and IPC-3 to vitronectin 96 well, flat bottom, microtiter plates were coated with two fold serial dilutions of vitronectin (20-0.156 ug/ml). Cells, prelabeled with 3H-thymidime were plated at a concentration of 4x10* (PC-3) and 8xl04 (IPC-3) cells/well, then incubated at 37\u00C2\u00B0C, 5.0% C0 2 for 1 h. Attached cells were removed with 50 pi 10 mM EDTA, 0.1% Triton-XlOO and the radioactivity counted in a B- scintillation counter. IPC-37PC-3 ADHESION ON FIBRONECTIN \u00E2\u0080\u0094 BSA-IPC-3 BSA-PC-3 PC-3 IPC-3 PERCENT ADHESION 100 | PROTEIN CONCENTRATION (ug/ml) PC-3/IPC-3 ADHESION TO VITRONECTIN PERCENT ADHESION -* - * \u00E2\u0080\u0094 f +D * T ^ 0 5 10 15 20 25 PROTEfJ CONCENTRATION (ug/ml) 60 Figure 12 Adhesion of PC-3 and IPC-3 to laminin 96 well, flat bottom, microliter plates were coated with two fold serial dilutions of laminin (20-0.156 ug/ml). Cells, prelabeled with 3H-thymidime were plated at a concentration of 4x10* (PC-3) and 8xl04 (IPC-3) cells/well, then incubated at 37\u00C2\u00B0C, 5.0% C0 2 for 1 h. Attached cells were removed with 50 |il 10 mM EDTA, 0.1% Triton-XlOO and the radioactivity counted in a B- scintillation counter. Figure 13 Adhesion of PC-3 and IPC-3 to type I collagen 96 well, flat bottom, microtiter plates were coated with two fold serial dilutions of type I collagen (20-0.156 Jig/ml). Cells, prelabeled with 3H-thymidime were plated at a concentration of 4xl04 (PC-3) and 8x10\" (IPC-3) cells/well, then incubated at 37\u00C2\u00B0C, 5.0% CO z for 1 h. Attached cells were removed with 50 ul 10 mM EDTA, 0.1% Triton-XlOO and the radioactivity counted in a B-scintillation counter. PC-3/IPC-3 ADHESION TO LAMININ - \u00E2\u0080\u0094 BSA-IPC-3 BSA-PC-3 ~*~ IPC-3 -a- PC-3 PERCENT ADHESION 120 r\u00E2\u0080\u0094 0 5 10 15 20 25 PROTEN CONCENTRATION (ug/ml) PC-3/IPC-3 ADHESION TO TYPE I COLLAGEN 62 Figure 14 Adhesion of PC-3 and IPC-3 to type IV collagen 96 well, flat bottom, microtiter plates were coated with two fold serial dilutions of type IV collagen (20-0.156 Hg/ml). Cells, prelabeled with 3H-thymidime were plated at a concentration of 4 X 1 0 4 (PC-3) and 8 X 1 0 4 (IPC-3) cells/well, then incubated at 37\u00C2\u00B0C, 5.0% CO z for 1 h. Attached cells were removed with 50 ul 10 mM EDTA, 0.1% Triton-XlOO and the radioactivity counted in a B-scintillation counter. PC-3/IPC-3 ADHESION TO TYPE IV COLLAGEN BSA-IPC-3 PERCENT ADHESION \u00E2\u0080\u00A2+- BSA-PC-3 - * - IPC-3 - S - PC-3 10 15 PRO TEN CONCENTRATION "Thesis/Dissertation"@en . "10.14288/1.0098671"@en . "eng"@en . "Pathology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Analysis of integrins and cell adhesion on invasive tumor cell lines using an in vitro invasion assay"@en . "Text"@en . "http://hdl.handle.net/2429/30317"@en .