Collagenase is Expressed by Rabbit VX2 Tumour Cells in Syngeneic and Xenogeneic Hosts

Matrix Vol. 911989, pp. 206-213

Collagenase is Expressed by Rabbit VX2 Tumour Cells in Syngeneic and Xenogeneic Hosts JELENA GAVRILOVIC 1, ROSALIND M. HEMBRY, JOHN J. REYNOLDS and GILLIAN MURPHY Cell Physiology Department, Strangeways Research Laboratory, Cambridge, CB14RN, UK.

Abstract Specific antisera for the connective tissue metalloproteinases, collagenase, gelatinase (type IV collagenase) and stromelysin were used to study their respective localizations in both rabbit primary VX2 tumours and in lung metastatic deposits (frozen immediately after excision). Collagenase was found within some cells of the primary tumour and also bound to the extracellular matrix at discrete sites. Previous studies suggest that this matrix staining represents active enzyme. Stromelysin and gelatinase had a more limited distribution, particularly the latter, but both showed cell and matrix staining. In the lung metastases collagenase and strom ely sin occurred less frequently, although both cell and matrix staining were observed; gelatinase was not seen. When rabbit VX2 cells were transplanted into nude mice they grew as a discrete nodule. Cells within this nodule stained with the antiserum to collagenase, which recognizes rabbit but not mouse enzyme, and thus demonstrated that cells of tumoural origin synthesize collagenase in vivo. Stromelysin was also co-localized with collagenase in some tumour cells. Key words: collagenase, metalloproteinase, TIMP, tumour.

Introduction Tumour invasion and metastasis have long been associated with remodelling of the extracellular matrix and proteinases of several classes have been implicated in these processes. In particular serine proteinases, including plasminogen activators and plasmin, and a family of metalloproteinases (MPs 2 ) are strongly implicated in a cascade leading to tissue resorption (Liotta, 1984; Woolley, 1984; Mignatti et al., 1986; Tryggvason et al., 1987).

1 Present address: Laboratoire de physiopathologie du developpement, Ecole Normale Superieure, 46, rue d'Ulm, 75230 Paris Cedex OS, France. 2 List of abbreviations: MP, metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; DMEM, Dulbecco's modification of Eagle's Medium; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate.

© 1989 by Gustav Fischer Verlag, Stuttgart

Invasion of the stroma by primary tumour cells is likely to involve the MP, collagenase, since this proteinase specifically degrades interstitial collagens under physiological conditions. Two studies using the amnion invasion system have concluded that collagenase is involved in tumour invasion in vitro, by the use of anti-collagenase antibodies and TIMP, the specific tissue inhibitor of MPs (Thorgeirsson et al., 1982; Mignatti et al., 1986). It has also been shown that collagenase can be directly extracted from tumours (McCroskery et al., 1975; Wirl, 1979) and collagenase and TIMP have been immunolocalized in tumour tissue (Woolley, 1982; Childers et al., 1987). Untransformed cells in culture do not synthesize collagenase constitutively but under appropriate stimulation this enzyme can be a major secreted product of many cell types including fibroblasts (McGuire et al., 1982) and chondrocytes (Trechsel et al., 1982). Some tumour cells synthesize collagenase without stimulation and primary rabbit VX2 tumour tissue has been shown by McCroskery et al. (1975) to contain readily

Collagenase in VX2 Tumours In Vivo extractable levels of this MP. Using a specific anti-collagenase antiserum (Hembry et al., 1986) we showed that in an in vitro model system cells derived from the primary VX2 carcinoma degrade type I collagen films by a collagenase-mediated mechanism (Gavrilovic et al., 1985). Two other MPs, stromelysin and gelatinase (also known as type IV collagenase), may also be important in tumour invasion, particularly because of their ability to degrade many basement membrane components (Galloway et al., 1983 ; Murphy et al., 1985): VX2 cells in culture synthesize these MPs (Gavrilovic, 1987). Stromelysin mRNA levels have been shown to increase following oncogenic transformation of rat embryo fibroblast cells and to be expressed more abundantly in invasive mouse skin squamous cell carcinomas than in benign papillomas (Matrisian et al., 1986a, b). Gelatinase, which has long been described as a connective tissue MP, and type IV collagenase, which was thought to be a tumour specific enzyme, were recently demonstrated to be identical by a comparison of sequences of these enzymic activities from human fibroblasts and transformed human epithelial cells (Collier et al., 1988 ). This work has been confirmed by studies by the present authors (Murphy et al., 1989 a). The actions of the MP family are in many situations regulated by TIMP (Murphy et al., 1981) which is a tightbinding inhibitor produced by many connective tissue cells in culture, and by some cells in vivo, in particular endothelial cells (Hembry and Ehrlich, 1986). Little is known of the distribution of TIMP within tumours, but evidence has been provided that invasive meningiomas produce lower amounts of TIMP than their less invasive counterparts (Halaka et al., 1983 ). We have previously shown, however, that VX2 cells in culture do not produce detectable TIMP activity (Gavrilovic et al., 1985) and do not synthesize TIMP (Gavrilovic, 1987). A major unanswered question in this field is whether tumour cells themselves produce MPs in vivo or whether their role is mainly to stimulate their production by host cells. In this paper we have used antisera to collagenase, stromelysin, gelatinase and TIMP to investigate the distribution of these proteinases and their endogenous inhibitor in the primary VX2 tumour grown in rabbits, and within metastatic deposits in rabbit lungs. By virtue of the species-specificity of the anticollagenase antiserum we have also been able to investigate the production of MPs by rabbit VX2 cells growing in nude mice.

Methods VX2 tumuur The rabbit VX2 carcinoma was transplanted in the thigh muscle of New Zealand White rabbits as described (Gavrilovic et al., 1985). Primary tumour tissue was dissected from three animals which had carried the tumour for

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between 2 and 3 weeks. Lungs with visible metastatic deposits were dissected from two rabbits. Two nude mice (DBA nu/nu, 3 months old, obtained from the National Institute for Medical Research, Mill Hill, London) were barrier maintained and injected subcutaneously in the flank with 105 primary VX2 cells from teased primary rabbit VX2 tumour tissue. The mice were killed after 18 days and 5 weeks respectively. Tissue was frozen in liquid nitrogen immediately after excision. Some of the primary rabbit tissue and metastatic lung deposits were also cultured in Dulbecco's modification of Eagle's Medium (DMEM) with monensin (5 11M, Sigma) for 16 h before freezing to allow intracellular accurriulation of enzymes and TIMP (Hembry et al., 1986). A portion of the nude mouse tissue was cultured similarly for 3 h with monensin before freezing.

Immunofluorescence Frozen tissue blocks (primary tumour or lungs with metastases in the rabbit, or tumour in the mouse) were sectioned on a cryostat at 7 Ilm, and the sections fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1 % Triton X-100 in PBS and stained as described (Hembry et al., 1986). In addition, the sections were treated for 10 min with 4-chloronaphthol (2.8 mM in methanol/PBS with 0.01 % HzOz) to prevent non-specific binding of fluorescein to eosinophils (Chowcat et al., 1988 ). Control sections were then stained with normal sheep serum IgG (50 Ilg/ml) and experimental sections with IgG (5 0 Ilg/ml) of either sheep anti-rabbit collagenase (Hembry et al., 1986), anti-rabbit stromelysin (Murphy et al., 1986), anti-rabbit ge1atinase (Murphy et al., 1989b) or anti-rabbit TIMP (Gavrilovic et al., 1987). After repeated washing, sections were incubated with the fluorescein-labelled second antiserum, pig anti-sheep Fab'FITC (Hembry et al., 1985), rewashed and nuclei counterstained with methyl green (1 mg/ml, 2 min). Some sections of primary VX2 tumour in nude mice were stained simultaneously for collagenase and stromelysin using directly conjugated antibodies as described previously (Murphy et al., 1986): the anti-collagenase antibody was labelled with tetramethylrhodamine isothiocyanate (sheep anti-collagenase-Fab'-TRITC) and the antistromelysin antibody was labelled with fluorescein isothiocyanate (sheep antistromelysin-IgG-FITC). Sections were viewed by fluorescence microscopy on a Zeiss Photomicroscope III and photographs taken on Kodak Ektachrome 400 ASA film uprated during processing to 1600 ASA. Sections were subsequently stained with Harris' haematoxylin and eosin and photographed on Kodak Ektachrome 50 film. The complete characterization of each of the antisera used, including species specificity, Western blots, inhibition curves and immunoabsorption experiments with purified antigen, are given in detail in each of the above references.

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Fig. 1. Collagenase in primary VX2 tumour in rabbit. Sections of primary rabbit VX2 tumour were fixed, permeabilized and stained with sheep anti-rabbit collagenase IgG followed by FITC-labelled pig anti-sheep Fab'. Nuclei were counterstained with methyl green. Some cells contain intracellular fluorescence (arrows) and active collagenase was also visible on some strands of collagenous matrix. Bar, 20 !-tm.

To study species cross-reactIvIty of the antisera Swiss mouse 3T3 cells stimulated with interleukin-Ia and phorbol 12-myristate 13-acetate and mouse osteoblasts stimulated with 1,25-dihydroxy vitamin D3 were used in immunolocalization studies (with monensin for the last 3 h and fixed and permeabilized as described above). Previous experience had shown that antisera cross-reactivity varied markedly according to the technique used (inhibition, Western blots, etc.) and that it is very important to carry out the analysis by immunolocalization under the same conditions. The 3T3 cells synthesized 0.1 units of collagenase, 0.2 units stromelysin and 2 units gelatinase per 105 cells in 24 h and the osteoblasts made 3.3 units of collagenase, 1.3 units stromelysin and 7.3 units gelatinase per 105 cells in 24 h. The antiserum to rabbit collagenase did not detect mouse collagenase, but the antisera to rabbit stromelysin and gelatinase detected the corresponding mouse enzymes, apparently concentrated in Golgi vesicles in both cell types.

Fig. 2. Stromelysin in rabbit lung metastasis. Sections of VX2 metastatic deposits within rabbit lungs were stained as in Fig. 1, but using sheep anti-rabbit stromelysin IgG as primary antibody. The central cell has bright intracellular fluorescence and a pleiomorphic nucleus. Bar, 10 !-tm.

Results

Primary VX2 tumour in rabbits

Sections of primary VX2 tumour stained with the antiserum to collagenase showed tumour cells with bright intracellular green fluorescence, indicating production of the enzyme (Fig. 1). Some areas of matrix also fluoresced, consistent with collagenase bound to matrix collagen (Hembry et aI., 1986). The positively stained cells were randomly distributed throughout the tumour mass, not necessarily adjacent to fluorescent matrix. Numbers of positive cells varied from area to area: overtly necrotic areas contained no positive cells but did have the occasional area of positively stained matrix. Adjacent sections stained with the antiserum to rabbit stromelysin showed a few cells with clear intracellular fluorescence scattered throughout the explant. There were generally fewer cells positive for stromelysin than for collagenase. Areas of fluorescent matrix were rare; usually one

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epidermis

hypodermis and muscle

tumour

A

8

Fig.3. Haematoxylin and eosin section of VX2 tumour in the nude mouse. The frozen section was stained with Harris' haematoxylin and eosin after immunostaining. a. Low power view of section . Bar, 0.2 mm. b. High power view of area outlined in Fig. 3 a, taken at same magnification as Figs 2,4 and 5. Tumour cells are large with pleiomorphic nuclei and bordered by a collagenous capsule of host tissue. Bar, 10 11m.

strand or less per section and less bright than that seen with collagenase. In sections stained for gelatinase (type IV collagenase), positive cells were rarely seen. Small areas of fluorescent matrix were present, but frequency varied considerably from section to section.

No tumour cells stained positively for TIMP in any of the sections, but where blood vessels were present the endothelial cells stained weakly positive. Incubation of the tissue in DMEM with monensin for 16 h, to block secretion and cause intracellular accumulation of enzyme, only increased the frequency of positive cells in the case of gelatinase, but

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the increase was only slight. Sections stained with nonimmune sheep IgG were always negative.

Metastatic deposits in rabbit lungs Sections of rabbit lung metastases stained with the antiserum to collagenase contained positive cells with intracellular fluorescence randomly distributed throughout the tumour mass, but less frequently than in the primary tumour. Positive cells were frequently large, with pleiomorphic nuclei. Matrix staining was rarely seen and no positive cells were seen within surrounding normal lung tissue. Sections stained with the antiserum to stromelysin also contained positive cells, but there were generally fewer than in the adjacent collagenase section. Again, positive cells were frequently large with pleiomorphic nuclei (Fig. 2) and matrix staining was infrequent. In sections stained with the antiserum to gelatinase no positive cells were seen nor was there matrix staining. In sections stained with anti-TIMP only blood vessel endothelial cells stained weakly. Sections stained with non-immune IgG were negative.

VX2 tumour transplanted into nude mice The tumour tissue grew as a discrete subcutaneous nodule within both nude mice (Fig. 3 a, b). In the mouse killed at 18 d the tumour tissue appeared as a mass of viable cells with little interstitial matrix, whereas in the mouse killed at five weeks the tumour mass had small foci of necrosis and interstitial collagenous matrix was present. The hypodermis in both animals contained many eosinophils. Sections of tumour and mouse skin incubated with the antiserum to rabbit collagenase contained positive cells with intracellular fluorescence within the tumour mass (Fig. 4). They appeared randomly distributed and numbers varied from area to area and from section to section. In the mouse killed at five weeks some, but not all, of the interstitial collagenous matrix within the tumour mass had bright fluorescence (Fig.4) , resulting in a picture similar to that seen in the primary VX2 tumour in the rabbit. No staining of cells or matrix components was seen in mouse epidermis, dermis or hypodermis. Adjacent sections stained with the antiserum to stromelysin also contained positive cells; at both time points these cells were only within the tumour mass and fewer in

number than seen in the collagenase-stained sections. There was no staining whatsoever of the host mouse tissue. Sections stained with the antisera to gelatinase and TIMP, and normal sheep serum, were always completely negative. As positive staining cells were seen within the tumour mass with both the anti-collagenase and the anti-stromelysin antisera, two questions arose: were separate cells secreting each enzyme or was secretion of both enzymes coordinate? Consequently dual localization experiments were carried out, incubating sections with both primary antisera together, as described in the Methods section. In tissue frozen directly, some cells were seen to have both rhodamine and fluorescein intracellular staining, indicating that they were synthesizing both collagenase and stromelysin simultaneously, but other cells contained only one fluorophore or none. However, the intensity of the staining was low and difficult to photograph (because the primary antiserum was labelled) so the experiment was repeated on tissue that had been incubated for 3 h in DMEM with monensin, to cause intracellular accumulation of secreted protein. Again, some tumour cells contained both fluorophores (Fig. 5 a, b) whereas others contained only one or none. The monensin treatment did not increase the overall number of positive cells, but merely increased the intensity of intracellular fluorescence.

Discussion By use of an anti-collagenase antiserum, which recognizes rabbit but not mouse collagenase, we have been able to show that rabbit VX2 tumour cells growing within nude mice synthesize collagenase. These cells were confined to a discrete structure and appeared completely characteristic of tumour cells, with large pleiomorphic nuclei quite distinct from host cells (Fig. 3 b). The frequency of normal rabbit cells was negligible. Stromelysin was only localized within the tumour mass and was also likely to be tumorigenic in origin (Fig. 5). Previous studies by Woolley (1982) have revealed the presence of collagenase in human melanoma tissue but in this case the cellular source of the enzyme could not be determined. Our observations do not exclude the possibility that host cells may in some circumstances also be induced to synthesize collagenase as suggested by Biswas et al. (1982). It is of interest to note that Childers et al. (1987) were not able to detect any cells with intracellular collagenase staining in human basal cell carcinoma. The finding

Fig. 5. Dual localization of both collagenase and stromelysin. ~ Sections of VX2 tumour grown within a nude mouse were fixed, permeabilized and stained with sheep anti-collagenase-Fab ' -T RITC and sheep anti-stromelysin-IgG-FITC in one incubation. a: Section viewed through FITC filter: the central cell has positive intracellular fluorescence while the rest are negative. Bar, 10 ~m. b: Section viewed through TRITC filter: the same cell has rhodamine fluorescence indicating that it is synthesizing both collagenase and stromelysin simultaneously. Bar, 10 ~m.

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.... Fig. 4. Collagenase in VX2 tumour in the nude mouse. Sections of tumour underlying epidermis and dermis were stained as in Fig. 1. Both cells synthesizing collagenase (arrows) and collagenase on collagenous matrix are visible. Bar, 10 ~m.

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of collagenase staining in VX2 cells in lung metastases of matrix, in contrast to the close apposition of collagenase rabbits indicate that synthesis of this MP may be involved in and TIMP synthesis seen in wound-healing (Chowcat et al., local invasion of the secondary metastatic site. Although 1988) and in remodelling in the growth plate (Brown et al., stromelysin has significant activity on basement membrane 1989). However, as yet no specific factors having such an components we did not find this proteinase in large effect have been defined. amounts in lung metastatic deposits. Collagenase, stromelysin and gelatinase (type IV collaIt is intriguing that only a relatively small proportion of genase) are synthesized and secreted from cells as prothe tumour cell population was observed to synthesize col- enzymes requiring extracellular activation (reviewed by lagenase and stromelysin. Although some cells made both Tryggvason et al., 1987). Thus a prerequisite for the proteinases, their localization was not always coincident. involvement of MPs in matrix degradation in tumour invaWe have previously shown that chondrocytes stimulated sion in vivo is their detection in an active form: our observawith mononuclear cell cytokines undergo an apparent co- tions of staining of matrix with the anti-collagenase antiordinate increase in the appearance of collagenase and body provide such evidence. We and others have previously stromelysin in the culture media. However, analysis of the shown that active but not latent collagenase binds to collacells by immunolocalization techniques (Murphy et al., gen fibres in vitro, including under the conditions used in 1986) showed that although many cells were making both immunolocalization studies (Welgus et aI., 1985; Hembry enzymes, this was not always the case. Hence, differential et al., 1986). The ability of TIMP to bind collagenase on regulation of enzyme expression can occur at the level of collagen, or for preformed enzyme-inhibitor complexes to bind to collagen requires further study. Woolley (1982) and individual cells. The very low level of gelatinase (type IV collagenase) Childers et al. (1987) have also observed collagenase bound expression we have observed in VX2 cells contrasts mar- to matrix in vivo and we have now shown that forms of kedly with the observations of Liotta, Tryggvason and gelatinase and stromelysin bind to matrix components. colleagues (reviewed in Liotta, 1984; Tryggvason et al., Whether these are latent, active or inhibitor complexed 1987) in which expression of this enzyme by mouse B16 species has not yet been established. Since gelatinase has melanoma cell lines apparently correlates with metastatic synergistic activity with collagenase on insoluble tendon potential. The expression of MPs by a tumour may reflect collagen in vitro (Murphy et al., 1985) it would be interestits collagenous nature, predominantly collagenase and ing to determine whether collagenase and gelatinase costromelysin being produced as part of the remodelling pro- localize on collagen. The model system we describe has allowed us to detercesses associated with growth. Collier et al. (1988) recently showed that gelatinase was expressed by a number of trans- mine unequivocally the tumour cell source of collagenase formed human cell lines as well as normal skin fibroblasts. and stromelysin in vivo. This system can now be exploited The precise role of gelatinase is still uncertain, although it is to determine precisely the individual roles of MPs in likely to be involved in collagen turnover as a collagenase tumour invasion and metastasis since both antisera and "helper" enzyme and its ability to specifically cleave type IV cDNA probes are available. collagen suggests a further role · in invasive processes. Gelatinase may be expressed in some tumours for this Acknowledgements purpose, whilst others may use alternative systems such as We thank Anne McGarrity for her work on the characterization cysteine proteinases (Baici et al., 1984; Sloane et al., 1986) of the antisera, Chris Green for the prints, Barry Halls and Chrisor membrane proteinases (Chen and Chen, 1987). It is pos- tine Tilley for animal care, and the Medical Research Council, UK sible that such enzymes would be expressed only transiently for financial support. by cells capable of metastasis as described by Liotta (1984) for type IV collagenase. Baici et al. (1984) have described References both collagenase and a cathepsin B-like cysteine proteinase in association with the rabbit VX2 carcinoma, the latter Baici, A., Gyger-Marazzi, M. and Strauli, P.: Extracellular cysteine being localised at the invasive front. A more detailed study proteinase and collagenase activities as a consequence of tumor of the VX2 tumour over the time of development might give host interaction in the rabbit V2 carcinoma. Invasion Metasa better indication of the expression of gelatinase or the tasis4: 13-27,1984. other metalloproteinase by tumour cells at the invasive Biswas, c., Bloch, K.J. and Gross, ].: Collagenolytic activity of rabbit V2 carcinoma implanted in the nude mouse. J. Nat/. front. Cancer Inst. 69: 1329-1336, 1982. The absence of TIMP synthesis by VX2 tumour cells Brown, C. c., Hembry, R. M. and Reynolds, ].J. : Immunolocalizaconfirms our in vitro observations (Gavrilovic et aI., 1985; tion of metalloproteinases and their inhibitor (TIMP) in the Gavrilovic, 1987). It is tempting to speculate that the lack rabbit growth plate. j. Bone Jt. Surg. 71-A: 580-593,1989. of staining for TIMP in and around the tumours in vivo may Chen, ].-M. and Chen, W.-T. : Fibronectin-degrading proteases reflect a tumour-induced down-regulation of TIMP synfrom the membranes of transformed cells. Cell 48: 193 - 203, 1987. thesis by host cells resulting in uncontrolled degradation of

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rheumatoid and normal synovial tissue in vitro and cells derived from them. Production of prostaglandins and collagenase in response to factors derived from cultured blood mononuclear cells and from synovium. Rheumatol. Int. 2: 113 -120,1982. Mignatti, P., Robbins, E. and Rifkin, D.B.: Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell 47: 487 -498, 1986. Murphy, G., Cawston, T.E. and Reynolds, J.J.: An inhibitor of collagenase from human amniotic fluid. Purification, characterization and action on metalloproteinases. Biochem. ].195: 167-170,1981. Murphy, G., McAlpine, C. G., Poll, C. T. and Reynolds, ].J.: Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen. Biochim. Biophys. Acta 831: 49 - 5 8, 1985. Murphy, G., Hembry, R.M. and Reynolds, J.].: Characterization of a specific antiserum to rabbit stromelysin and demonstration of the synthesis of collagenase and stromelysin by stimulated rabbit articular chondrocytes. Collagen Re!. Res. 6: 351-364, 1986. Murphy, G., Ward, R., Hembry, R.M., Reynolds,].]., Kuhn, K. and Tryggvason, K.: Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase. Biochem.]. 258: 463-472, 1989a. Murphy, G., Hembry, R. M., McGarrity, A. M., Reynolds, J. J. and Henderson, B.: Gelatinase - type IV collagenase - immunolocalisation in cells and tissues. Use of an antiserum to rabbit bone gelatinase that identifies high and low Mr forms.]. Cell Sci. 92: 487-495, 1989b. Sloane, B.F., Rozhin, J., Johnson, K., Taylor, H., Crissman, ].D. and Honn, K. V.: Cathepsin B: association with plasma membrane in metastatic tumors. Proc. Natl. Acad. Sci. USA 83: 2483-2487,1986. Thorgeirsson, U. P., Liotta, L. A., Kalebic, T., Margulies, I. M., Thomas, K., Rios-Candelore, M. and Russo, R.G.: Effect of natural protease inhibitors and a chemoattractant on tumor cell invasion in vitro.]' Natl. Cancer Inst. 69: 1049-1054,1982. Trechsel, U., Dew, G., Murphy, G. and Reynolds, ].].: Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture. Biochim. Biophys. Acta 720: 364- 370,1982. Tryggvason, K., Hoyhtya, M. and Salo, T.: Proteolytic degradation of extracellular matrix in tumor invasion. Biochim. Biophys.Acta907: 191-217, 1987. Welgus, H. G., Jeffrey,].]., Eisen, A. Z., Roswit, W. T. and Stricklin, G.P.: Human skin fibroblast collagenase: interaction with substrate and inhibitor. Collagen Rei. Res. 5: 167 -179,1985. Wid, G. and Frick, J.: Collagenase - a marker enzyme in human bladder cancer? Urol. Res. 7: 103-108,1979. Woolley, D. E.: Collagenase immunolocalization studies of human tumours. In: Tumor Invasion and Metastasis, ed. by Liotta, L. A. and Hart, 1. R., Martinus Nijhoff Publishers, The Hague, Boston and London, 1982, pp. 391-404. Woolley, D. E.: Collagenolytic mechanisms in tumor cell invasion. Cancer Metastasis Rev. 3: 361-372, 1984. Dr. Gillian Murphy, Cell Physiology Department, Strangeways Research Laboratory, Worts Causeway, Cambridge, CB14RN, UK.