Expression of Laminin Receptor in Normal and Carcinomatous Human Tissues As Defined by a Monoclonal Antibody

[CANCER RESEARCH 45, 2713-2719, June 1985]

Expression of Laminin Receptor in Normal and Carcinomatous Human Tissues as Defined by a Monoclonal Antibody

P. Horan Hand,1 A. Thor, J. Schlom, C. N. Rao, and L. Liotta

Laboratory of Tumor Immunology and Biology [P. H. H., A. T., J. S.] and Laboratory of Pathology [C. N. R, L L], National Cancer Institute, NIH, Bethesda, Maryland 20205

ABSTRACT phages (9,10), human monocytes (10), as well as human breast cancer cells (4), metastatic murine BL6 melanoma cells (11), and It has been hypothesized that epithelial and endothelial cells murine fibrosarcoma cells (12, 13). Laminin affinity chromatog- interact with the laminin component of basement membranes via raphy studies have demonstrated that both the murine and a cell surface laminin receptor molecule. It has also been pro human receptor molecules have a molecular weight of approxi posed that the expression of this molecule may be involved in mately 67,000 to 69,000 (11,12). the invasion of carcinoma cells from their tissue of origin and We have recently generated MAb2 by using purified human their subsequent penetration through blood vessel basement laminin receptor as immunogen. Initial characterization of these membranes. We report here the use of a monoclonal antibody, MAb (14) demonstrated their reactivity to purified laminin recep LR-3, to define the expression of laminin receptor in normal, tor. The discovery of a laminin receptor and the availability of a dysplastic, and carcinomatous human tissues. Monoclonal anti MAb which binds this molecule now make possible many diverse body LR-3 is shown by immunoblotting to recognize the M, avenues to study the roles of laminin receptor in tumor cell 67,000 laminin receptor protein, to bind to the carcinoma cells, mÃ©tastases and in the differentiation and function of various and to constitute approximately 0.1% of total cellular protein. normal human cell types. Numerous normal human epithelial and endothelial cell types, as well as pulmonary macrophages, are shown to express laminin receptor to varying degrees. Selected human mammary carci MATERIALS AND METHODS nomas and colon carcinomas are shown to bind more monoclo nal antibody LR-3 than normal or dysplastic counterparts. A Purfication of Laminin Receptor Antigen. Laminin receptor was purified from extracts of plasma membranes from pooled human breast monoclonal antibody to laminin receptor now makes possible carcinoma tissue, as described (4, 7,11). the study of the role of laminin receptor in tumor cell mÃ©tastases Immunizations. Four-week-old BALB/c mice were immunized by i.p. and in the differentiation and function of various normal human inoculation of 5 /Â»gof laminin receptor purified from human breast epithelial and endothelial cell types. carcinomas, mixed with an equal volume of complete Freund's adjuvant. Seven days later, 2 /Â¿gof the laminin receptor mixed with an equal volume of incomplete Freund's adjuvant were inoculated i.p. On Day 14, INTRODUCTION 2 fig of the laminin receptor were injected into the mice i.v. Three days The mechanism of tumor cell invasion appears to be a highly later, spleens were removed for cell fusion. complex and multistage phenomenon. One necessary step in Hybridoma Methodology. Somatic cell hybrids were prepared using the method of Herzenberg ef al. (15) with some modifications (16). the invasive process is the movement of tumor cells through the Hybridoma cell lines were cloned twice by limiting dilution. Ascites fluid extracellular matrix, which is composed of basement membranes was prepared in pristane-primed BALB/c mice inoculated i.p. with 1 x and interstitial stroma and forms a barrier between tissues of 107hybridoma cells (16). different types. One of the components of the basement mem Cells. The MCF-7(5A9) cell line (17) is a clone of the MCF-7 human brane is laminin, a glycoprotein with a molecular weight of mammary adenocarcinoma cell line. The Flow-4000 normal human fetal approximately 106 (1-3). A 50-fold greater binding of laminin to kidney cell line, the A204 human rhabdomyosarcoma cell line, the WI-38 unoccupied receptors of human breast carcinoma cells versus normal human fetal lung cell line, and the A375 human melanoma cell normal and benign breast lesions has been reported (4). It has line were obtained and maintained as previously described (18,19). Solid-Phase RIA. Ascites fluid of one of the MAb, designated MAb been proposed that laminin receptor may aid in the attachment Laminin Receptor (LR>3, was used in solid-phase RIA with 5 /ig of cell of tumor cells to laminin in basement membranes, thereby facil extracts or 25 ng of purified laminin receptor as described (16) with the itating (a) the exit of tumor cells from their tissue of origin and exception that polyoxyethylene sorbitan monolaurate (Tween-20) (o) the penetration of tumor cells through other basement mem (0.05%) was added to wash buffer. The Tween-20 was required to branes, including those of blood vessels, to result in metastatic reduce the nonspecific binding of the MAb observed in control wells, i.e., lesions (5, 6). phosphate-buffered saline in place of extract. The existence of a laminin receptor was established when a Suspension Live-Cell RIA. The suspension live-cell RIA was per high-affinity receptor for laminin was first identified on the surface formed as described (17) with the exception that cells were seeded into of the MCF-7 human breast carcinoma cell line (5-7). Since this round-bottomed, 96-well polyvinyl plates at 2 x 105 cells/well. The plates were then incubated under constant agitation at 37Â°C for 1 h in a initial observation, the presence of laminin receptor has been demonstrated on the surface of several normal and neoplastic humidified incubator containing 5% C02 before addition of the primary tissues; these are rodent striated muscle (8), rodent macro- antibody. All subsequent incubations were performed using identical conditions. 1To whom requests for reprints should be addressed, at the Laboratory of Immunoperoxidase Method. Five-^m sections of formalin-fixed tis- Tumor Immunology and Biology, Building 10, Room 8B07, National Cancer Insti tute, NIH, Bethesda, MD 20205. 2The abbreviations used are: MAb, monoclonal antibody(ies);RIA, radioimmu- Received 11/26/84; revised 3/1/85; accepted 3/4/85. noassay.

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sues were stained using a modification of methodology defined previ the immunoblot method to the breast carcinoma membrane ously (20) and the avidin:biotin:peroxidase complex method (21). To extract. reduce nonspecific binding of the MAb to the tissue sections, ascites Reactivity of MAb LR-3 to Occupied versus Unoccupied fluid containing MAb LR-3 was diluted in phosphate-buffered saline Laminin Receptors. The MAb generated versus lamininreceptor containing 0.1% bovine serum albumin and 0.1% Tween-20. MAb LR-3 differ in their ability to block the binding of laminin to laminin was incubated with tissue sections for 30 min at room temperature. This was followed by a 10-min rinse in phosphate-buffered saline containing receptors (14). Although some of the MAb inhibit 100% of the 0.1% Tween-20 and a 10-min rinse in phosphate-buffered saline prior to binding of laminin to its receptor on isolated breast carcinoma application of the biotinylated second antibody. membranes or live cells (MCF-7), MAb LR-3 had no effect on laminin binding under identical conditions (data not shown). MAb LR-3 therefore recognizes a site on the laminin receptor which RESULTS is distinct from the binding site and is therefore capable of detecting both occupied and unoccupied laminin receptors. Generation of MAb. Mice were immunized with lamininrecep Expression of Laminin Receptor in Cell Extracts. Experi tor purified from plasma membranes of pooled human breast ments were then conducted in an attempt to quantitate the level carcinoma tissues (Fig. 1, Lane B). Splenic lymphocytes from of expression of laminin receptor in a human breast carcinoma the immunized mice were fused with NS-1 mouse myeloma cells. cell line versus a normal human cell line. Using a solid-phase RIA, Supernatant fluids from these hybridomas were assayed using MAb LR-3 was reacted with various concentrations of purified a solid-phase RIA for immunoglobulins reactive with the laminin laminin receptor and protein extracts of the MCF-7(5A9) and WÃ•- receptor immunogen. Positive cultures were then expanded and 38 cells. As shown in Table 1, 1000-fold more MCF-7(5A9) cloned twice by limiting dilution. Thirty-nine double-cloned cul extract than purified laminin receptor is required to achieve tures representing 6 primary hybridoma cultures were selected approximately the same level of binding of MAb LR-3; it thus for further study. Immunoglobulins from all 39 cultures were appears that approximately 0.1% of the MCF-7(5A9) cellular demonstrated to be IgM and demonstrated reactivity in solid- protein represents laminin receptor. In other experiments per phase RIA with purified laminin receptor. One of the MAb, formed with different extracts of the same MCF-7(5A9) cell line, designated LR-3, was chosen for further studies based on high the level of expression of laminin receptor in the breast carcinoma titer and efficacy in immunohistochemical assays using formalin- cells ranged from 0.05 to 0.1% of the total extract protein. fixed tissue sections (see below). A differential of approximately 1.6- to 3.6-fold was observed Reactivity of MAb LR-3 to Human Breast Carcinoma Biopsy between the level of laminin receptor expressed by the MCF- Tissue. To define the specificity of the MAb LR-3 reaction, 7(5A9) cells versus the WI-38 normal fetal lung cells at the protein extracts of membranes of breast carcinoma tissue were different amounts of protein extracts tested (Table 1). The levels reacted with MAb LR-3 using the immunoblot method; as seen of laminin receptor observed in MCF-7 cells were also approxi in Fig. 1C, a single protein component with a molecular weight mately 3-fold higher than those observed in the A204 human of approximately 67,000, characteristic of laminin receptor, is rhabdomyosarcoma and Flow-4000 kidney cell lines (data not observed. No binding of a MAb which reacts with a human shown). Using similar concentrations of LR-3 MAb, minimal reac thymus antigen (data not shown) or tissue culture supernant tivity was observed to bovine serum albumin (Table 1), thyro- fluid of NS-1 mouse myeloma cells (Fig. 1D) was observed using globulin, and ovalbumin (data not shown), at concentrations as high as 10,000 ng of each per well. Cell Surface Expression of Laminin Receptor. Initial at tempts to bind MAb LR-3 to the surface of MCF-7 cells grown in monolayer proved unsuccessful. It has previously been shown 200K- (7, 11, 12, 22), however, that enzymatic digestion of rodent fibrosarcoma cells with trypsin, followed by a 1-h incubation period for regeneration of the laminin receptor, enhanced binding of 125l-laminin to the cell surface. Such a treatment was thus necessary to expose the laminin receptor determinant. MCF- 67K- 7(5A9) and Flow-4000 cells were removed from plastic culture flasks with 0.1% trypsin containing 0.5 HIMEDTA. The cells were 43K-

Table 1 Reactivity of MAb LR-3 in solid-phase RIA Solid-phase RIA was performed as described in "Materials and Methods" using a 1:3,000 dilution of MAb LR-3 ascites fluid. Numbers represent cpm bound minus 20K-J nonspecific binding (cpm obtained with MAb LR-3 at 1:3,000 ascites dilution versus phosphate-buffered saline dried to assay wells, approximately 500 cpm). A D Protein input (ng) Fig. 1. Immunoblot of human breast carcinoma plasma membrane extract with ProteinLaminin MAb LR-3. Lane A, molecular weight markers. Lane B, iodinated purified human breast carcinoma laminin receptor. Lane C, MAb LR-3 blotted to total protein receptor MCF-7(5A9) 5,804 3,936 1,776 352 NT extract. A single protein component is bound by the MAb. Lane D, tissue culture WI-38 2,717 2,513 491 0 supernatant fluid of NS-1 mouse myeloma cells blotted to total protein extract. No NT Bovine serum albumin10,000NT* 6501,000NT 0100NTNT106,032NT14,425 NT reactivity is observed. Extract and immunoblot methods have been detailed previ a NT, not tested. ously (11, 14).

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. LAMININ RECEPTOR EXPRESSION IN HUMAN TISSUES then incubated in their respective growth media for 1 h at 37Â°C Table 2 Reactivity of MAb LR-3 with normal human tissues in immunoperoxidase assay (5% C02) with constant agitation and then maintained in suspen sion for the duration of the live-cell RIA. As seen in Chart 1, Tissue Cell type Reactivity Kidney Glomerular capillary endothelium approximately 3-fold more laminin receptor was detected by Parietal epithelium of Bowman's space MAb LR-3 on the surface of the MCF-7(5A9) cells than on the Renal tubular, collecting duct, and transitional Flow-4000 cells. In contrast to these findings, approximately 7- epithelium fold more HLA-A,B,C antigen, as detected by MAb W6/32 (23), Glomerular mesangial cells, interstitial stroma was observed on the Flow-4000 cells than on the MCF-7(5A9) Skin Epidermis, dermal capillary endothelium cells (Chart 1, inset). Sebaceous and merocrine gland epithelium Analysis of Normal Human Tissue. Formalin-fixed 5-^m sec Dermis, hair matrix tions of adult human tissues were assayed for expression of Lung and trachea Respiratory epithelium, type 1 pneumocytes laminin receptor using MAb LR-3 and the avidinibiotin complex- Macrophages (intraalveolar) Hyaline cartilage, interstitial stroma immunoperoxidase method. Many normal tissues tested dem onstrated at least one cell type with laminin receptor expression Esophagus Squamous epithelium, submucosal glands, smooth muscle (Table 2). In the majority of tissues, the cell types (epithelium or endothelium) positive for laminin receptor were intimately asso Stomach Mucous and parietal cells ciated with a basement membrane. Laminin receptor expression Chief cells was demonstrated in several renal cell types, including the Small intestine Enterocytes Goblet and Paneth's (granular) cells majority of glomerular capillary endothelial cells which stained most intensely (Table 2; Fig. 2, A to C). The interstitial stroma Large intestine Mucous cells, ganglion cells of Meissner's and glomerular mesangial cells were routinely negative (Table 2). plexus The epidermis (squamous epithelium) of human skin was Interstitial stroma, smooth muscle routinely positive for laminin receptor expression (Table 2; Fig. Breast Ductal and lobular epithelium 2D); expression was transepidermal and heterogeneous. Nega Interstitial stroma tive control slides using dilution buffer in place of MAb LR-3 Liver Hepatocytes, biliary epithelium demonstrated melanocytes containing brown melanin pigment. Interstitial stroma These were examined concurrently with MAb LR-3 assays to prevent misinterpretation of the diaminobenzidine-peroxidase Gall bladder Biliary epithelium Interstitial stroma color reaction (Fig. 2Â£). Several pulmonary cell types were routinely positive for laminin receptor (Table 2) with pulmonary Spinal cord White matter, ependymal lining of central macrophages reacting most intensely with MAb LR-3. The en canal Peripheral nerves dothelium of all blood vessels (arteries, capillaries, veins) also Grey matter demonstrated strong laminin receptor expression within all tis Bladder Transitional epithelium sues examined. The other cell types listed as positive in Table 2 Smooth muscle, interstitial stroma all demonstrated a heterogeneity in laminin receptor expression, Cervix Squamous and endocervical glandular epithe usually with only a small percentage (<10%) scoring positive. lium Interstitial stroma

BloodvesselsSkeletal SmoothmuscleStriated

muscleSmooth cellsSmoothmuscle

muscleProstateTonsilTestisFallopianmuscleGlandular

epithelium InterstitialstromaLymphocytesGerminal

epithelium InterstitialstromaTubular

tubeOropharynxLymph epithelium SmoothmuscleSquamous

epitheliumLymphocytes,

nodesSpleenAdrenal histiocytesLymphocytes,

histiocytesCorticalplasma cells, I/LOG LR-3 ASCITES DILUTION glandThymusSalivary cellsLymphocytesGlandand medullary Chart 1. Cell surface expression of laminin receptor on MCF-7(5A9) human breast carcinoma and Flow-4000 normal human fetal kidney cell lines. Using a suspension live-cell RIA, increasing concentrations of MAb LR-3 were tested for binding to the MCF-7(5A9) (â€¢)andFlow-4000 (O) cell lines. Inset: binding of purified glandThyroid epitheliumFollicularand duct immunoglobulin of MAb W6/32, a MAb reactive with HLA-A.B.C (23), to the surface of MCF-7(5A9) (A) and Flow-4000 (A) cell lines using the suspension live-cell RIA. glandEndothelium cells, colloid++-+-+++------

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Analysis of Malignant and Benign Human Breast. Three that observed in infiltrating ductal carcinomas (Chart 2A). infiltrating ductal carcinomas were examined using various con Analysis of Malignant and Benign Human Colon. The vast centrations of MAb LR-3. Two of the 3 tumors demonstrated majority of normal colonie epithelial cells scored negative for laminin receptor on 70 to 80% of tumor cells at the highest MAb laminin receptor, with the exception of rare mucous-secreting LR-3 concentration used (Chart 2A; Fig. 2F). A linear decrease cells. Three adenocarcinomas and 2 tubulovillous adenomas of in laminin receptor detection was demonstrated with MAb LR-3 the colon were examined using multiple concentrations of MAb dilution. Apparently normal mammary epithelium (when seen LR-3 (Fig. 2, J and K; Chart 2C). All 3 adenocarcinomas dem within the same section containing carcinoma, fibroadenoma, or onstrated laminin receptor expression (Fig. 2J), with the per fibrocystic disease) was either negative or demonstrated very centage of positive cells ranging from 20 to 65% at the highest low levels of laminin receptor; breast stroma was also routinely MAb LR-3 concentration used (Chart 2C). Both tubulovillous negative. Most fibrocystic disease and fibroadenoma specimens adenomas examined demonstrated weak (<1%) staining (Fig. demonstrated <10% of cells staining at all MAb LR-3 concentra 2K; Chart 2C). The colonie stroma including smooth muscle was tions (Chart 2A; Fig. 2, G and H). negative in all assays. Twenty-four infiltrating ductal carcinomas from different pa Twenty adenocarcinoma specimens from patients were then tients were then examined using MAb LR-3 (Chart 3A). Eight of examined for laminin receptor (Chart 3C). Six of 20 adenocarci 24 breast carcinomas (33%) clearly showed enhanced expres noma specimens (33%) were strongly positive (>10% of carci sion of laminin receptor (>10% of carcinoma cells scoring posi noma cells scoring positive). Fifteen (75%) demonstrated some tive). Twenty (85%) demonstrated some laminin receptor expres laminin receptor expression (>1% carcinoma cells positive), and sion (with at least one carcinoma cell scoring positive), and only 5 (25%) were completely negative in this assay. Twenty benign 4 specimens were completely negative in this assay (Chart 3/4). colon specimens (tubular adenomas, tubulovillous adenomas, Twenty specimens from different patients with fibrocystic dis and a hamartomatous polyp) were also assayed (Chart 3C). ease, fibroadenomas, or chronic inflammation of the breast were Benign colon specimens did not demonstrate significant laminin also examined (Chart 3B). Three of 20 specimens (15%) dem receptor expression (I.e., >10% of benign epithelial cells posi onstrated significant laminin receptor expression (>10% carci tive), and only 4 (20%) demonstrated minimal positivity to MAb noma cells positive). Two of these 3 were from patients with a LR-3 in this assay. clinical history of multiple fibroadenomas (Fig. 21; Chart 28; Chart 36, a and b). These 2 samples were from 21- and 24-year-old DISCUSSION females who collectively have had 7 fibroadenomas removed to date. Titration studies demonstrated that laminin receptor It has been hypothesized that all endothelial and epithelial cells expression in these fibroadenomas (Chart 2ÃŸ)was similar to attach to basement membranes via the laminin receptor (8, 24-

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J l L _L l l l l L, 1: 1:400 1:500 1:6001:7001:300 1:400 1:500 1:600 1:7001:300 1:400 1:500 1:6001:700 LR-3 ASCITES DILUTION Chart 2. Titration of MAb LR-3 with formalin-fixed colon and breast tissues. In A, serial sections of infiltrating ductal carcinoma of the breast (â€¢),fibroadenomaof the breast (e), fibrocystic disease of the breast (O), and lymphoma (A) were assayed for laminin receptor using the avidin:biotincomplex-immunoperoxidasetechnique and MAb LR-3. The percentageof positive cells scored for carcinomaand fibroadenomais the total epithelialtumor cells (malignantfor carcinomaand benignfor fibroadenoma) scoring LR-3 positive divided by the total tumor epithelialcells observed multiplied by 100; for fibrocystic disease, scoring is the percentage of benign breast epithelium scoring positive for laminin receptor divided by the total number of breast epithelialcells multiplied by 100. In 6, serial sections of fibroadenomas (from 2 patients with multiple fibroadenomas, Â®)wereassayed for lamininreceptor using an identical assay as in A. In C, serial sections of adenocarcinomaof the colon (â€¢)andtubulovillous adenomas(D) assayed for lamininreceptor are shown. The percentageof positive cells is a semiquantitativevalue for tumor cells scoring lamininreceptor positive divided by the total epithelialtumor cells (malignantfor carcinomas and benignfor adenomas).

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parallels that observed with the breast carcinomas and benign 80w lesions. Not all colon carcinomas expressed detectable levels of 70Ã¶ the laminin receptor, and higher levels were detected in malignant colon samples versus benign lesions (Chart 3). *>Â£ A consistent difference in the distribution of immunoreactivity 50w â€¢BAaAVAVC...; between the benign and malignant cells was noted. The benign cells appeared to express laminin receptor immunoreactivity at 40 the basal location adjacent to the formed basement membrane. o. â€¢;â€¢ K30Ã¶ In contrast, the carcinoma cells exhibited staining over the entire surface of the cell, including more pronounced cytoplasmic reac 20Â°- -D--â€¢â€¢â€¢A tivity compared to the benign cells. This observation suggests 100A- that the surface distribution, state of internalization, and/or proc TAB essing of the receptor may all be altered in the actively invading cells. Studies are now in progress to delineate the role of MAb LR-3, and the other MAb reactive with laminin receptor, on the MALIGNANT BENIGN MALIGNANT BENIGN functional activity of the laminin receptor. These studies should BREAST COLON lead to a better understanding not only of the fine specificity of the MAb, but also of the possibility and route of internalization Chart 3. Laminin receptor expression in various breast and colon disease states. All samples were reacted with a 1:400 dilution of MAb LR-3 ascites fluid using the of the laminin receptor and its role in cell growth and migration. avidin:biotin complex-immunoperoxidase method. -A. infiltrating ductal carcinomas The role of the laminin:laminin receptor complex in the various (â€¢).B, fibrocystic disease (T), fibroadenomas (A), fibroadenomas from patients nonneoplastic disease stages is thus far unknown. It may play a with multiple fibroadenomas (A) and other benign inflammatory diseases of the breast (â€¢).C,adenocarcinomas of the colon (â€¢).D,tubular adenomas (A), tubulo- role in normal growth, differentiation, or morphogenesis. Autoim villous adenomas (â€¢).anda hamartomatous polyp (T). Each symbol represents a mune states such as Goodpasture's syndrome and Chagas' tumor from a different patient. The percentage of positive cells denotes for adenocarcinoma or the benign tumors the number of epithelial tumor cells reactive disease both have been shown to involve the laminin molecule with MAb LR-3 divided by the total number of tumor cells multiplied by 100. (27, 28). Thus, MAb against laminin receptor may also have potential use in the study of a variety of immunologically mediated 26). Our results with MAb LR-3 define the expression of the disease states which may destroy epithelium, endothelium, or laminin receptor molecule in a wide variety of cell types (endo- the integrity of the basement membranes. thelial and epithelial) and thus substantiate these theories. The studies reported here have shown that more than 33 epithelial ACKNOWLEDGMENTS and endothelial normal human cell types, virtually all adjacent to The authors gratefully acknowledge the expert technical assistance of Earl basement membranes, are positive for laminin receptor (Table Smith, Amy Sloan, Michelle Ballard, Jackie Knapp, and Larry Otsby. 2). We have also observed that pulmonary macrophages (cells not bound to basement membrane) were uniformly positive for REFERENCES laminin receptor expression. These results complement the find ings of Wicha ef al. (9) and Huard ef al. (10) who demonstrated 1. Chung, A. 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laminin from murine fibrosarcoma cells. J. Cell Biol., 96; 1475-1479, 1983. bution of a novel tumor associated antigen in human mammary carcinoma cell 13. Varani, J., LoveÂ«,E. J., McCoy, J. P., Shibata, S., Maddox, D. E., Goldstein, populations. Int. J. Cancer, 29: 539-545,1982. I. J., and Wicha, M. Differential expression of a laminin-like substance by high 21. Hsu, S. M., Raine, L., and Fanger, H. J. Use of avidin-biotin-complex (ABC) and low metastatic cells. Am. J. Pathol., 111: 27-34,1983. and unlabeled antibody (PA) procedures. J. Histochem. Cytochem., 29: 577- 14. Liotta. L. A., Horan Hand, P., Rao, C. N., Bryant, G., Barsky, S. H., and 580, 1981. Schlom, J. Monoclonal antibodies to the human laminin receptor inhibit binding 22. Malinoff, H. L., Varani, L., McCoy, P., and Wicha, M. S. Metastatic potential of laminin to breast carcinoma cells. Exp. Cell Res., 756. 117-126, 1985. of murine fibrosarcoma cells correlates with endogenous surface receptor 15. Herzenberg, L. A., Herzenberg, L. A., and Milstein, C. Cell hybrids of myelomas bound laminin (abstract). J. Cell Biol., 95:126a, 1983. with antibody forming cells and B-lymphocytes in T cells. In: D. M. Weir (ed.), 23. BarnstaWe, C. J., Bodmer, W. F., Brown, G., Galfre, C., Milstein, C., Williams, Handbook of Experimental Immunology, Chap. 25. London: Blackwell Scientific A. F., and Ziegler, A. Production of monoclonal antibodies to Group A eryth- Publications, 1979. rocytes, HLA, and to the human cell surface antigensâ€”new tools for genetic analysis. Cell, 14: 9-20, 1978. 16. Colcher, D., Horan Hand, P., Nuti, M., and Schlom, J. A spectrum of monoclonal antibodies reactive with human mammary tumor cells. Proc. Nati. Acad. Sci. 24. Kefalides, N. A., Alper, R., and Clark, C. C. Biochemistry and metabolism of USA, 78.: 3199-3203.1981. basement membranes. Int. Rev. Cytol., 67. 167-288, 1979. 17. Horan Hand, P., Nuti, M., Colcher, D., and Schlom, J. Definition of antigenic 25. Terranova, V. P., Rohrbach, D. H., and Martin, G. R. Role of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen. Cell, heterogeneity and modulation among human mammary carcinoma cell popu 22:719-728,1980. lations using monoclonal antibodies to tumor associated antigens. Cancer Res., 43.: 728-735. 1983. 26. Timpl, R., and Martin, G. R. Components of basement membranes. In: H. Furthmayr (ed.), Immunochemistry of the Extracellular Matrix, Vol. 2, pp. 119- 18. Colcher, D., Zalutsky, M., Kaplan, W, Kufe, D., Austin, F., and Schlom, J. 150. Boca Raton, FL: CRC Press, 1982. Radiolocalization of human mammary tumors in athymic mice by a monoclonal 27. Szarfman, A., Terranova, V. P., Rennard, S. I., Foidart, J. M., Lima, M. D. F., antibody. Cancer Res., 43: 736-742, 1983. Scheinman, J. I., and Martin, G. Antibodies to laminin in Chagas' disease. J. 19. Colcher, D., Horan Hand, P., Nuti, M., and Schlom, J. Differential binding to Exp. Med., 755: 1161-1171, 1982. human mammary and non-mammary tumors of monoclonal antibodies reactive 28. Yaar, M., Foidart, J. M., Brown, K. S., Rennard, S. I., Martin, G. R., and Liotta, with carcinoembryonic antigen. Cancer Invest.. 7: 127-138, 1983. L. The Goodpasture-like syndrome in mice induced by intravenous injections 20. Nuti, M., Teramoto, Y. A., Mariani-Constantini, R., Horan Hand, P., Colcher, of anti-type IV collagen and anti-laminin antibody. Am. J. Pathol., 707: 79-91, D., and Schlom, J. A monoclonal antibody (B72.3) defines patterns of distri 1982.

Fig. 2. Expression of laminin receptor in normal human kidney, skin, and lesions of the breast and colon. MAb LR-3 (at a 1:400 dilution of ascites fluid) was reacted with formalin-fixed, paraffin-embedded tissues using the avidinibiotin complex-immunoperoxidase method (20, 21). A, laminin receptor expression demonstrated in capillary loop endothelial cells (c) and Bowman's space (bs) parietal epithelial cells (p) of a renal glomerulus. Also visualized but unreactive with MAb LR-3 is basement membrane (6m) glomerular mesangium (m). The dark staining reflects the reaction of the diaminobenzidene substrate and, thus, MAb LR-3 binding. The lighter nuclear staining is a hematoxylin counterstain, i.e., no MAb LR-3 reactivity. x540.8, renal tubules with intense tubular epithelial laminin receptor expression. x330. C, transitional epithelium from the renal pelvis demonstrating MAb LR-3 reactivity and heterogeneity of laminin receptor expression. x540. D, normal skin with MAb LR-3 reactivity to the squamous epithelium (epidermis, e) and dermal capillaries (c). The dermis as well as the keratin layer (k) are negative. x220. E. normal skin control using phosphate- buffered saline instead of primary MAb LR-3. Melanin pigment is present in the basal epithelial layer of the epidermis (m), and this is readily distinguished from the MAb reactivity of D. x220. F, infiltrating ductal carcinoma of the breast with strong laminin receptor expression. x130. G, fibroadenoma of the breast demonstrating no reactivity. x130. H, fibrocystic disease of the breast with weak laminin receptor expression in scattered lobules. x130. /, fibroadenoma of the breast from a patient (see Chart 38, Patient a) with multiple fibroadenomas. Positivity of epithelial cells demonstrates laminin receptor expression. x130. J, adenocarcinoma of the colon (invasive) with significant laminin receptor expression. x130. K, tubulovillous adenoma of the colon negative for reactivity with MAb LR-3. x54.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. Expression of Laminin Receptor in Normal and Carcinomatous Human Tissues as Defined by a Monoclonal Antibody

P. Horan Hand, A. Thor, J. Schlom, et al.

Cancer Res 1985;45:2713-2719.

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