Serum and Plasma Mr 92,000 Progelatinase Levels Correlate with Spontaneous Metastasis of Rat 13762NF Mammary Adenocarcinoma

The expression of metalioproteinases, such as type IV collagenase/ gelatinase, enables tumor cells to degrade type IV collagen present in the basement membrane and correlates with metastatic potential of several tumor types. We found that increased levels of rat serum type IV collagenolytic activity are associated with increased 13762NF mammary adenocarcinoma metastases in lungs and lymph nodes of syngeneic rats. To investigate serum metalloproteinases responsible for type IV collagenolysis, we performed zymography and Western blot analysis of rat sera. A Mr 92,000 progelatinase (progelatinase B, Mr 92,000 type IV procollagenase, MMP-9) was detected on zymograms of rat sera within 16 days after intramammary fat pad inoculation of highly metastatic MTLn3 cells. The activated serum Mr 92,000 progelatinase degraded sodium dodecyl sulfate-denatured type I and IV collagens but was not active on casein, albumin, hemoglobin, and immunoglobulin. Sera from rats with fat pad tumors and lung metastases formed from highly metastatic clones pos. sessed greater than 7 times higher levels of serum Mr 92,000 progelatinase than control sera or sera from rats bearing similar size fat-pad tumors of low or nonmetastatic clones. The results were confirmed by Western blot analysis of rat sera using rabbit anti-human Mr 92,000 progelatinase antibodies. Similar results were obtained from the analysis of rat plasma samples. The serum and plasma Mr 92,000 progelatinase levels correlated with the extent of metastases in the lung and lymph nodes. The results hulicate that high levels of serum and plasma Mr 92,000 progelatinase are associated with the presence of disseminated metastatic adenocarcinoma cells and suggest that this enzyme may facilitate the invasion of bloodborne tumor cells through vascular basement membranes. Using zymography we have identified two major metalloproteinases in the serum-free conditioned medium of highly metastatic mammary adenocarcinoma cell cultures (11). The metalloproteinases were Mr 64,000 and Mr 88,000 progelatinases as determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis under nonreducing conditions. These gelatinases specifically degraded type IV collagen and heat-denatured type I collagen (gelatin) but not serum albumin, casein, hemoglobin, or immunoglobulin (11). The production of the progelatinases by rat mammary adenocarcinoma cells was suppressed by retinoic acid treatment, paralleling a decrease in cell invasion through reconstituted basement membranes (11). These enzymes are rat homologues of the human metalloproteinases known as M r 72,000 progelatinase/Mr 72,000 type IV procollagenase/progelatinase A/MMP-23 (12) and M~ 92,000 progelatinase/Mr 92,000 type IV procollagenase/progelatinase B/MMP-9 (13). Since rat mammary adenocarcinoma cells secrete type IV collagenolytic metalloproteinases in direct relation to their metastatic potentials (10), we assayed sera from rats bearing mammary adenocarcinoma cells of differing metastatic potentials for the collagenotytic metalloproteinases using metabolically [3H]proline-labeled type IV collagen. We found that serum type IV collagenolytic activity correlated with the extent of metastases in the lung and lymph nodes (14). Here, we demonstrate that the expression of serum and plasma Mr 92,000 progelatinase correlates with the metastasis formation of rat 13762NF mammary adenocarcinoma cells. I N T R O D U C T I O N The penetration of basement membranes by malignant cells is an important step in the formation of tumor metastases. Basement membranes, formed from type IV collagen, laminin, heparan sulfate proteoglycan, nidogen, and fibronectin, are relatively rigid barriers between different tissues. Several proteinases implicated in the process of tumor invasion and metastasis are capable of degrading basement membrane components (1-9). An important class of these enzymes are the metalloproteinases, such as the type IV collagenases/ gelatinases (2--4). Type IV collagenolytic activity itseff has been correlated with the metastatic potential of tumor cells in a variety of malignant tumors (4). For example, we found an excellent correlation between the type IV collagenolytic enzyme activities in rat 13762NF mammary adenocarcinoma cells and their spontaneous lung metastatic potentials (10). These metalloproteinases secreted from the rat mammary adenocarcinoma cells degraded both a-subunits of type IV procollagen and produced characteristic high molecular weight fragments (10). Received 6/28/93; accepted 9/30/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported in part by National Cancer Institute RO1-CA41524 [M. N.], R35-CA44352(OIG) (G. L. N.), National Foundation for Cancer Research (G. L. N.), the Jake Gittlen Memorial Golf Tournament (D. R. W.), and the Ministry of Education, Science and Culture, Japan (M. N., T. T.). 2 To whom requests for reprints should be addressed, at Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-i Yayoi, Bunkyo-ku, Tokyo, 113 Japan. MATERIALS AND METHODS Cells and Cell Culture. Doubly cloned cell lines were derived from the 13762NF mammary adenocarcinoma growing at local mammary fat pad implant sites (MTC and MTF7) and from spontaneous lung metastases (MTLn2 and MTLn3) (15). Subclones MTLn3(T44).5 and MTF7(T35).3 were selected by in vitro cloning from MTLn3 and MTF7, respectively (16). Cells were grown on 100-mm plastic tissue culture plates (Coming Glass Works, Corning, NY) containing a-minimal essential medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Biocell, Carson, CA) at 37~ in a humidified incubator (5% CO2; 95% air). Cells from subconfiuent cultures were used for all assays. Spontaneous Metastasis Assay. 13762NF Mammary adenocarcinoma cell clones were harvested from subconfiuent cultures by 0.25% trypsin treatment, and their viability was determined by trypan blue dye exclusion. Only cell suspensions with viability greater than 95% were used for animal inoculations. Age-matched F344 rats received 1 • 10 6 tumor cells suspended in 0.5 ml phosphate-buffered saline s.c. in the left posterior inguinal mammary fat pad. Blood was collected by cardiac puncture at various periods after tumor cell injection. Rats were killed using Metofane anesthesia and were subjected to complete gross necropsies. The numbers of metastases were related to enzyme activities using a one-way analysis of variance and the Student-Newman-Keuls procedure for group significance analysis. Type IV Collagenolysis Assay. The type IV collagenolysis assay was performed as previously reported (10, 11). Briefly, [3H]proline-labeled type IV collagen was purified from EHS tumors that were cultured for 48 h in prolinefree Dulbecco's modified Eagle's medium containing 100/xg/ml ascorbic acid, 100 p~g/ml /3-aminopropionitrile fumalate, and 25 /xCi/rrd L-[2,3,4,5-3H]pro 3 The abbreviations used are: MMP, matrix metalloproteinase; EHS, Engelbreth-HolmSwarm; ELISA, enzyme-linked immunosorbent assay.