The central aim of investigations into molecular carcinogenesis is the identification of gene products involved in cancer progression during which tumour cells acquire the capacity for invasion with resulting metastasis . Metastasis is an active process involving the altered attachment of the tumour cell to the basement membrane, localised degradation of connective tissue, and migration through stromal tissue [28, 29]. Such matrix degradation is mediated by members of several proteinase families (the serine, cysteine, aspartate proteinases and MMPs), with substantial tissue destruction being carried out by members of the MMP family . The expression of members of the MMP family and their inhibitors, the TIMPs, have been examined in both physiological and pathological conditions by many methods both in vitro and in vivo, including the use of total RNA from tissues and cell lines, Northern analysis, RT-PCR in situ and standard in situ hybridisation (ISH) [13, 21, 24, 30–33]. Xenografts of cell lines representing increased progression of breast cancer were examined in the present study using the IS -RT-PCR technique. The samples ranged from the poorly invasive, estrogen-receptor positive cells (MCF-7), through to examples of more aggressive, estrogen-receptor negative human breast carcinoma (MDA-MB-231, MDA-MB-435, Hs578T) . We analysed and compared the localised expression of MT1-MMP, MMP-1, MMP-3 and β-actin, in both the tumour parenchyma and the surrounding stroma. Both the MDA-MB-231 splenic and MDA-MB-435 xenografts demonstrate no detectable MMP gene expression in the stromal compartment. This highlights that although significant sequence homology exists between the human and mouse MMPs examined, minor differences may influence the detection of gene expression. Localised gene expression in both the epithelial tumour cells and the stroma compartments in the remaining xenografts however, demonstrate the detectable homology between human and mouse for the IS -RT-PCR primer sequences used.
MT1-MMP analysis of the HBC xenografts demonstrated mRNA in the tumour cells of all of the samples examined. In vitro, we have previously demonstrated MT1-MMP expression in MCF-7 cells by IS -RT-PCR , however Northern analysis of MCF-7 cells failed to find MT1-MMP in MCF-7 cells, whereas more invasive HBC cell lines showed MT1-MMP expression in concordance with their ability to be induced by Con A to activate MMP-2 [12, 34, 35]. MT1-MMP has also been shown to correlate with increased invasion and to enhance migration of MCF-10A epithelial cells [6, 12, 36], and transfection of MT1-MMP into MCF-7 cells stimulates higher migration and invasiveness [37, 38], and also stimulates VEGF production, angiogenic stimulation, and xenograft take rate in nude mice [39, 40]. Presumably the IS -RT-PCR method is more sensitive than Northern analysis, as may be expected. Indeed, we detected lower levels of MT1-MMP in the MCF-7 xenografts than in the majority of other xenografts derived from more invasive HBC cells. More recently, using isolated RNA for quantitative analyses, we were unable to detect MT1-MMP in MCF-7 derived xenografts, but consistent with our observations here, increasing levels of MT1-MMP was detected in MDA-MB-231 derived xenografts as compared to the parental cells . The reasons for these differences are not clear and require further experimentation, but it is important to note that the cultured MCF-7 cells would have received estrogenic stimuli from both the phenol red and foetal calf serum in the medium .
MT1-MMP overexpression in the mammary gland results in abnormalities including hyperplasia, fibrosis, lymphocytic infiltration and adenocarcinoma , suggesting a pivotal role for MT1-MMP in carcinogenesis. However, IS H analyses of human breast carcinomas have primarily localised MT1-MMP mRNA expression to the stromal cells [11, 29, 43–45], although one study found localised MT1-MMP in the tumour . An immunocytochemical analysis showed expression of MT1-MMP in both tumour and stromal cells . More recently, one study by Bisson et al, combining IS H and IHC data has co-localised MT1-MMP to the α-smooth muscle positive myofibroblast cells in close contact with tumour cells . Our studies certainly show the potential for the cell lines examined to make MT1-MMP, even the relatively well-conserved cell lines such as MCF-7. Although it is acknowledged that in vitro propagation of cell lines may alter some genetic pathways, it is also apparent that cell lines and tumour samples have distinctive gene expression patterns in common . Being ER-positive, and predominantly epithelial, MCF-7 cells far better represent what one may find clinically in breast cancers than the other cell lines examined here . The invasive HBC cell lines show a mesenchymal-like phenotype that may have resulted from epithelial-mesenchymal transition (EMT) in breast carcinoma. EMT is thought to have occurred in the MDA-MB-231, MDA-MB-435 and Hs578T cell lines . The demonstration of MT1-MMP expression observed in the stromal component is not totally unexpected. MT1-MMPs ability to stimulate invasion and metastasis in in vitro systems along with the estrogen receptor status of the cells is well documented as described above. Also, the basal levels observed between the MCF-7 and more invasive HBC cell lines may also be of impact in respect to the higher invasive potential of the cells.
MMP-1 mRNA was detected in the tumour cells of five of the six xenografts examined, the exception, surprisingly, being the MDA-MB-231 splenic metastasis. This is unexpected since MDA-MB-231 cell overexpress MMP-1 in culture , and these cells have a mutation/polymorphism in the promotor region which drives strong MMP-1 expression . This transcriptional repression has not been found to occur extensively in breast cancer . The reasons for suppression of the MMP-1 expression in splenic metastasis are not clear, but could include a strong repression, possibly due to altered signalling from the stroma. The splenic metastasis stroma was notably also lacking all three MMPs examined, but showed a strong β-actin signal in both compartments. One cannot rule out the possibility of selective aberrant loss of MMP mRNA rather than the β-actin, however, this is unprecedented. Further analysis of splenic metastases of the MDA-MB-231 cells, and primary human tumours, would be warranted. MMP-1 has been previously demonstrated to be equally expressed in vitro in both MCF-7 and MDA-MB-231 cells , although in a similar study using Northern analysis we found selective expression in MDA-MB-231 cells but not MCF-7 . Increased production and secretion of MMP-1 has been correlated with increased metastatic potential [52, 54]. More recently, Bachmeir et al have demonstrated a cell density-dependent regulation of gene expression of several MMPs in HBC cell lines . Increased cell density resulted in decreasing levels of both MMP-1 and MMP-3 expression levels, with a resultant decreased invasion concurrent with invasive potential [31–33]. Increased expression of MMP-1 has been reported on contact with Matrigel , and also in response to fibronectin fragments , attesting to the potential regulation of this MMP by the microenvironment. In vivo, MMP-1 expression has been demonstrated in the stroma of nine of thirty-four breast carcinomas  when examined by IS H, localised to stromal constituents during tumour formation , and to be upregulated in the stromal component of ductal carcinomas when examined by IS H and IHC [57–59]. Thus, again, our observation that MMP-1 expression was absent in the stroma of three out of six HBC xenografts appears to contrast the clinical situation. This may reflect the increased sensitivity of IS -RT-PCR and may also reflect the stage in tumour formation of the MCF-7 and MDA-MB-231 mesenteric xenografts that we examined.
MMP-3 mRNA was localised to the tumour component in all six xenograft samples examined. In vitro, MMP-3 mRNA has been demonstrated in the MDA-MB-231 HBC cell line , where it is stimulated by fibroblasts via the extracellular matrix metalloproteinase inducer (EMMPRIN) , and can enhance tumourigenicity and migration . In vivo, MMP-3 is centrally involved in mammary gland development , and has been demonstrated to promote tumour initiation and formation in the tetracycline-regulated mouse mammary model [62, 63]. IHC and IS H in vivo analysis has demonstrated MMP-3 in both the tumour and stroma cellular compartments of both invasive and non-invasive tumours, with the level of stromal expression increasing with tumourigenicity [59, 64, 65], and in the extracellular matrix adjacent to breast tumours . Our observation of MMP-3 gene expression in the stroma of four out of six of the xenografts examined, and in particular in the Hs578T xenograft, further support a role for this MMP in breast cancer.
Although HBC cell lines have been demonstrated to express certain MMPs, this is less evident in vivo where, as detailed above, the surrounding stromal cells contribute to much of the MMP activity. Important differences in MMP expression between the stromal cells recruited by each cell line and the tumour cells themselves, were detected in the current study. The MCF-7 xenograft stroma demonstrated low stromal MT1-MMP, MMP-1 and MMP-3 expression. MT1-MMP expression was observed at a lower level in the stroma associated with the MDA-MB-231 mesenteric metastasis. In the Hs578T xenograft stroma, MMP-1 was absent while MMP-3 was observed at a higher level in the stroma as compared to the tumour cells. No stromal expression of the three MMPs examined was observed in either the MDA-MB-231 spleen metastasis or the MDA-MB-435 xenografts. The level of stromal gene expression will depend on signals from the different HBC lines in the primary site, and also on the different responsivities in the different host sites for the splenic and mesenteric metastases. In regard to the primary site, overall recruitment of the stroma will differ among the HBC lines; however, β-actin mRNA detection was used to normalise the data. We did detect lower levels of β-actin in the Hs578T xenograft, and indeed higher levels of β-actin in the tumour cell component of the MCF-7 xenograft. However, this does not appear to have influenced the detection of the MMPs as indicated by the moderate levels of all MMPs examined in both compartments in the MCF-7 xenograft, and the moderate level of MMP-3 in the Hs578T tumour compartment.