In this translational study, we showed association between CTC with EMT phenotype (CTC_EMT) and MMP1 expression in primary breast cancer. This association was observed for MMP1 expression in cancer cells as well as in cancer associated stroma. MMP1 expression was increased in primary tumors with poor prognostic features such as high grade tumors with increased proliferation (Ki 67 > 20%) which is consistent with previous observations
. We also found association between ductal carcinoma histology and MMP1 expression as described previously
; however, in our study, this association was statistically significant only for tumor associated stroma.
Breast cancer represents highly complex tissue composed of cancer cells and stromal cell compartments containing different types of mesenchymal cells. MMPs, in breast cancer, are produced by cancer cells as well as cancer associated stroma and are involved in cancer progression through multiple mechanisms. Degradation of ECM by MMPs facilitates movement of cancer cells through ECM. MMPs also disrupt cell-cell and cell-ECM adhesions that result in the release of individual tumor cells from epithelial sheets, and initiating signaling pathways that lead to widespread changes in gene expression patterns that are responsible for increased migration and invasion of breast cancer cells
[7, 11, 17]. MMPs could also impact cancer cell behaviour due to their ability to cleave growths factors, cell surface receptors, cell adhesion molecules and chemokines
[7, 16]. Therefore, MMPs are promising therapeutic targets in breast cancer and several trials evaluating drugs that interfere with MMPs function are ongoing [reviewed by 11].
EMT is believed to play an important role in intravasation and the release of CTCs, and the expression of EMT-inducing TF gene transcripts in breast cancer has been associated with poor prognosis
. EMT has been previously linked with cancer stem cell properties
 which have been associated with increased therapeutic resistance
[20–22]. MMPs have been associated with EMT in cancer progression by several mechanisms
. Elevated levels of MMPs in the tumor microenviroment can directly induce EMT in epithelial cells. Cancer cells that undergo EMT could produce more MMPs further supporting cell invasion and metastasis and finally, EMT can generate activated stromal like cells that drive cancer progression via further MMPs production
. The most important of these is MMPs mediated activation of EMT that was seen in variety of epithelial tumors and has been best characterized in mammary epithelial cells
. Our observation thus further support association between MMPs and EMT in breast cancer.
EMT of tumor cells could produce stromal-like cells that could further facilitate tumor progression through the production of MMPs. Myofibroblasts are key components of cancer associated stroma in breast cancer and these cells have important tumor promoting activity. Myofibroblasts can be derived through activation of stromal fibroblast or circulating fibrocytes; however, recent studies in mouse models have shown, that myofibroblasts can be derived from epithelial cells by EMT, as well
[23–26]. In our study, we observed an association between CTC_EMT and MMP1 expression in both cancer cells as well as in cancer associated stroma. Expression of MMP1 in the stromal compartment of breast carcinomas possibly represents two populations of cells: EMT transformed neoplastic cells and stromal fibroblastic cells that undergo activation of EMT induced TFs due to growth factors produced by the tumor
. However, we don’t know exactly, if an observed association between CTC_EMT and MMP1 in tumor stroma, was due to expression of MMP1 in stromal fibroblasts, or stromal like cancer cells that underwent EMT.
The flow of cancer cells may not be unidirectional. Experimental data suggest, that CTCs can be released from metastatic tumors and then rejoin the tumor of origin, a process termed ‘self seeding’
[28, 29]. It seems, that self-seeding is a major driver of tumor progression in solid tumors and that CTCs are mediators of tumor self-seeding
. CTCs are an independent prognostic factor in breast cancer and probably represent surrogate marker for tumor self-seeding ability. In seminal work, Kim et al. observed that MMP1 is a mediator of CTCs infiltration into mammary tumors
 and are involved in tumor self-seeding in an animal model. Our translational data are consistent with these observations and further support role of MMP1 in tumor self-seeding.
The EMT is a dynamic and progressive process, and it can also be reversible. Therefore, it usually produces cells with a spectrum of intermediate phenotypic states. Cells can advance to differing extents through an EMT program, progressively acquiring mesenchymal features as they shed epithelial ones
. Cells that have entered an EMT program rarely shed all of their pre-existing epithelial features. In the context of carcinoma pathogenesis, neoplastic cells may reside in a state in which they coexpress newly acquired mesenchymal markers together with retained epithelial ones
[13, 30, 31]. For these reasons our distinction of two CTCs subpopulation (CTC-EP, CTC-EMT) may represent some study limitations.