In this report, we have shown that both premalignant (vHMEC-ras0.5) and malignant (MDA-MB-231) mammary epithelial cells can assume an aligned mesenchymal morphology when co-cultured with CAFs but not RMFs. This phenotype differs from the classic epithelial-to-mesenchymal transition that is accompanied by a down-regulation of E-cadherin. Instead, this mesenchymal phenotype appears to be governed at least in part by the organization of the ECM deposited by CAFs in that it was not induced by CAF-conditioned media or CAFs in transwell co-cultures with the epithelial cells where the two cell populations were not in contact with one another, but it was induced when the epithelial cells were overlaid on top of ECM deposited by CAFs, and it was blocked in co-cultures when deposition of ECM was blocked. Thus, while it is well known that some CAF-induced phenotypes can be transmitted by secreted factors [24
], this particular CAF-induced phenotype requires contact with deposited ECM proteins.
Importantly, our data indicate that the phenotype induced by CAFs in vitro
is biologically relevant in vivo
in that the aligned ECM deposited by CAFs in vitro
could be detected in the mammary glands of mice following co-injection with premalignant cells as well as in tumors following co-injection with malignant cells. This ECM phenotype is also clinically relevant in that studies have shown that the radial alignment of collagen fibers relative to tumors facilitates local invasion [15
], and correlates with poor disease-free survival, suggesting that quantifying collagen alignment could be a viable, novel strategy for the prediction of human breast cancer survival [16
]. Our results reinforce this notion and, in addition, suggest that the presence of aligned collagen could also predict dissemination in the earliest stages of breast cancer development.
There is increasing evidence that in some cancers, tumor cells start spreading long before the primary tumor is detected and removed [2,28
]. Our data suggest that alterations in the composition and organization of the ECM surrounding premalignant lesions could account for this. Alterations in the ECM component of the stroma that resemble those observed in malignant lesions are often observed in the context of wound healing, radiation response, pregnancy-associated involution, and high mammographic density [29–33
]. These alterations manifest even in the absence of malignancy and may thus provide a favorable environment for early dissemination of premalignant cells. This might explain why certain cancers that are driven by alterations in ECM, such as postpartum breast cancers, have such poor prognosis [32
] and why metastatic dissemination can occur in women with small tumors.
Our data suggest that alterations in stromal ECM that result in the induction of an aligned mesenchymal phenotype are both necessary and sufficient to enhance dissemination because a CAF that failed to induce the phenotype did not enhance dissemination (Figure W8
) and an RMF that did induce the mesenchymal phenotype did (). However, unlike the induction of the aligned mesenchymal phenotype by a CAF, which enhanced both dissemination and metastasis, the aligned mesenchymal phenotype induced by an RMF enhanced dissemination but not metastasis. These data indicate that reorganization of the ECM plays a key role in the early stages of the metastatic cascade but is not sufficient to bestow on tumor cells a fully enhanced metastatic potential and suggest that CAFs induce additional alterations in tumor cells that endow them with the ability to metastasize more efficiently. Notably, the alterations that drive metastatic dissemination are uncoupled from those that drive primary tumor growth as all three CAFs co-injected with tumor cells increased pulmonary metastatic burden ( and ) whether they stimulated, inhibited, or failed to affect primary tumor growth ( and ). These data highlight the heterogeneity of signals emanating from the tumor microenvironment.
Importantly, the composition and organization of the ECM cannot only promote the tumorigenic and metastatic potential of malignant cells, but it can also constrain it, as RMFs isolated from four different disease-free individuals inhibited primary tumor growth ( and ) and one of these also inhibited metastatic spread to the lung ( and ). In addition, RMFs also inhibited the dissemination of premalignant cells (). These data imply that maintaining a normal ECM environment can be therapeutically beneficial. Along these lines, others have shown that normalizing a matrix-rich desmoplastic stroma can enhance the delivery and efficacy of chemotherapy [34,35
]. In one study, this was achieved with the antihypertensive drug losartan, which depletes ECM in part by suppressing active TGFβ levels [35,36
]. Consistent with this, we observed that blocking TGFβ signaling as well as other signaling pathways through which TGFβ induces a mesenchymal phenotype and ECM remodeling prevented the CAF-induced mesenchymal phenotype in co-culture assays. TGFβ's role in promoting metastasis is well established; however, targeting TGFβ in the clinic has been a challenge due to the complex nature of TGFβ's tumor suppressive and tumor promoting actions. Losartan may represent a safe alternative to indirectly inhibit some of the ECM-related pro-metastatic effects of TGFβ. In fact, losartan has been used clinically to abrogate excess TGFβ in patients with Marfan's syndrome, a genetic disorder caused by mutations in the ECM protein fibrillin-1, which binds to the large latent TGFβ complex [37,38
]. Antihypertensive therapeutics such as losartan could potentially also be used prophylactically, as there is evidence that women on anti-hypertensive therapy have a lower risk of developing breast cancer [39
In conclusion, our data underscore the potential of the ECM as a prognostic and therapeutic target. Targeting ECM proteins, such as biglycan and LOX, that are involved in the organization and cross-linking of collagen may prove effective in limiting dissemination, an event that may occur much earlier than previously appreciated. Hence, the possibility that cells may have disseminated long before the detection of the primary tumor must be considered when designing experiments and testing new therapeutics. To make personalized cancer medicine a success, companion diagnostics aimed at identifying the right patient populations for the right therapeutics should also include markers to assess early systemic cancer spread.