Tumors systemically initiate metastatic niches in distant target metastatic organs. These niches, composed of bone marrow–derived hematopoietic cells, provide permissive conditions for future metastases. However, the mechanisms by which these cells mediate outgrowth of metastatic tumor cells are not completely known. Using mouse models of spontaneous breast cancer, we show enhanced recruitment of bone marrow–derived CD11b+Gr1+ myeloid progenitor cells in the premetastatic lungs. Gene expression profiling revealed that the myeloid cells from metastatic lungs express versican, an extracellular matrix proteoglycan. Notably, versican in metastatic lungs was mainly contributed by the CD11b+Ly6Chigh monocytic fraction of the myeloid cells and not the tumor cells or other stromal cells. Versican knockdown in the bone marrow significantly impaired lung metastases in vivo, without impacting their recruitment to the lungs or altering the immune microenvironment. Versican stimulated mesenchymal to epithelial transition of metastatic tumor cells by attenuating phospho-Smad2 levels, which resulted in elevated cell proliferation and accelerated metastases. Analysis of clinical specimens showed elevated versican expression within the metastatic lung of patients with breast cancer. Together, our findings suggest that selectively targeting tumor-elicited myeloid cells or versican represents a potential therapeutic strategy for combating metastatic disease. Cancer Res; 72(6); 1384–94. ©2012 AACR.
Malignant tumors colonize distal target organs to establish metastases (1, 2) causing more than 90% of human cancer-related deaths (3). Many studies have investigated the cancer cell intrinsic molecular mechanisms and the extrinsic microenvironmental factors that enhance the metastatic potential of primary tumor cells (3, 4). Notably, activation of epithelial to mesenchymal transition (EMT), a developmental program, endows metastatic properties upon cancer cells to promote invasion, migration, and subsequent dissemination (5).
Following dissemination, the establishment of metastatic lesions depends on the organ-colonizing properties of disseminated tumor cells as well as on permissive conditions or the “metastatic niche” that may be present in the microenvironment of target organs (3, 6). Notably, bone marrow–derived cells are recruited to the metastatic organs to support initiation of metastases (7), and angiogenesis-mediated progression of micrometastases to macrometastases (8). Although some of these stromal contributors have been identified, effective therapies still require a more comprehensive understanding of the complex molecular and cellular network of tumor–stroma interactions in the metastatic organ that contribute to the formation of macrometastases. Transitions between epithelial and mesenchymal states have crucial roles in both embryonic development and cancer. An apparent contradiction of the association between EMT and metastasis comes from repeated observations that distant metastases derived from primary carcinomas are largely composed of cancer cells showing an epithelial phenotype closely resembling that of the cancer cells in the primary tumor. This has led to speculation that the disseminated mesenchymal tumor cells recruited to the target organs may undergo mesenchymal to epithelial transition (MET) which would favor metastases formation (9). However, the MET cascade has not been recapitulated in animal models, and the cellular and molecular regulators that promote MET remain unknown. In this study, we have focused on events in the distal metastatic organ and sought to identify the microenvironmental factors that affect metastatic outgrowth.
Materials and Methods
FVB/n, FVB.Cg-Tg (ACTB-EGFP) B5Nagy/J, and MMTV-PyVT mice were obtained from The Jackson Laboratory. Male MMTV-PyMT transgenic mice were bred with wild-type FVB/N females. Female offsprings (3 weeks) were genotyped to identify mice carrying the PyMT transgene. The positive mice spontaneously develop mammary tumors by 6 to 7 weeks of age and pulmonary metastases by 10 to 12 weeks of age. To quantify lung metastases in MMTV-PyMT mice, serial lung sections (at least 10) were prepared and stained with hematoxylin and eosin. Within the stained sections, areas depicting metastatic lesions and total lung were measured with ImageJ software.
The human breast cancer cell line (MDA-MB-231) was obtained from American Type Culture Collection (ATCC), a kind gift from Dr. Randy Watnick (Harvard Medical School, Boston, MA). Cultures were resuscitated from stocks frozen at low passage within 6 months of purchase. Cell authentication was conducted at ATCC by short tandem repeat profiling, cell morphology monitoring, karyotyping, and the ATCC cytochrome c oxidase I (COI) assays. The morphology and metastatic behavior of MDA-MB-231 cells were tested in our laboratory and the laboratory of Dr. Watnick. Cells were cultured in Dulbecco's Modified Eagle's Media with 10% FBS, 5 mmol/L glutamine, and 1% penicillin/streptomycin. Versican-expressing cells (MDA-Vcn) were generated by transfection with a construct carrying the secreted form of human versican cDNA (pSecTag-V1, a gift from Dr. Zimmermann, University of Zurich, Zurich, Switzerland; ref. 10). Stably transfected cells were obtained by selecting cells with Zeocin (200 μg/mL). MDA-Cont cells were obtained by transfection of MDA-MB-231 cells with control empty vector and selected through the same procedure. MDA-MB-231 cells were also labeled with luciferase-RFP (red fluorescent protein) fusion protein for bioluminescent imaging in vivo.
Human normal lung tissues (n = 5) were obtained from ILSbio LLC. Human lungs bearing metastases from patients with breast cancer were obtained from Department of Cardiothoracic Surgery, Weill Cornell Medical College (New York), consented according to approved Institutional Review Board (IRB) protocols from the institution. Metastatic tissues from the lungs of patients with breast cancer (n = 11) were obtained in the form of a tissue microarray from the group of P. Argani and S. Sukumar at Johns Hopkins University School of Medicine, Baltimore, MD.
Short hairpin RNA design and lentiviral vector generation
Mir30-based short hairpin RNAs (shRNA) targeting mouse versican (V0/V1 isoform) were designed and cloned into the XhoI/EcoRI site of the lentiviral construct pGIPZ (OpenBiosystem). Multiple hairpin constructs were screened for effective knockdown of versican. The 22-mer targeting sequence that resulted in efficient knockdown included 5′-ACACCAGAATTAGAAAGTTCAA-3′ (shVcn1) and 5′-AGCACCTTGTCTGATGGCCAAG-3′ (shVcn2), shRNA targeting firefly luciferase served as a nonspecific control. Lentivirus was generated and concentrated using standard protocols. Lentiviral transductions of Lin− bone marrow cells were conducted as described (8).
Bone marrow transplantation of mice
Bone marrow cells were harvested by flushing femurs and tibias of donor animals. Bone marrow transplantation was conducted by injecting 1 × 107 total bone marrow cells via tail vein into lethally irradiated (900 radians) recipients as described (8).
Immunostaining and microscopy
Fixed tissue sections (30 μm) were stained with fluorescence-labeled antibodies against CD11b (clone ICRF44), CD33 (Cat#555459, BD), Gr1 (Clone RB6-8C5), versican (Cat#V5639, Sigma and Clone 2B1 Seikagaku), and PyMT (NB 100-2749, Novus) according to standard protocols. Fluorescent images were obtained using an Axiovert 200M fluorescent microscope (Carl Zeiss Inc.). For versican staining, sections were first treated with chondroitinase ABC (Sigma) overnight and then incubated with an anti-versican antibody (Cat#V5639, Sigma). For immunohistochemistry, the antibody labeling was visualized using the DAKO Envision system (DAKO). Images were taken with an Olympus BX51 microscope coupled with QCapture software (Olympus).
Conditioned medium, purified versican, and cell proliferation assay
Flow cytometry–sorted CD11b+Gr1+ cells from MMTV-PyMT mice (10 weeks old) were cultured in RPMI with 10% FBS with a density of 100,000 cells/mL in 6-well plates. Conditioned medium was harvested after 2 days. For versican purification, 293T cells were transfected with construct carrying 6× His-tagged human versican (V1 isoform). Supernatant was harvested 5 days after transfection. Versican was purified with Ni-NTA Fast Start Kit (Qiagen Inc). For cell proliferation assay, cells were treated with the conditioned medium, purified versican (2.5 μg/mL), or normal growth medium for 3 days. EdUrd (5-ethynyl-2′-deoxyuridine; 10 nmol/L) was administered to culture medium for 30 minutes. The incorporation of EdUrd was detected using the Click-iT EdUrd Cell Proliferation Assay Kit (Invitrogen Inc.) and analyzed by flow cytometry.
Results are expressed as mean ± SD. Analyses of different treatment groups were conducted using the Student T test using the GraphPad Prism statistical program. P values <0.05 were considered significant. Error bars depict SD, except indicated otherwise.
Supplementary methods include flow cytometric analysis, real time PCR analysis including primer sequences (Supplementary Table S1), and Western blot analysis.
Bone marrow–derived CD11b+Gr1+ myeloid cells are recruited in the metastatic lungs
To determine the contribution of the bone marrow–derived cells to the metastatic lung, we transplanted MMTV-PyMT transgenic mice (11) with syngeneic GFP+ bone marrow as previously described (8, 12). In these animals, spontaneous breast tumors (6–7 weeks of age) metastasize to the lungs and form micrometastases (11–12 weeks of age) and macrometastases by week 16 (8, 12). Flow cytometric analysis showed increased recruitment (∼3-fold) of GFP+ bone marrow–derived cells in the MMTV-PyMT metastatic lungs compared with wild-type (36.3% ± 4.3% and 12.4% ± 4.2% of total lung cells, respectively; Fig. 1A). Notably, in the metastatic lung, the recruited GFP+ bone marrow–derived cells were predominantly CD11b+Gr1+ myeloid progenitor cells (>50%; Fig. 1A) as determined by flow cytometry and immunostaining (Fig. 1B).
We next conducted a kinetic analysis to determine the recruitment of these cells as a function of metastases progression. Notably, the recruitment of CD11b+Gr1+ cells was observed in the premetastatic lung of 8-week-old MMTV-PyMT mice before the appearance of metastases, and increasing numbers of CD11b+Gr1+ cells were associated with the progression of metastases (Fig. 1C and Supplementary Fig. S1). Conspicuously, compared with the metastatic lung, the numbers of CD11b+Gr1+ myeloid cells were less abundant in the primary tumor. The CD11b+ cells in primary tumor tissue were predominantly Gr1−F4/80+ macrophages (approximately 80%) in contrast to the Gr1+F4/80− myeloid cells in metastatic lung (Fig. 1D). Such enhanced recruitment of myeloid cells specifically in the metastatic lungs suggests that they may be involved in promoting outgrowth of tumor cells.
Versican is expressed by myeloid cells in the metastatic lung
To determine the molecular mechanisms by which the CD11b+Gr1+ myeloid cells may contribute to lung metastasis, we conducted gene expression profiling of flow cytometry–sorted CD11b+Gr1+ cells from metastatic and wild-type lungs. A cluster of differentially upregulated genes was identified in the myeloid cells from metastatic lungs (Fig. 2A). Of the candidate genes, we focused on versican, an extracellular matrix chondroitin sulfate proteoglycan (13), expressed by tumor stromal cells (14–19). However, the biologic function of versican in vivo particularly in the metastatic organs has not been elucidated. RT-PCR analysis showed an approximately 5-fold increase in versican expression in the metastatic lung (ML, total) compared with controls (WT, total; Fig. 2B). In the metastatic lungs, versican expression was confined to CD11b+Gr1+ cells and not to the CD11b−Gr1− stromal cells including subsets of T and B cells (Fig. 2B). Consistent with RT-PCR, Western blot analysis showed elevated versican protein in the metastatic lung compared with wild-type controls (Fig. 2C).
Flow cytometric analysis showed that the CD11b+Gr1+ cells are composed of CD11b+Ly6Chigh and CD11b+Ly6Ghigh subpopulations (Fig. 2D; refs. 20–22) and their recruitment increased as a function of metastatic progression (Supplementary Fig. S2A and S2B). Interestingly, versican expression was confined to the Ly6Chigh cells (Fig. 2E). Nuclear morphology analysis showed that the CD11b+Ly6Chigh cells are mononuclear, whereas the CD11b+Ly6Ghigh cells are polymorphonuclear (Fig. 2F). Consistently, versican protein was also detected in the mononuclear CD11b+Ly6Chigh cells by immunohistochemical (IHC) and Western blot analyses (Fig. 2F and G). In the primary tumors, versican was also confined to the low abundance CD11b+Ly6Chigh myeloid cells, whereas the abundant CD11b+F4/80+ myeloid cells did not express versican (Supplementary Fig. S3).
Contrary to previous studies using cultured cell lines (23–25), endothelial cells and the fibroblasts did not show significant versican levels compared with CD11b+Ly6Chigh cells in the metastatic lungs (Supplementary Fig. S4A). Importantly, the contribution of fibroblasts to the metastatic lung was about 10-fold lower compared with CD11b+Ly6Chigh cells, and no significant increase in numbers was observed in the metastatic lungs compared with controls (Supplementary Fig. S4B). Furthermore, analysis a panel of tumor cell lines showed significantly lower versican expression compared with sorted myeloid cells (Supplementary Fig. S4C). Taken together, these results suggest that the Ly6Chigh myeloid cells are the major contributors of versican in metastatic lungs.
Versican suppression in myeloid cells impairs lung metastases
To explore the role of myeloid cell–derived versican in pulmonary metastasis, we conducted versican knockdown in bone marrow cells in vivo. Given that versican is confined to the CD11b+Ly6Chigh myeloid cells, we reasoned that versican knockdown in total bone marrow cells would only impact the Ly6Chigh cells, thereby providing exquisite specificity. Two shRNAs specifically targeting exon 8 (V0/V1-specific exon) were generated (Supplementary Fig. S5A and S5B), which effectively reduced endogenous versican (V0/V1) expression as compared with nonspecific shRNA control (Supplementary Fig. S5C).
Versican-specific shRNA (shVcn) or nonspecific shRNA (shNS) was introduced via lentiviruses into lineage wild-type–negative (Lin−) bone marrow progenitor cells and then transplanted into lethally irradiated MMTV-PyMT recipient mice (4 weeks old) as described in our previous studies (8, 12). Successful bone marrow reconstitution was confirmed by flow cytometric analysis of transplanted recipient mice (Supplementary Fig. S6A). In the lungs, as expected, versican expression was upregulated (>3-fold) in shNS-BMT MMTV-PyMT mice compared with controls (Supplementary Fig. S6B). However, versican expression was inhibited in shVcn-BMT mice (Supplementary Fig. S6B). Versican knockdown in the recruited Gr1+ cells in the lungs of shVcn-BMT mice (Fig. 3A and B), resulted in impaired lung metastases compared with shNS-BMT controls (0.60% ± 0.25% vs. 12.8% ± 3.2%, respectively; Fig. 3C and D). IHC examination of the lungs from shVcn-BMT animals showed severe impairment of macrometastases while micrometastases remained unaffected (Fig. 3E, Supplementary Fig. S7), whereas the primary breast tumors remained unaffected in these mice. Taken together, these results show that versican deficiency in the recruited myeloid cells significantly impaired tumor outgrowth at the metastatic site.
Myeloid cell–derived versican enhances proliferation of metastatic tumor cells to promote outgrowth
Versican knockdown did not affect the recruitment of CD11b+Gr1+ myeloid cells in the lung microenvironment as determined by immunostaining (Fig. 3A) and flow cytometry (15.8% ± 1.9% in shRNA-BMT vs. 15.3% ± 4.2% in shVcn-BMT mice; Supplementary Fig. S8A and S8B), nor did it perturb the recruitment of other bone marrow cells including B cells (B220+) or T cells (CD3+; Supplementary Fig. S8A and S8B). As expected, the immune microenvironment of the lungs remained unperturbed as a result of versican knockdown as evaluated by expression of key mediators including TNF-α, interleukin (IL)-1, IL-6, IL-4, IL-10, arginase 1, arginase 2, and NOS2 (Supplementary Fig. S8C), suggesting that versican deficiency did not affect myeloid-derived suppressor cell (MDSC) activity of myeloid cells. Taken together, these results suggest a novel function for these myeloid progenitor cells in promoting metastases.
Previous studies have shown that versican V1 promotes cell proliferation in NIH3T3 fibroblasts (25). Therefore, we posited that versican expressed by recruited myeloid cells might enhance proliferation of metastatic tumor cells to promote tumor outgrowth. Consistent with this hypothesis, abundant Ki67+ proliferating cells were observed in metastatic lesions in shNS-BMT mice where versican was available, compared with metastatic lesions from shVcn-BMT mice where versican expression was suppressed (Fig. 3F and G; Supplementary Fig. S9A).
Taken together, these results show that versican deficiency in the bone marrow does not affect the recruitment of CD11b+Gr1+ cells and other immune cells to the lung microenvironment. However, versican expressed by these myeloid cells promotes metastatic tumor outgrowth by enhancing cell proliferation.
Versican promotes proliferation and induces MET of metastatic tumor cells
To further evaluate the cell proliferation promoting function of versican, we used metastatic human breast cancer cells, MDA-MB-231. To mirror the paracrine effects of versican secreted by myeloid cells on metastatic tumor cells in vitro, we generated conditioned media from flow cytometry–sorted CD11b+Gr1+ cells from the lungs of tumor-bearing mice. Administration of the Gr1 conditioned media to MDA-MB-231 cells increased cells in S-phase compared with controls (Fig. 4A). Consistently, the proliferation rate of MDA-MB-231 cells was enhanced following expression of a secreted form (10) of versican V1 isoform (12.1% and 5.4% of S-phase cells with and without versican, respectively; Supplementary Fig. S9B).
Interestingly, versican-induced proliferation increase in MDA-MB-231 cells treated with Gr1 conditioned media was associated with the acquisition of an epithelial phenotype as indicated by upregulation of epithelial cell markers including E-cadherin and occludin and a concomitant inhibition of the mesenchymal marker vimentin (Fig. 4B). These results suggested that an MET had occurred in these cells, presumably mediated by versican present in the Gr1 conditioned media. To confirm this, we treated MDA-MB-231 cells with biochemically purified versican (V1 isoform; Supplementary Fig. S10A and S10B), and observed induction of MET (Fig. 4B). In agreement with this observation, MDA-MB-231 cell expressing a secreted form of versican (V1 isoform) established aggregated cobblestone-like colonies (Fig. 4C, phase-contrast; top and bottom) and showed induction of epithelial and suppression of mesenchymal markers as determined by immunostaining, Western blot, and RT-PCR analysis (Fig. 4C and D; Supplementary Fig. S11). Importantly, versican attenuated phospho (p)-Smad2 levels in MDA-MB-231 cells, whereas the levels of total Smad2/3 remained unchanged (Fig. 4D). Given that p-Smad2 is a regulator of key EMT-promoting transcription factors including Snail, it is plausible that versican-mediated attenuation of p-Smad2 levels (Fig. 4D) and suppression of Snail (Supplementary Fig. S11) in MDA-MB-231 cells may have inhibited the TGF-β/Smad2/3 signaling pathway, a well-known stimulator of EMT in various tumors (26–29). These results suggest that versican-mediated blockade of the TGF-β/Smad2 pathway may stimulate MET, resulting in increased cell proliferation which collectively promotes focal tumor outgrowth at the metastatic site.
Versican deficiency inhibits metastases in vivo by blocking MET
In tumor progression, EMT confers an invasive and metastatic phenotype that supports escape of tumor cells from the primary tumor site. It is speculated that subsequently, the disseminated mesenchymal tumor cells must undergo the reverse transition, MET, at the site of metastasis, as metastases recapitulate the pathology of their corresponding primary tumors (9, 30, 31). However, MET has not been accurately recapitulated in breast cancer metastasis. To assess if MET occurs in vivo, we inoculated MDA-MB-231 cells that exhibit a typical mesenchymal phenotype (E-cadherin−/vimentin+) in severe combined immunodeficient (SCID) mice and allowed metastases to develop in the lungs. Notably, MDA-derived metastases exhibited an E-cadherin+ epithelial phenotype (Supplementary Fig. S12A and S12B), suggesting that MDA-MB-231 cells have undergone MET in the lung environment. In this context, analysis of lung metastases from patients with breast cancer also showed E-cadherin+ and vimentin− metastases (Supplementary Fig. S12C). On the basis of these observations, we hypothesized that myeloid cells recruited in the premetastatic lungs produce versican which induces MET in tumor cells to promote tumor outgrowth.
To determine whether versican-mediated MET is necessary for metastases formation in vivo, we injected luciferase-RFP–labeled MDA-MB-231 cells via tail vein in SCID mice and monitored metastases progression either in the presence of versican (control IgG–treated mice) or in lack of versican-producing myeloid cells (anti-Gr1–treated mice). Depletion of Gr1+ myeloid cells significantly impaired progression of metastases by more than 5-fold (Fig. 5A and B). Of note, the anti-Gr1 antibody treatment significantly inhibited the upregulated versican expression in the metastatic lungs (Fig. 5C). Further evaluation of the lungs showed that macroscopic E-cadherin+/vimentinlow metastatic lesions were generated by MDA-MB-231 cells in the presence of versican (Fig. 5D, top) as expected. However, versican deficiency resulted in microscopic vimentinhigh/E-cadherin− lesions (Fig. 5D, bottom) indicating a failure of MET. To confirm that the MET-induced accelerated metastasis was mainly due to versican, we carried out a versican gain-of-function experiment. We injected MDA-MB-231 cell expressing a secreted form of versican V1 isoform (MDA-Vcn) and MDA-Cont cells via tail vein in SCID mice. Bioluminescence imaging analysis revealed accelerated progression (>4-fold) of lung metastases with MDA-Vcn cells compared with MDA-Cont cells (Fig. 5E and F).
Taken together, these results suggest that versican promotes MET of metastatic tumor cells and enhances progression into macrometastatic lesions.
Myeloid cells express versican in the metastatic lungs of breast cancer patients
Next, we asked whether metastatic lungs of patients with breast cancer exhibit elevated levels of versican as observed in mouse models. IHC analysis showed enhanced versican in the metastatic lungs of patients with breast cancer but not in lungs of normal healthy controls (Fig. 6A). IHC analysis of metastatic lungs showed that versican expression was confined to the vicinity of CD11b+ myeloid cell clusters (Fig. 6B). Using a cohort of patients with breast cancer who have developed lung (n = 6) or liver (n = 11) metastases, we quantified versican expression by RT-PCR. Significantly higher versican expression was detected in metastatic organs than in normal tissues (5.8 ± 1.3- and 6.5 ± 1.5-fold in the liver and lung metastasis, respectively; Fig. 6C). We further evaluated whether a subset of CD11b+ cells expressed versican in patients with cancer. Given that Gr1 is not expressed in humans, myeloid cells are defined by the coexpression of CD11b and CD33 (20). Indeed, flow cytometric analysis showed that the CD11b+CD33+ cells that comprise the monocytic population expressed versican, whereas the CD11b+CD33− fraction or the pure tumor cells did not (Fig. 6D and E). These results suggest that as observed in mouse models, versican expressed by tumor-elicited myeloid cells may contribute to metastasis in patients with cancer.
We have shown that bone marrow–derived CD11b+GR1+ myeloid progenitor cells recruited to the premetastatic lungs promote tumor outgrowth. Various protumorigenic activities have been associated with Gr1+ myeloid cells, including expression of proangiogenic factor BV8 (32), metastasis-promoting lysyl oxidase and matrix metalloproteinase 9 (MMP9; ref. 33), contribution to TGF-β–mediated metastasis (34), and immune tolerance and suppression by virtue of innate MDSC activity (35, 36). Here, we have identified a novel role for the recruited Gr1+ myeloid cells in mediating tumor outgrowth in metastatic lungs. By secreting versican, the Ly6Chigh monocytic subpopulation of Gr1+ myeloid cells, in a paracrine fashion, promoted cell proliferation and induced MET of tumor cells (Fig. 7). Importantly, versican deficiency in the Gr1+ cells did not block their recruitment to the lung microenvironment; neither did it alter the immune microenvironment of the lung as determined by evaluation of key cell types and immune mediators suggesting that the MDSC activity of these cells was not compromised. However, versican knockdown in myeloid cells or specific depletion of versican-producing myeloid cells significantly impaired macrometastases formation.
In the metastatic lungs, the major contributors of versican were CD11b+Ly6Chigh cells. While the fibroblasts expressed versican as observed in previous studies (23–25), the contribution of fibroblasts to the metastatic lung was about 10-fold lower compared with CD11b+Ly6Chigh cells, and no significant increase in numbers was observed in the metastatic lungs compared with controls (Supplementary Fig. S4B). Consistent with these observations, versican knockdown specifically in the bone marrow significantly impaired lung metastasis in vivo. However, in one report, versican expression by Lewis lung carcinoma (LLC) cells promoted metastasis (37). Our analysis of a panel of tumor cell lines from breast, prostate, colon, and lung cancer showed that versican expression in tumor cells is significantly lower compared with myeloid cells. On the basis of all these results, we conclude that the Ly6Chigh myeloid cells are the major contributors of versican in metastatic lungs in both mouse and human (Figs. 2 and Fig. 6 and Supplementary Fig. S4C). MET plays an important role in development (38); however, the MET cascade has not been shown in tumor metastasis. In this study, we have observed E-cadherin+ and vimentin− metastatic lesions in both mice and human. Consistently, in a recent study, IHC analysis of many human primary lung tumors and their brain metastases showed increased epithelial to mesenchymal ratios in metastatic lesions compared with advanced primary tumors (39). Thus, our observation that versican stimulates MET of metastatic tumor cells explains, in part, the observations that cancer cells in distant metastases exhibit an epithelial phenotype resembling the primary tumor from which they arose. Our study has also addressed the speculation in the field that metastatic tumor cells recruited to distal target organs undergo a reverse EMT often referred to as MET (9, 30, 31). However, technological limitations, such as lack of robust markers, to track single disseminated tumor cells that have undergone EMT to the metastatic site in patients preclude a detailed analysis of MET in humans, further exacerbated with limitations in noninvasive dynamic imaging at the single-cell level.
We have also explored the mechanism by which versican induces MET. Versican attenuated the Smad-mediated EMT signaling pathway as determined by the reduction of p-Smad2 levels and suppression of transcription factor Snail. The suppression of Smad2 pathway–induced MET increased cell proliferation consistent with previous reports (40, 41). In addition, versican did not impact apoptosis (data not shown), suggesting that versican-mediated stimulation of MET and enhanced proliferation may be the main mechanisms of increased tumor outgrowth and formation of focal macrometastases. Analysis of a cohort of patients with breast cancer from whom distal metastases were available showed clusters of myeloid cells expressing versican in the metastatic lungs. Consistent with immunohistochemistry, RT-PCR analysis showing enhanced versican expression in metastatic lungs, brain, and liver in patients with breast cancer compared with control normal lungs. These observations suggest that versican expression in the metastatic organ may be used to predict survival, given the relationship between metastases and poor survival in patients with breast cancer. However, such an analysis is challenging at the present time due to limited availability of human metastatic lesions, and future studies are required to expand the analysis in a much larger cohort of patients with breast cancer to determine with statistical confidence whether increased levels of versican expression in the lungs correlate with poor survival.
Given that the initial colonization by disseminated tumor cells has occurred in many patients at the time of primary tumor diagnosis, our results suggest that selectively targeting tumor-elicited myeloid cells or versican-mediated proliferation pathways, perhaps in combination with conventional chemotherapeutics, may represent a potential therapeutic strategy for combating metastatic disease.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interests were disclosed by other authors.
The authors thank Drs. Zimmermann (University of Zurich) for human versican cDNA clone and Anna Durrans for valuable comments on the manuscript.
The work was partly supported by Neuberger Berman Lung Cancer Laboratory, the Robert I. Goldman Foundation, and Cornell Center on the Microenvironment and Metastasis through Award Number U54CA143876 from the National Cancer Institute (V. Mittal).
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.