Tumor-associated Macrophages Regulate Colorectal Cancer Stem Cell Properties

In normal tissues, stem cells harbor in a specialized niche that regulates their stem cell properties and fate. The niche components provide context signals to keep stem cells in a balance between self-renewal and differentiation. Tumors are perceived as abnormal organs and only a small fraction of tumor cells, termed cancer stem cells (CSCs), have the capacity to initiate a new tumor. Emerging evidence shows that CSCs also shelter in a malignant tumor microenvironment which named CSC niche. Similar to the regulation of normal stem cells by their niches, CSCs are regulated by, and in turn regulate, surrounding cells within the tumor microenvironment. These stromal cells, including cancer-associated fibroblasts, endothelial cells, infiltrating leukocytes, macrophages, myofibroblasts and mesenchymal stem cells, interact with CSCs directly or via growth factors and cytokines. Among the stromal cells, tumor-associated macrophages (TAMs) represent almost half of the tumor mass. High levels of tumor-associated macrophages are always correlated with cancer metastasis and poor patient outcomes. Although numerous studies have highlighted the links between TAMs and tumor initiation, proliferation, immunosuppression, angiogenesis and metastasis, the role of TAMs on colorectal CSC activities remains largely unknown.In this study, we isolated CD44-positvie colorectal cancer cells and TAMs from patient-derived xenograft tumors, and then co-injected these two population into nude mice, and found that TAMs, but not the peritoneal macropahges, have a great contribution in promoting tumorigenesis of CD44-positive colon cancer cells. We next extracted the total RNA from peritoneal macropahges and TAMs, and then performed the quantitative real-time PCR assays, we found that osteopontin, a secreted glycoprotein, was highly expressed in TAMs. Immunohistochemistic assays showed that, in both xenograft tumors and patient samples, the grade of tumor-infiltrating macropahges were higher than normal tissues, as well as the expression level of osteopontin. Double immunofluorescent staining with anti-OPN and anti-CD68antibody, which is a marker of macrophages, revealed the colocalization of OPN and CD68at the stromal area of tumor sections. These results suggest that OPN are mainly derived from TAMs.To investigate if colorectal cancer cells have any influences on the expression of OPN in macropahges, we established a co-culture system and demostrated that CD44-positive colorectal cancer cells, from patient tumor samples or established cancer cell lines, were able to promote OPN secretion in TAMs, and the induction of OPN is closely associated with CD44expression on cancer cells. Knockdown of CD44expression in cancer cells caused the reduction of OPN expression in macrophages. In vitro colony forming assay shows that TAM-derived OPN could promote soft agar clone formation, and a CD44v6monoclonal antibody could inhibit the OPN-promoted clonogenicity. We next constructed several different OPN mutants and purified the protein from E.coli, and found that the C-terminal of OPN, especially CD44v6/v7-binding domain, is mainly responsible for OPN-promoted clonogenicity. Western blotting assay reveals that OPN can activate JNK signaling pathway via binding to CD44receptor. In addition, a CD44v6neutralizing antibody could attenuate the OPN-induced JNK signaling activation. Knockdown of JNK expression by using lentivirus-based RNA interference resulted in the inhibition of OPN-promoted clonogenicity.To determine the clinical relevance of our findings, we next collected190colorectal cancer samples from patients, and performed tissue microarray. Immunohistochemistry results demonstrated that the OPN expression level and the tumor-associated macrophages were significantly higher in poorly differentiated than in moderately and well-differentiated samples. We also test whether there were prognostically significant associations between the expression of OPN, CD44v6or CD68and overall patient survival, and found that there were no significant differences between overall survival and the expression of OPN, CD68or CD44v6. However, if we analyzed overall patient survival with the combination of OPN with CD44v6, a significant negative correlation was observed.Taken together, our results show that OPN-CD44v6interaction activates JNK signaling pathway and promotes colon cancer clonogenicity, thus provide an explanation for tumor microenvironment regulates cancer progression, and the combination of OPN with CD44v6may be served as a potential prognostic indicator for patient survival.