EPCs cultured for 7C14 days demonstrated a cobblestone appearance on collagen-coated plates and cell-cluster formation on methylcellulose. coinjection increased the tumor volume and vessel formation. Moreover, IL-3, stromal cell-derived factor-1, and vascular endothelial growth factor A in the c-Kit+ASCs + 4T1/EPCs coinjection group were higher than those in the 4T1, EPCs + 4T1, CD38 inhibitor 1 and c-Kit?ASCs + 4T1/EPCs groups. Conclusions c-Kit+ ASCs may promote breast cancer growth and angiogenesis by a synergistic effect of c-Kit and IL-3. Our findings suggest that c-Kit+ subpopulations of ASCs should be eliminated in fat grafts for breast reconstruction of cancer patients following mastectomy. 1. Introduction Adipose tissue-derived mesenchymal stem cells (ASCs) CD38 inhibitor 1 with autologous fat improve the regenerative ability and retention of fat grafts and are increasingly being used for breast reconstruction of breast cancer patients following mastectomy . However, increasing evidence has shown that ASCs may promote the growth and metastasis of breast cancer cells [2C5], and several studies have demonstrated that ASCs CD38 inhibitor 1 inhibit the growth of breast cancer [6, 7]. These contradictory observations may be due to different sources of ASCs, tumor models, and biomarkers for identifying ASCs. To enhance the safety of ASC application in breast CD83 reconstruction, it is very important to identify specific biomarkers to distinguish the breast cancer CD38 inhibitor 1 cell growth-promoting ASC subpopulation from other ASC subpopulations that do not enhance the growth and metastasis of breast cancer cells. c-Kit is a protooncogene located at chromosome 4q12, and its encoding protein is a transmembrane receptor tyrosine kinase [8, 9]. c-Kit is expressed in many cells of the tumor microenvironment, including mesenchymal, mast, and progenitor cells. In breast cancer, the c-Kit/Kit ligand (KitL) signaling pathway promotes the proliferation, survival, and metastasis of tumor cells . Moreover, the expression level of c-Kit is closely related to triple-negative breast cancer . Recently, it was found that c-Kit+ ASCs display a higher differentiation potential in comparison to c-Kit? ASCs [12, 13]. These facts suggest that c-Kit may be a potential biomarker that could distinguish the breast cancer cell growth-promoting ASC subpopulation from other ASC subpopulations. The growth and metastasis of tumor cells is dependent on vessel formation in the tumor mass . It has been shown that tumor cells recruit bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) by increasing the expression of hypoxia-inducible factor-1(HIF-1= 5), the tumor size was measured twice/week, and the tumor volume was calculated according to the following formula: tumor volume = 0.5 (value 0.05 was considered as a significant difference. Differences were considered highly significant when 0.01. 3. Results 3.1. Isolation and Characterization of ASCs and EPCs from Mice To investigate whether c-Kit+ ASCs promote the growth of breast cancer cells, we isolated ASCs from mouse inguinal adipose tissues. The isolated ASCs appeared as a spindle shape, and oil red O staining showed that adipogenic differentiation of sorted ASCs contained lipid drops inside their cytoplasms, a feature of mature adipocytes (Figure 1(a)). The cells obtained from mouse adipose tissues were mostly CD90+ cells and included a CD38 inhibitor 1 c-Kit+ subpopulation (Figures 1(b), 1(c), and 1(d)). Nevertheless, there were very few cells that were positive for the endothelial progenitor cell marker CD34, and no CD45+ subpopulation was found by immunofluorescence staining (Figure 1(e)). These results indicated that the isolated and expanded cells including c-Kit+/CD90+ ASCs were not contaminated with endothelial or hematopoietic cells. Open in a separate window Figure 1 The characterization of isolated ASCs and EPCs. ASCs were isolated from inguinal adipose tissue of Balb/c female mice and cultured in DMEM. The cells were placed on EZ slides for detection of biomarker expression using immunofluorescence. BM-EPCs were isolated from the femurs of Balb/c female mice and cultured in EGM-2. A total of 1 1 103?EPCs were plated on methylcellulose containing EGM-2 medium for 8C10 days, and colony formation units were defined as cluster-like collections of cells. (a) The morphology and differentiation potential of ASCs. ASCs appeared as a spindle.