Tao J, Jiang MM, Jiang L, Salvo JS, Zeng HC, Dawson B, Bertin TK, Rao PH, Chen R, Donehower LA, Gannon F, Lee BH. is to use functional properties to enrich for sarcoma TPCs, such as the side population (SP) assay [2, 9]. This assay is based on the ability of stem-like and progenitor cells to efflux Hoechst dye. Cells that can exclude the dye from their nucleus are termed SP cells, and have been shown to have both increased tumorigenicity and self-renewal ability compared to self-renewal ability compared to non-side population (NSP) cells that make up the bulk of the tumor. However, dye efflux is a dynamic process, and the lack of specific criteria and guidelines for delineating the SP fraction can lead to large variability between studies [10]. As such, a cell surface marker would be of important utility for sarcoma TPC research. Self-renewal is a defining characteristic of TPCs and is associated with tumor recurrence [4, 11]. Expressions Donepezil of genes that regulate self-renewal of normal stem cells are significant predictors of disease relapse [12C14]. Currently, the clinical outcome of patients with recurrent or metastatic sarcoma remains poor [15]. The inhibition of self-renewal in sarcoma TPCs may offer valuable targets of therapy. Here, we used a flow cytometry screen to identify cell surface markers enriched on SP cells compared to bulk tumor cells. We found CD146 (also known as MCAM or MUC18), can reliability enrich for TPCs in osteosarcoma and UPS. Importantly, we showed that CD146+ and SP cells are independently tumorigenic and represent overlapping and distinct populations of sarcoma TPCs. Furthermore, Donepezil pathway analysis revealed that Notch signaling is activated in both of these two TPC populations in osteosarcoma. Treatment with a -secretase inhibitor significantly reduced the tumor growth and self-renewal capacity of human osteosarcoma (NSG) mice. After 20 weeks, the mice were sacrificed, and the tumors that formed were weighed and examined by histologic examination. CD31+, CD66+, CD104+ and CD144+ cells did not show higher tumor initiating ability compared to their respective marker negative populations or bulk tumor cells (data not Donepezil show). In contrast, CD146+ cells enriched for TPCs close to 50-folds higher than CD146? cells. We then analyzed the expression of CD146 using flow cytometry in an independent cohort of 10 human UPS samples and 5 human osteosarcoma samples. The mean percentage of SP and CD146 cells in UPS is 0.70% (0.16%SEM) and 3.63%(0.95%SEM) respectively, per tumor. The expression of CD146 was significantly enriched in the SP population compared to the NSP cells (< 0.001), with 53.2% (9.51% SEM) of SP cells expressing CD146, and 2.98% (0.90% SEM) of NSP cells expressing CD146 (Figure 1A, 1B). We observed 1 UPS sample (UPS106) with higher percentage of CD146+ cells in the NSP populations than the SP population (Supplementary Table S1). This was likely due to the heterogeneity among different patient tumor biopsies. In osteosarcoma, the mean percentage SP and CD146+ cells is 0.68% ( 0.28 SEM) and Donepezil 4.92% (0.90 SEM) respectively. Similar to UPS, 49.37% (15.48% SEM) of SP cells express CD146, as compared to 4.73% (0.87% SEM) of NSP (< 0.05, Figure ?Figure1B,1B, Supplementary Table S2). Overall, the enrichment of CD146+ cells in SP suggests that there is an overlapping population of CD146+ cells and SP cells. Open in a separate window Figure 1 CD146 expression is enriched on the surface of SP cells in human UPS and osteosarcomaA. Mouse monoclonal to ROR1 Representative flow cytometry analysis of SP, NSP, and enrichment of CD146 on SP cells in human sarcoma. The NSP is labeled with a box in the upper right quadrant, and SP is in the lower left quadrant. Treating the cells with verapamil inhibits Hoechst dye exclusion, and is used as a negative control for SP analysis. Expression of CD146 is gated on the SP and NSP cells. B. Analysis of CD146 expression on SP and NSP cells in 10 primary human UPS samples and 5 primary human osteosarcoma samples, showing CD146 is significantly enriched on the sarcoma SP cells. *< 0.05; *< 0.01. The location of CD146+ cells in UPS and osteosarcoma was visualized using immunofluorescence. Since CD146 Donepezil is also a marker of pericytes [18], we stained.