2c, d), CXCR7 knockdown may lead to an almost complete abrogation of in OT7 cells, in turn causing a significant downregulation of is finely regulated by the levels of SDF-1 and CXCR7 and, in turn, that CXCR4 regulates the expression of the ligand. CXCR4 knockdown significantly reduced the proliferation of PD/S cells in both lineages, whereas CXCR7 knockdown only inhibited the proliferation of OT7 PD/S cells, in accordance with the strong downregulation of observed in these cells (Fig. invasion, promoting in vivo lung metastasis. Validation of these findings in patient samples of skin SCCs shows a strong correlation between the expression of and receptors in early WD-SCCs and advanced PD/S-SCCs of two different lineages of mouse skin SCC progression (OT7 and OT14). In each of these lineages, PD/S-SCCs were generated after the serial engraftment of their WD-SCC precursors in immunodeficient mice [11]. We found that expression, which was weakly detected in early SCCs, was upregulated in PD/S-SCCs (Fig. ?(Fig.1a).1a). In WD-SCCs, was expressed by fibroblasts (Fig. ?(Fig.1b)1b) and immunodetected in stromal cells (Fig. ?(Fig.1c),1c), as previously described [32, 35], whereas tumor cells and CD45+ immune cells (deficient in T-cell in nude immunodeficient mice) exhibited a faint or undetectable expression (Fig. ?(Fig.1b).1b). In contrast, SDF-1 was strongly upregulated in tumor cells of PD/S-SCCs (Fig. ?(Fig.1c),1c), reaching similar levels to those expressed by fibroblasts (Fig. ?(Fig.1b1b). Open in a separate window Fig. 1 CSCs of mouse advanced skin Dronedarone Hydrochloride SCCs express CXCR4 and CXCR7 and up-regulate the expression of mRNA relative to mRNA levels relative to in E-CSCs and L-CSCs (three different samples per group) isolated by FACS-sorter from the indicated tumors. h Flow cytometry quantification of CXCR4+ and CXCR7+ cells (red numbers) into the 6-integrin+/CD34+ Dronedarone Hydrochloride CSC population (blue numbers) of the indicated tumors. i Mean percentage (SE) of the indicated cell populations in WD-SCCs and PD/S-SCCs, as quantified in h. *, significant differences between compared groups (and expression was upregulated in advanced SCCs relative to WD-SCCs (Fig. ?(Fig.1d).1d). An expansion of CXCR4-expressing tumor cells was detected by immunohistochemistry in PD/S-SCCs, as compared to WD-SCCs (Supplementary Fig. 1A). Flow cytometry analysis showed that around 3C7% of tumor cells expressed CXCR4 (6-integrin+/CXCR4+ cells) in WD-SCCs, and this frequency was significantly increased in PD/S-SCCs (Fig. 1e, f). CXCR7+ cells were more frequent than CXCR4-expressing cells in early and advanced SCCs (Supplementary Fig. 1A and 1B). Accordingly, 60C70% of tumor cells expressed CXCR7 (6-integrin+/CXCR7+ cells) in WD-SCCs and this frequency was not significantly increased in PD/S-SCCs (Fig. 1e, f). Dronedarone Hydrochloride These results suggest that increased levels of mRNA detected in advanced tumors may be associated with a different stromal/tumor cells ratio in WD-SCCs and PD/S-SCCs [36, 37]. Analysis of the ligand and receptors in the CSC population (6-integrin+/CD34+ cells) showed that CSCs isolated from PD/S-SCCs (L-CSCs) strongly expressed and upregulation was observed in primary PD/S-SCCs (spontaneously developed in K14-HPV16 mice [11]) and in PD/S-SCCs that were engrafted in syngeneic immunocompetent mice, relative to their respective WD-SCCs (Supplementary Fig. 1C and 1D). SDF-1 expression was significantly induced in tumor and stromal cells (Supplementary Fig. 1E), and an expansion of CXCR4-expressing CSCs was detected in PD/S-SCCs generated in immunocompetent mice (Supplementary Fig. 1FC1H), indicating that SDF-1 signaling may be activated in CSCs of advanced tumors independently of the immune status of the mice. Autocrine SDF-1 signaling promotes PD/S-SCC CSC proliferation and invasion To determine whether an autocrine SDF-1 signaling is induced in L-CSCs, we isolated tumor cells from WD-SCCs (WD cells) and PD/S-SCCs (PD/S cells), which were maintained Mouse monoclonal to ATXN1 in culture. We previously demonstrated that these primary cultures were enriched in tumor-initiating cells that conserved the molecular traits of parental SCC CSCs [11]. In this regard, PD/S cells expressed and secreted higher levels of SDF-1 than WD cells (Fig. 2a, b). In addition, PD/S cells expressed and than (Fig. ?(Fig.2c).2c). Accordingly, 40C60% of 6-integrin+/CD34+-CSCs in PD/S cell cultures expressed CXCR7, whereas 6C16% of 6-integrin+/CD34+-CSCs expressed CXCR4 (Fig. ?(Fig.2d;2d; Supplementary Fig. 2A and 2B), and most CXCR4+-CSCs expressed CXCR7 (Supplementary Fig. 2C). Open in a separate Dronedarone Hydrochloride window Fig. 2.