Fig. 5. COX2-PGE₂ signaling is required for MSC-induced increase in ALDH^high CSC-enriched population and tumor initiation.

(A) ALDH1 protein expression in LoVo cells treated as indicated for 5 days. (B) ALDH activities of LoVo cells treated with vehicle, PGE₂ (100 nM) or GW627368X (20 µM) was analyzed by flow cytometry. The percentages indicate the percentage of ALDH^high LoVo cells; i.e., LoVo cells with ALDH activity beyond the indicated thresholds. The gray line at the right side of the plot indicates the threshhold of the high ALDH activitiy. (C) ALDH1 protein levels in various carcinoma cells treated with vehicle or PGE₂. The numbers indicate relative protein levels. (D) PGE₂ increases LoVo TICs. LoVo cells pre-treated with vehicle or PGE₂ (100 nM) were injected into SCID mice (5 × 10⁴ cells/injection). After 6 weeks, tumors were isolated and weighted. Bars are means ± SE. (E) LoVo cells were cultured with tdTomato-MSCs, PGE₂, NS398 or GW627368X, as indicated. After 5 days, the LoVo cells were isolated by cell sorting and injected into SCID mice (5 × 10⁴ cells/injection). After 7 weeks, the tumors were isolated and weighted. Filled circles indicate individual tumor weights; Open circles indicate no tumor grew at the site of injection. Bars are means ± SE. (F) Panel (a), levels of PGE₂ secreted by LoVoCM-treated MSCshsc and MSCshcox2. Data are means ± SE, n = 3. Panel (b), Weights of tumors derived from LoVo cells (5 × 10⁴ cells/injection) injected into SCID mice either alone, with MSCshsc or with MSCshcox-2 (2 × 10⁵ cells/injection). Filled circles indicate individual tumor weights; Open circles indicate no tumor grew at the site of injection. Bars are means ± SE.