Fig. 4. MSC-induced increase in tumor initiation is reflected in an increase in ALDH^high CSC-enriched populations.

(A) ALDH1 protein expression in LoVo cells cultured either alone or with MSCs. The numbers indicate relative protein levels. (B) Unsorted (whole), ALDH^high and ALDH^low LoVo cells were cultured either alone or with dTtomato-MSCs. After 5 days, ALDH activity of LoVo cells was analyzed by flow cytometry (panel a) and dTtomato-MSCs were removed from the cultures by flow sorting. After removing MSCs, the LoVo cells were cultured alone for another five days. The ALDH activities were again analyzed by flow cytometry (panel b). The percentages indicate the percentage of ALDH^high LoVo cells; i.e., percent of LoVo cells with ALDH activity beyond the indicated thresholds. (C) a: Weights of tumors derived from ALDH^high or ALDH^low LoVo cells injected into SCID mice, either alone or with MSCs. Solid-filled and hash-filled circles indicate individual tumor weights; open circles indicate no tumor grew at the site of injection. Red circles: injection of ALDH^high LoVo cells. Blue circles: injection of ALDH^low LoVo cells. Hash-filled circles: injection of LoVo cells, Solid-filled circles: injection of LoVo cells and MSCs. Bars are means ± SE, b: The ranges of the estimated tumor-initiating frequencies evaluated by ELDA. (D) LoVo cells cultured with tdTomato-MSCs have increased numbers of TICs. Cells were cultured as in panel B. After isolating LoVo cells by sorting, cells (5 × 10⁴ cells/injection) were injected into mice. After 6 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.