Figure 2. TLR4 Expression Negatively Correlates with Stemness.

(A and B) Experimental paradigm to assess the effect of LPS on tumor growth in vivo (A). Mice (n = 7 per arm) were injected with cells from xenograft T3832. After 14 days, tumors were injected with LPS (150 or 300 μg), and tumor volume (B) was measured using electronic calipers 3 days later. Tumor volume at day 3 was normalized to the volume of the tumor at day 0 when LPS was injected.

(C) Bulk tumors derived from patient specimens were sorted for TLR4 surface expression using fluorescence-activated cell sorting (FACS). The 20% of cells with the highest expression and the 20% of cells with the lowest expression were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods.

(D) CSCs were differentiated in DMEM containing 10% fetal bovine serum (FBS) for the indicated lengths of time, and protein levels of TLR4, SOX2, OCT4, NANOG, GFAP, and Actin were assessed by western blotting.

Experiments in (C) and (D) were performed at least three times. Data are represented as mean ± SEM. ***p < 0.001 as assayed by one-way ANOVA. See also Figure S2.