Figure 1. HS06 selectively inhibits CSCs growth.
a. Scatter plot showing IC50 of primary (1°) spheroid growth of HT29 cells as a function of the degree of polymerization of sulfated GAGs. Error bars represent ±1 SD (n ≥ 3). b. Results of the secondary (2°) and tertiary (3°) spheroid growth in the absence of further GAG treatment in HT29 cells (p53 mutant, K-RAS wild type, microsatellite stable) for most promising agents (IC50 < 100 μM) identified in the 1° screen. HS06 was the only GAG that satisfied the predefined cutoff of >50% inhibition in both 2° and 3° spheroids growth. Data presented refers to percent of the vehicle control. Error bars represent ±1 SEM. c. Comparative effect of 100 μM HS06 on 1° spheroid formation in colon (HCT116, HT29, LS174T), pancreas (Panc1), and breast (MDA-MB-231) cancer cells. Check boxes indicate the mutational status of each of the cancer cell line (from http://cancer.sanger.ac.uk/cosmic). d. Western blot analyses showing expression of markers CD44 and LGR5 on HT29 CSCs in the presence of 100 μM of HS02, HS06, or HS10. Only HS06 showed robust (>50%) inhibition of these markers. e. Bar graph representing relative expression of CSC markers normalized to vehicle control. f. Flow cytometric evaluation of LGR5 following treatment with 100 μM HS06 or 100 nM CHIR99021 or 40 nM iCRT 14, a stimulator and an inhibitor of LGR5 expression, respectively.