Figure 7.

Acquired resistance to the combination of LB-100 and adavosertib is tumor-suppressive. A, Long-term viability assays show HT-29 and SW-480 parental and resistant cells treated with LB-100, adavosertib, or the combination at the indicated concentrations. Cultures were refreshed every 2–3 days, and the cells were grown for 10–14 days before fixing, staining, and imaging. B, Western blots show selected oncogenic signaling and stress response pathways in HT-29 and SW-480 parental and resistant cells. Parental cells were exposed to the combination for 24 hours, whereas for resistant cells, that grow in the presence of the combination, the drugs were washed out (w/o) 24 hours before harvesting. LB-100 was used at 4 μmol/L and adavosertib was used at 400 nmol/L. Vinculin was used as a loading control. C, UMAP representations of HT-29 (top) and SW-480 (bottom) parental and resistant cells colored by a sample of origin (left) or by clusters (right). Parental cells were harvested untreated and resistant cells were harvested 24 hours after the washout of the drugs. D, Tumor growth curves of SW-480 parental and resistant cells in the absence of drugs. Per cell line, 10 mice were injected subcutaneously with 3 million cells and we measured tumor size 3 times per week. Graph, mean and SEM of the measurements until the first mouse reached 1,500 mm³ (ethical sacrifice). E, Kaplan–Meier survival curves of the experiment above along 3 months considering the 1,500 mm³ ethical sacrifice.