Figure 6.

(A) Combinatorial efficacy of DDR1/BCR-ABL1 multi-tyrosine kinase inhibitors (dasatinib, imatinib, and nilotinib) and compound drugs GMB475 and KB-SRC4 when administered with EGFRi (lapatinib and afatinib) on multi-spheroids of HCT116. Representative images (left) and bar graphs (right) indicate spheroid viability and relative caspase 3/7 activity (normalized to viability), with percent inhibition listed in the table at the bottom. (B) Clonal cell proliferation assay performed for HCT116 cells for drugs bafetinib and nilotinib in combination with lapatinib validated the combinatorial efficacy. Clonal cell growth was measured as percent confluence, using IncuCyte. Representative images (right) for clonal cell proliferation obtained at time-points day 0, day 3, and day 5 are pseudo-colored utilizing IncuCyte’s inbuilt clonal dilution module. (C) Western blotting for HCT116 spheroid-lysates prepared 48 h after administration of drugs indicated significant downregulation of phospho-BCR and phospho-AKT levels in DDR1/BCR-ABL with EGFR/ERBB2 inhibitor combination treatment as compared with either of the drugs administered alone. Change in phospho-ERK levels was minimal, indicating the prime signaling affected in combination treatments is BCR-AKT axis. (D) Efficacy of nilotinib in combination with lapatinib validated on viability of multi-spheroids from SW480 (KRAS^G12V). (E) Representative bright-field images of U251 (GBM) spheroids treated with drugs lapatinib and nilotinib at day 5 post-treatment. Graph (middle) shows reduction in spheroid total area over 5 days post-treatment. Bar graph (right) indicates loss of spheroid cell viability measured on day 5. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.