Figure 5. LUAD cells in the HPCS show high growth potential in vitro and in vivo, and are chemoresistant in vivo.

(A) Experimental design. TIGIT⁺ HPCS/cluster 5 cells (blue) and all non-HPSC TIGIT⁻ cells (grey) were sorted from 17–22 week LUAD tumors, and grown as tumor spheres for 11 days, as in Figure 4B. (B) Number of tumor spheres per 500 cells plated (y axis) arising in individual tumor spheres (dots) from TIGIT⁺ vs. TIGIT⁻ KPT LUAD cells after 11 days in 3D culture (x axis). Data plotted as mean ± S.D. Two independent biological replicates are shown. ** p < 0.01; *** p < 0.001 (unpaired t-test). (C) Experimental design. TIGIT⁺ HPCS/cluster 5 cells (blue) and all non-HPCS TIGIT⁻ LUAD cells (grey) expressing firefly luciferase were sorted from 18–21-week tumors and orthotopically allotransplanted into immunodeficient NSG mice. Bioluminescence imaging and tumor harvest were performed at 39 days post-transplantation. (D) Average radiance (y axis) in allotransplanted tumors derived from TIGIT⁺ and TIGIT⁻ sorted cells. Data plotted as mean ± S.D. * p < 0.05 (t-test; n = 4 TIGIT⁺ vs 11 TIGIT⁻ allotransplants). (E) Number of surface tumors per 10,000 transplanted cells (y axis) for TIGIT⁺ or TIGIT⁻ cells in lungs of recipient mice. Data plotted as mean ± S.D. *** p < 0.001 (t-test). (F) Experimental design. Mice with 20-week LUAD tumors were subjected to treatment with vehicle or cisplatin (7 mg/kg); tumors were harvested after 72 hours. (G) tSNE of scRNA-Seq profiles from 20-week KPT LUAD tumors, collected 72 hours after administration of vehicle or cisplatin, colored by predicted membership (STAR Methods) in cluster 5 (blue) or 8 (green). Two independent mice were used per condition. (H) Relative enrichment (y axis, Pearson’s residual: $\mathrm{(observed\; number\; of\; cells\; -\; expected\; number\; of\; cells)}/\sqrt{\mathrm{expected\; number\; of\; cells}}$, STAR Methods) of cells in different clusters (x axis), after cisplatin treatment in KPT LUAD tumors in vivo. See also related Figure S4.