Fig 1: DCLK1-IN-1 downregulates immune checkpoint marker PD-L1 and enhances anti-tumor immunity in co-culture. (A) Immunoblotting from ACHN, 786-O, and CAKI-1 cells treated with DMSO or DCLK1-IN-1 demonstrating a dose-dependent decrease in the expression of tumor immune checkpoint ligand PD-L1. (B) Immunoblotting of proteins isolated from naïve and T-cell expanded PBMCs confirming increased expression of T-cell markers CD8, PD-1, and CD3. (C) Flow cytometry results for PBMCs and T-cell expanded PBMCs confirming successful T-cell expansion via strongly increased expression of membrane CD25 (manufacturer-recommended confirmation marker) and PD-1 (immune checkpoint marker). (D,E) 786-O was selected for co-culture assays based on its characteristically strong PD-L1 expression, pre-treated with vehicle (DMSO) or DCLK1-IN-1, counted by trypan blue exclusion, stained with Calcein-AM (green fluorescence), and seeded into co-culture with PBMCs or T-cell expanded PBMCs. Following, 3 h co-culture, 786-O cells demonstrated significant sensitivity to anti-tumor immunity from PBMCs after 10 µM pre-treatment with DCLK1-IN-1 (D) and from T-cell expanded PBMCs after 5 and 10 µM pre-treatment with DCLK1-IN-1 (E) (** p < 0.01 vs. DMSO at corresponding time; representative images included adjacent to graphs and merged with brightfield images; 10× magnification). (F) Co-culture assay of 786-O RCC cells pretreated with vehicle (DMSO) or DCLK1-IN-1 (5 or 10 µM), and PBMCs pre-treated with isotype antibody or anti-PD1, demonstrating a statistically significant effect on 786-O cell viability at 3 h for DCLK1-IN-1 pre-treated 786-O cells (* p < 0.05 vs. DMSO at corresponding time; 10× magnification).