Figure 4. KCL-HO-1i and chemotherapy create an immunologically hot TME.

KCL-HO-1i and/or 5-FU or gemcitabine and/or vehicle were administered to MMTV-PyMT mice bearing 500-700 mm³ tumors. At 36 h post treatment initiation of treatment mice were sacrificed and tumors were harvested and analyzed. (A) Schematic representing the dosing strategy. (B) The fold change of tumor growth over 36 h post initiation of treatment. (C-G) Tumors were enzyme-digested to release single cells which were analyzed for their live (7AAD^-) stromal cell composition using flow cytometry (n=5 tumors per group) (C) and CD8⁺ T-cells (D). Gating strategy (left panel) and quantitation of CD8⁺ T-cell subsets based on CD62L and CD44 expressions (right panel; TCM; T central memory cells, TEM; T effector memory cells, Teff; T effectors cells) (E), and the ability of the tumoral CD8⁺ T-cells to secrete IFNγ post ex vivo exposure to PMA/ionomycin treatment (F). (G) Representative image of a frozen section of MMTV-PyMT tumor treated with KCL-HO-1i and gemcitabine stained with DAPI (nuclei;blue) and antibodies against CD8 (red) and CD31 (green). Scale bar is 100 µm and white arrows indicate infiltrating CD8⁺ T-cells. (H) Schematic of the perivascular niche assay (left) and the relative transendothelial migration of CD8⁺ T-cells in the presence or absence of M₍₀₎ or M_(IL-6) cells on the basolateral surface with or without 25 µM KCL-HO-1i as indicated. Image in panel (A) was created using BioRender. Bar charts show the mean, error bars SD, and the dots show individual data points from individual tumors and mice. Gemc.; gemcitabine. * P<0.05, ** P<0.01, *** P<0.001.