Fig. 7

HELA-Exos profoundly enhance cDC1 antigen cross-presentation and tumor-reactive CD8 ⁺ T cell generation in a patient-derived tumor organoid coculture system. PBMCs were cocultured with autologous tumor organoids derived from breast cancer for 2 weeks and administered medium, Hiltonol or HELA-Exos during this two-week period. After two weeks of coculture, immune infiltration in organoids was evaluated by flow cytometry and IF, and the growth-inhibitory effects of HELA-Exos in organoids were evaluated by a cell viability assay and live/dead cell staining. A Gating strategy for analysis of CD11c⁺ DCs and the percentage of CD141⁺ cDC1s in organoids. B Gating strategy for analysis of CD3⁺ T cells and the percentages of CD4⁺ and CD8⁺ T cells in organoids. C Organoid-infiltrating CD11c⁺ CD141⁺ cDC1s and CD3⁺ CD8⁺ T cells were detected by IF. Blue: DAPI; Green: CD141 or CD8; Red: CD11c or CD3. Scale bar, 50 μm. D Organoid-infiltrating CD8⁺ T cells were analyzed for the expression of the cytotoxicity markers granzyme B and perforin. E Cell viability of organoids was measured with MTS reagent. F Representative bright field images of organoids from various treatment groups. The figure below shows representative three-dimensional images of organoids from various treatment groups stained with LIVE/DEAD reagents. Scale bar, 50 μm. The data are presented as the mean ± SD; n = 6. A t test was performed for statistical analysis (****: P < 0.0001; ***: P < 0.001)