Activated/memory T cells are able to infiltrate tumor spheroids upon IDX treatment
(A) To enable an unbiased analysis of data, we selected live cells and then used t-SNE to reduce dimensionality, followed by a graph-based heat-map clustering analysis. Representative t-SNE analysis of concatenated mononuclear cells (2,000 cells/sample) from “IN” compartment done by pooling 8 wells/condition of infiltrating tumor immune cells upon IDX treatment. Merged t-SNE plot (left panel) for drug treated and untreated cocultures, with each cluster indicated by a color (DMSO in pink; IDX2 in blue). Data were further clustered according to criteria used to define known T cell subsets: CD3, CD4, CD8, CD62L, CD27, CCR7, CD45RO, CD45RA and PD1. Automatic gating by Flowjo performed multivariate clustering of cells based on self-organized map algorithm and categorized all individual cells into 100 distinct clusters based on the surface expression marker proteins. Most prominent differential protein expressions CD3 a), CD62L b) and PD1 c) are highlighted in representative rose-charts boxes. (B) Flow cytometry analyses of T cells (respectively gated CD3+ ) as well as CD4+, CD8+ an double-positive T cells subsets (respectively gated CD4+ CD8-, CD4-CD8+ and CD4+ CD8+ among CD3+) percentages in the IN and OUT compartments, in the presence or without IDX at 72 hours. (C) Flow cytometry analyses of migratory T cells (respectively gated CD62L+/CCR7+ of CD3+) on CD4+, CD8+ an double-positive T cells subsets, percentages in the IN compartments, in the presence or without IDX at 72 hours. (D) Flow cytometry analyses of exhaustion (PD1+) T cells phenotypes percentages (respectively gated CD4+ CD8+ of CD3+) on CD45RO+CD27+ (memory T cells) and CD45RO-CD27+ (naïve T cells ) subsets, in the IN compartments, in the presence or without IDX at 72 hours. (E) Quantitation of flow cytometric data on the percentage of PD+ and PD1- memory T cells and naïve T cells in the presence of IDX relative to control (DMSO). * p≤0.05, ** p<0.01 when compared to DMSO control.