Figure 4 from Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

CCL2 produced by fibroblasts activated CCR2/4⁺ CAR T cells to induce antigen-dependent cytotoxicity. A, Cytokine profile of FB-, G164-, and OvCAR3-conditioned medium using proteome profiler array. FBs from two donors were used for this experiment. Each cytokine is in duplicate. Dots that are not labeled are reference points. B, ELISA showing CCL2 concentrations in monolayers and spheroids of malignant cells with and without FB. Two FB donors were used for this experiment. Data plotted as mean ± SD of two replicates. C and D, IFNγ concentration measured by ELISA (C) and Incucyte CAR T-cell killing assay of G164 spheroids with no treatment (NT) or with recombinant CCL2 (D). E and F, ELISA data showing IFNγ concentration (E) and Incucyte killing assay of G164 spheroids cultured in FB-M with anti-CCL2 (aCCL2) antibody (F). G, Fold change analysis of the expression of T-cell activation markers HLA-DR and CD69 on CCR2⁺ (top) and CCR4⁺ (bottom) CAR T cells after CCL2, FB-M, or FB-M with aCCL2 treatments. C, E, and G, Data plotted as mean ± SD for three CAR T-cell donors. Statistics performed using two-way (C and E) and one-way ANOVA (G). D and F, Data shown for one CAR T-cell donor. Representative images at day 3 (left) and quantification (right). Red, dead cells. Scale bar, 400 μm.