Figure 5 from Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

Dense ECM prevented CAR T-cell migration and cytotoxicity in G164/FB collagen gels. A, CD3⁺ CAR T cells at the peripheral and core regions of OvCAR3/FB and G164/FB collagen gels. Two different CAR T-cell and FB donors were used in this experiment. B, IHC staining and quantification for caspase-3 (Casp3) on OvCAR3/FB and G164/FB collagen gels. C, Behavior of CAR T cells in OvCAR3/FB and G164/FB collagen gels. Representative images from three repeats. Blue, fibronectin; green, CAR T cells; red, EpCAM. Scale bar, 50 μm. Proportion of static (st), wobbling (wob), migrating (mig), and long migrating (Lmig) CAR T cells. Speed and length of movement of wobbling (OvCAR3: 172, G164: 112) and migrating (OvCAR3, 145; G164, 83) CAR T cells. D, Masson’s trichrome (M3C), COL1A1, and fibronectin (FN1) staining and quantification of OvCAR3/FB and G164/FB collagen gels. E, TWOMBLI analysis showing the heatmaps and percentage of HDM for M3C, COL1A1, and FN1 in OvCAR3/FB and G164/FB collagen gels. Scale bar, 100 μm. F, Expression of FAP and αSMA on OvCAR3/FB and G164/FB collagen gels. G, Barplot illustrating significantly enriched canonical pathways (FDR < 0.05) in G164 vs. OvCAR3 cells in monolayer. A, B, D, and F, Data plotted as mean ± SD of three gels per two replicates. Scale bar, 50 μm. Statistics performed using two-way ANOVA (A and B) and unpaired t test (C–F).