Journal: bioRxiv
Article Title: Actionable spatial prostanoid barriers constrain BiTE-driven adoptive T cell immunity in intact human tumors
doi: 10.64898/2026.03.26.713601
Figure Lengend Snippet: A, Schematic of the tiered benchmarking assay comparing anti-MSLN CAR-T cells and anti-MSLN BiTE T cells in OVCAR-8 2D monolayers and matrix-free 3D spheroids after 24 hrs coculture at a 1:1 effector-to-target (E:T) ratio. B, Representative flow cytometry plots and quantification of CD8 + CD137 + cells among CD8 + T cells after coculture of MSLN-CAR T cells alone (T cell only), with 2D OVCAR-8 cells, or with 3D OVCAR-8 spheroids. C, Representative flow cytometry plots and quantification of CD8 + CD137 + cells among CD8+ T cells after coculture of MSLN-BiTE T cells alone (T cell only), with 2D OVCAR-8 cells, or with 3D OVCAR-8 spheroids. D, Supernatant concentrations of granzyme B, TNFα, and IFNγ after 24-hour coculture of MSLN-CAR (top) or MSLN-BiTE (bottom) T cells with 2D OVCAR-8 cells or 3D spheroids. E, Representative confocal immunofluorescence images of embedded 3D spheroids stained for nuclei (DAPI), CD8, and MSLN at baseline and at 5 and 24 hrs after exposure to MSLN-CAR or MSLN-BiTE T cells. Scale bars, 100 μm. F, Quantification of intratumoral CD8 density in 3D spheroids across conditions. Statistical analysis using one-way ANOVA (+Kruskal-Wallis) G, Quantification of intratumoral CD8 density in 3D spheroids across conditions. H, Interpatient heterogeneity of MSLN expression in PDTs assessed by flow cytometry, schematic of Matrigel-free PDT coculture with MSLN-BiTE T cells at a 1:1 E:T ratio, and correlation between epithelial target abundance (%CK + MSLN + of CK + cells) and CD8 activation (FC of CD8 + CD137 + over UT control). I , Multiparametric functional profiling of PDT responses to MSLN-BiTE T cells. Upper part, CD8 activation (%CD8⁺CD137⁺) under untreated versus +MSLN-BiTE conditions. Below, heatmap summarizing changes in secreted mediators (log2 fold-change over UT control) following co-culture across the indicated tumoroid lines. One-way repeated-measures ANOVA (+Tukey) was performed in GraphPad Prism v10.2 (n=3-4 technical replicates per patients). Exact P values and comparisons are indicated in the panels. In B and C , representative plots are shown and quantification reflects 3 to 4 technical replicates per condition. In D , n = 3 technical replicates per condition. In F , n = 12 to 22 regions of interest per condition. Exact P values and comparisons are indicated in the panels. Figure created with BioRender.
Article Snippet: When indicated, tumor cells were identified using anti EpCAM (BioLegend Cat# 324232, RRID:AB_2564301) and anti-MSLN (Santa Cruz Biotechnology Cat# sc-33672, RRID:AB_627930).
Techniques: Flow Cytometry, Immunofluorescence, Staining, Expressing, Activation Assay, Control, Functional Assay, Co-Culture Assay
