Patient-derived organotypic tumor spheroids (PDOTS)

PDOTS were generated as previously described (12,22). Briefly, fresh MPM tumor specimens (n=37; Supplementary Tables S1 and S2) were minced in a 15 mL falcon tube in prewarmed to 37°C full media (DMEM from Thermo Fisher Scientific #10013CV + 10% FBS) + 100 U/mL collagenase type IV (Thermo Fisher Scientific #17104019) and 50 μg/mL DNase I (Roche #10104159001) for approximately 20 minutes using sterile scissors and pipetting. Dissociated material was strained over 100-μm filter and 40-μm filters to generate S1 (>100 μm), S2 (40–100 μm), and S3 (<40 μm) spheroid fractions, which were subsequently maintained in ultralow-attachment (ULA) tissue culture plates (Corning). S1 fractions were treated with 50 μM ADU-S100 for cytokine analysis and single-cell RNA sequencing (scRNA-seq) (see below). S2 fractions were used for ex vivo culture by resuspending them in type I rat tail collagen (Corning #354236) at a concentration of 2.8 mg/mL prior to loading into the center gel region of the 3-D microfluidic culture device (AIM Biotech #DAX-1) and incubation for 40 minutes at 37°C in humidity chambers to allow for polymerization. Collagen hydrogels containing PDOTS were hydrated with media with or without indicated treatments. TAK-676 was provided by Takeda (23) and diluted in dH20. Recombinant human IFNβ (100 ng/mL; R&D Systems #8499-IF) was used as a positive control downstream of STING for STAT1 pathway activation. CD8a was blocked with 50 μg/mL InVivoMAb antibody (Bio X Cell #BE0004–2) vs. IgG control (Bio X Cell #BE0092). CXCR3 was blocked with 5 μg/mL human CXCR3 antibody (R&D MAB160).