Fig 2: 15 ml of supernatant was isolated from 16.5 × 106 cells and was processed for exosome isolation using various methods. Real-time assay measurement results for Compact Surface Plasmon Resonance Biosensor (SPR) detection of PD-L1. (a) The clone used, MIH1, is as effective as 28–8 for use in SPR assay when normalized by number of exosomes (1 × 1010). (b) Linear regression analysis of PD-L1 positive exosomes using MIH1 clone (R2 = 0.9818). When normalized by number of exosomes (1 × 1010), PD-L1 expression of exosomes in (c) 888-mel HMEX and (d) 2183-Her4 HMEX is as shown (n = at least 6).

Fig 3: Sulfisoxazole (SFX) reinvigorates T cell activity by inhibiting of cancer exosome (EXO) biogenesis. a) Gating strategy used to identify the purified CD8+ T cells from human peripheral blood mononuclear cells (PBMCs). b) Representative contour plots of granzyme B (GzmB) in human CD8+ T cells with the indicated treatments. EXOs obtained from equal number of cells (2 × 107 cells) were used. c) Quantification of CD8+GzmB+ cells in CD8+ T cells (n = 3). d) CD8+ T cells from human PBMCs were co-cultured with MDA-MB231-luc (+) cells and then treated with or without SFX and aPD-L1. Tumor cell death was evaluated by luciferase activity using an Alpha microplate reader (n = 3). e) CD8+ T cell-mediated tumor killing assay in MDA-MB231-luc (+) cells incubated with SFX, exosomes, or PD-L1- blocked EXOs by aPD-L1 (n = 3). Significance was determined using an unpaired two-tailed Student's t-test. ***p < 0.001, **p < 0.01, and *p < 0.05. Error bar, standard deviation (SD).

Fig 4: Sulfisoxazole (SFX) inhibits cancer exosome (EXO) biogenesis and suppresses exosomal PD-L1. a) TEM images of MDA-MB231-derived EXOs immunogold-labeled with aPD-L1 antibodies. Arrowheads indicate 5 nm gold particles. Scale bar, 50 nm. b) Quantification of secreted EXOs in the presence of different concentrations of SFX (n = 3). c) Immunoblot for the indicated proteins in cell lysates and EXOs from MDA-MB231 cells. Exosomal proteins, obtained from equal number of cells (1 × 107), were loaded per lane (n = 3). d) PD-1 binding to MDA-MB231-derived EXOs, obtained in the presence of SFX (200 × 10-6 m) or IFN-? (10 ng mL-1). e,f) Immunoblot of human CD63 and PD-L1 in circulating EXOs and tumor lysates from MDA-MB231 xenograft models. ß-actin was used as the loading control for tumor lysates. (Vehicle; n = 5 and SFX; n = 4). Significance was determined using an unpaired two-tailed Student's t-test. ***p < 0.001, **p < 0.01, and *p < 0.05. Error bar, standard deviation (SD).

Fig 5: Schematic illustration depicting the mechanism of action of combination therapy using sulfisoxazole (SFX) and anti-PD-1 (aPD-1). SFX, an FDA-approved ETA antagonist, inhibits cancer exosome biogenesis and synergistically enhances the antitumor effect of aPD-1. 1) Tumors actively secrete exosome with PD-L1 (exosomal PD-L1), which inhibits T cell activation as an immune escape mechanism in aPD-1 monotherapy. 2) SFX inhibits exosome biogenesis in tumors, leading to enhanced antitumor efficacy of aPD-1.