Extended Data Fig. 4: Tumor-derived sEVs transfer tumor antigens to CAR T cells.
a, Western blot showing the MSLN expression in human MSLN-CAR T cells treated with sEVs derived from EM cells and MSLN-expressing EM cells (EM-MSLN cells). Experiments consisted of three biologically independent samples with similar results. b, EM-MSLN cell-derived sEVs increased the percentage of MSLN-positive human MSLN-CAR T cells in a dose-dependent manner as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. c, Western blot showing the PSMA expression in human PSMA-CAR T cells treated with sEVs derived from PC3 cells and PSMA-expressing PC3 cells (PC3-PSMA cells). Experiments consisted of three biologically independent samples with similar results. d, PC3-PSMA cell-derived sEVs increased the percentage of PSMA-positive human PSMA-CAR T cells in a dose-dependent manner as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. e, Flow cytometry showing the PSMA expression on the surface of human PSMA-CAR T cells with or without sEVs from PC3-PSMA cells or PC3 cells. The percentages of PSMA-positive CAR T cells under indicated treatments are shown at the right. Experiments consisted of three biologically independent samples. f, Western blot showing the HER2 expression in human HER2-CAR T cells treated with sEVs derived from SKOV3 cells with or without HER2 knockout. Experiments consisted of three biologically independent samples with similar results. g, SKOV3 cell-derived sEVs increased the percentage of HER2-positive human HER2-CAR T cells in a dose-dependent manner as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. h, Flow cytometry showing the HER2 expression on the surface of human HER2-CAR T cells with sEVs from SKOV3 cells or HER2 KO SKOV3 cells. The percentages of HER2-positive CAR T cells under indicated treatments are shown at the right. Experiments consisted of three biologically independent samples. i, Western blot showing treatment with 4662 cell-derived sEVs increased the level of MSLN in murine MSLN-CAR T cells in a dose-dependent manner. GAPDH was used as loading control. Experiments consisted of three biologically independent samples with similar results. j, 4662 cell-derived sEVs increased the percentage of MSLN-positive murine MSLN-CAR T cells as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. k, Western blot showing MOC1 cell-derived sEVs increased the level of MSLN in murine MSLN-CAR T cells. Experiments consisted of three biologically independent samples with similar results. l, MOC1 cell-derived sEVs increased the percentage of MSLN-positive in murine MSLN-CAR T cells as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. m, Western blot showing the MSLN expression in murine MSLN-CAR T cells with or without sEVs derived from MOC1 or Msln KO MOC1 cells. Experiments consisted of three biologically independent samples with similar results. n, The percentage of MSLN-positive murine MSLN-CAR T cells with or without sEVs from MOC1 or Msln KO MOC1 cells as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. o, Flow cytometry showing the percentage of MSLN+ MSLN-CAR T cell or TRP1-CAR T cells after 4662 sEV treatment. Experiments consisted of three biologically independent samples. p, Flow cytometry showing the percentage of TRP1+ TRP1-CAR T cell or MSLN-CAR T cells after B16-F10 sEV treatment. Experiments consisted of three biologically independent samples. q, Western blot showing B16-F10 cell-derived sEVs increased the level of TRP1 in TRP1-CAR T cells in a dose-dependent manner. Experiments consisted of three biologically independent samples with similar results. r, B16-F10 cell-derived sEV treatment increased the percentage of TRP1-positive TRP1-CAR T cells as analyzed by flow cytometry. Experiments consisted of three biologically independent samples. s, Western blot showing the TRP1 expression in TRP1-CAR T cells after treatment with sEVs from B16-F10 cells or Trp1 KO B16-F10 cells. Experiments consisted of three replicates with similar results. t, Flow cytometry showing the TRP1 expression on the surface of TRP1-CAR T cells with or without treatment of sEVs from B16-F10 cells or Trp1 KO B16-F10 cells. Quantification of TRP1+ CAR T cells with indicated treatment is shown to the right. Experiments consisted of three biologically independent samples. u, Schema of C57BL/6 mice bearing wide type or Rab27a KO B16-F10 tumors with indicated treatments after tumors reach ~150 mm3. Five mice were used in each group. v, Flow cytometry showing TRP1 expression on the surface of TRP1-CAR T cells in B16-F10 or Rab27a KO B16-F10 tumors from C57BL/6 mice with indicated treatments. Quantification of TRP1+ CAR T cells with indicated treatments is shown to the right. Five mice were used in each group. (n = 5). Data represent mean ± SD (n = 3 or indicated). Statistical analysis is performed using one-way ANOVA with Sidak’s multiple comparison tests (b, d, g, j, l, r and v), one-way ANOVA with Dunnett’s multiple comparison tests (e, h, n and t), or using two-sided unpaired t-test (o, p).
