Extended Data Fig. 1: Increased secretion of tumor antigens via sEVs in response to CAR T cell treatment.
a, Flow cytometry showing the purity of T cells isolated from mouse spleen used in the study. Experiments consisted of three biologically independent samples with similar results. b, Transfection efficiency of mouse MSLN-CAR T cells. Experiments consisted of three biologically independent samples with similar results. c, Representative electron microscopic images of sEVs isolated from tumor tissues and plasma from mice bearing 4662 tumors. Scale bar, 100 nm. Experiments consisted of three biologically independent samples with similar results. d, NTA of sEVs isolated from tumor tissues from mice bearing 4662 tumors. The X-axis indicates the diameters of the isolated vesicles; the Y-axis indicates the concentration of isolated vesicles (particles/ml). Experiments consisted of six biologically independent samples with similar results. e, NTA of sEVs isolated from plasma of mice bearing 4662 tumors. Experiments consisted of six biologically independent samples with similar results. f, Western blot showing the baseline levels of MSLN in plasma sEVs in tumor-free mice and untreated 4662 tumor-bearing mice. The blot for MSLN was overexposed so that the MSLN in plasma sEVs from tumor free mice could be detected. Relative levels of MSLN on plasma sEVs were quantified and shown to the right. Six mice were used in each group. g, Transfection efficiency of mouse TRP1-CAR T cells. Experiments consisted of three biologically independent samples with similar results. h, TRP1 co-fractionated with exosome marker proteins (CD63, TSG101 and CD9) on iodixanol density gradients from B16-F10 tumor tissue-derived sEVs. Experiments consisted of three biologically independent samples with similar results. i, TRP1 co-fractionated with exosome marker proteins on iodixanol density gradients from the plasma of B16-F10 tumor bearing mice. Experiments consisted of three biologically independent samples with similar results. j, Western blot showing the baseline levels of TRP1 in plasma sEVs in tumor-free mice and untreated B16-F10 tumor-bearing mice. The blot for TRP1 was overexposed so that the TRP1 in plasma sEVs from tumor free mice could be detected. Relative levels of MSLN on plasma sEVs were quantified and shown to the right. Six mice were used in each group. k and l, Western blots showing TRP1 and exosome marker proteins in the sEVs isolated from B16-F10 tumor tissues (k) and plasma (l) of B16-F10 tumor bearing mice. All lanes were loaded with an equal amount of proteins. Six mice were used in each group. Quantification of TRP1 expression in sEVs is shown to the right. m and n, ZetaView analysis showing the percentage of TRP1+ sEVs derived from tumor tissues (m) or the plasma (n) of B16-F10 tumor-bearing mice with indicated treatments. In the left panel, each green dot represents a sEV, while each blue ‘×’ represents TRP1 a positive sEV, as generated from manufacturer’s software. Quantification is shown to the right. Six mice were used in each group. o, Western blots showing MSLN and exosome marker proteins (CD63, TSG101 and CD9) in the sEVs isolated from MOC1 tumor tissues and the plasma of tumor-bearing mice. All lanes were loaded with an equal amount of proteins. Six mice were used in each group. p and q, Quantification of MSLN levels in sEVs from tumor tissues (p) and the plasma of tumor-bearing mice (q). Six mice were used in each group. Data represent mean ± SD (n = 3 or indicated). Statistical analysis was performed using two-sided unpaired t-test (f, j-n, p and q).
