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. Author manuscript; available in PMC: 2015 Oct 24.

Published in final edited form as: Nat Commun. 2015 Apr 24;6:6956. doi: 10.1038/ncomms7956

(A–C) C57BL/6 mice were orthotopically implanted with MC38-luc cells with exosomes by colonic submucosal injection. Colonoscopy image of implanted MC38-luc cells at day 18 after injection (A), photon flux from C57BL/6 mice harboring MC38-luc tumors (B), and H&E-stained colon sections (C). The data (B) represent means ± SEMs ^**p < 0.01 (Student’s t-test).

(D) Colonic MC38 tumor leukocytes were isolated at day 18 post treatment from mice treated with ET-Tumor^EXO or NT-Tumor^EXO. TCRβ⁺ CD4⁺ IL-17A⁺ and FoxP3⁺ Treg cell populations were examined by FACS.

(E) RT-PCR analysis of the gene expression in colonic MC38 tumor leukocytes isolated at day 18 from mice treated with ET-Tumor^EXO or NT-Tumor^EXO.

(F–G) Confocal images of colon sections stained with p-Stat3 and CD11b from naïve mice treated with ET-Tumor^EXO or NT-Tumor^EXO (F); Western blotting the lysates of colonic CD11b⁺ cells for S1PR1 (G).

(H and I) Mice were orthotopically implanted with MC38 cells mixed with exosomes from MC38 cells with or without SK1-I. Colonoscopy image of MC38 tumor size (H); confocal images of colon sections stained with p-Stat3 and E-cadherin (I).

(J) Western blotting showing p-Stat3 proteins in LPLs stimulated by anti-CD3 (5 µg/ml) and CD28 (2 µg/ml) antibodies in the presence of NT-Tumor^EXO, ET-Tumor^EXO (50 µg/ml) or S1P (100 nM) with or without W146 (20 nM) for 24 h.

(K) ELISA analysis of IL-6 and IL-17A in the supernatants of LPLs stimulated with anti-CD3 and CD28 antibodies in the presence of ET-Tumor^EXO (50 µg/ml) with or without W146 (20 nM) after 4 days in culture.

(L–N) Mice were orthotopically implanted with MC38 cells mixed with ET-tumor exosomes and treated with or without W146. Tumor size (L), number of CD4⁺IL-17A⁺ cells in the colon (N), expression of COX2 in the colon (N). The data (E, K–N) represent means ± SEMs (n = 5). ^*p < 0.05, ^**p < 0.01 (Student’s t-test).

(A–C) C57BL/6 mice were orthotopically implanted with MC38-luc cells with exosomes by colonic submucosal injection. Colonoscopy image of implanted MC38-luc cells at day 18 after injection (A), photon flux from C57BL/6 mice harboring MC38-luc tumors (B), and H&E-stained colon sections (C). The data (B) represent means ± SEMs ^**p < 0.01 (Student’s t-test).

(D) Colonic MC38 tumor leukocytes were isolated at day 18 post treatment from mice treated with ET-Tumor^EXO or NT-Tumor^EXO. TCRβ⁺ CD4⁺ IL-17A⁺ and FoxP3⁺ Treg cell populations were examined by FACS.

(E) RT-PCR analysis of the gene expression in colonic MC38 tumor leukocytes isolated at day 18 from mice treated with ET-Tumor^EXO or NT-Tumor^EXO.

(F–G) Confocal images of colon sections stained with p-Stat3 and CD11b from naïve mice treated with ET-Tumor^EXO or NT-Tumor^EXO (F); Western blotting the lysates of colonic CD11b⁺ cells for S1PR1 (G).

(H and I) Mice were orthotopically implanted with MC38 cells mixed with exosomes from MC38 cells with or without SK1-I. Colonoscopy image of MC38 tumor size (H); confocal images of colon sections stained with p-Stat3 and E-cadherin (I).

(J) Western blotting showing p-Stat3 proteins in LPLs stimulated by anti-CD3 (5 µg/ml) and CD28 (2 µg/ml) antibodies in the presence of NT-Tumor^EXO, ET-Tumor^EXO (50 µg/ml) or S1P (100 nM) with or without W146 (20 nM) for 24 h.

(K) ELISA analysis of IL-6 and IL-17A in the supernatants of LPLs stimulated with anti-CD3 and CD28 antibodies in the presence of ET-Tumor^EXO (50 µg/ml) with or without W146 (20 nM) after 4 days in culture.

(L–N) Mice were orthotopically implanted with MC38 cells mixed with ET-tumor exosomes and treated with or without W146. Tumor size (L), number of CD4⁺IL-17A⁺ cells in the colon (N), expression of COX2 in the colon (N). The data (E, K–N) represent means ± SEMs (n = 5). ^*p < 0.05, ^**p < 0.01 (Student’s t-test).