Fig 1: Local injection of iMSC/CCL19 augments the antitumor effect by anti-PD-L1 blockade therapy. (A) PD-L1 expression on CT26 cells that were cultured in the presence or absence of recombinant 50 ng/mL IFN-? for 48 hours. Gray: isotype control, black dotted: PE anti-PD-L1 antibody. (B) CT26-bearing mice were i.t. injected with iMSC or iMSC/CCL19 on days 14 and 16; on day 17 after tumor inoculation, TILs were collected and stained with anti-CD3, anti-CD8a, and anti-PD-1 antibodies. Splenic cells from CT26-bearing mice were analyzed as control cells. The numbers represent percentages of PD-1+ CD8+ cells among CD3+ CD8+ T cells. (C) CT26-bearing mice were i.t. injected with iMSC/CCL19 on days 13 and 15 after tumor inoculation (arrowhead). Control IgG (200 µg) or anti-PD-L1 (200 µg) antibody were i.p. injected on days 15 and 17 (arrow). The tumor growth in individual mice is shown. (D) The mean (±SEM) tumor size. *P<0.05. **P<0.01 by two-tailed t-test. N.S., not significant. (E) Photograph of tumor tissues is shown. Scale bar=10 mm. CCL19, chemokine (C-C motif) ligand 19; IFN, interferon; iMSC, immortalized MSC; i.t., intratumoral; MSC, mesenchymal stem/stromal cells; TILs, tumor-infiltrating lymphocytes.
Fig 2: Local injection of iMSC/CCL19 alone can regress tumor growth. (A) BALB/c mice were inoculated s.c. into the right flank with CT26 (5×105 cells). Thereafter, 100 mg/kg CP was administered by i.p. injection on day 13, and iMSC or iMSC/CCL19 (5×105 cells) were used for i.t. injection on days 14 and 16. Tumor size was measured every 4 days. Numbers of cured mice are shown. The mean tumor size is indicated in (B). Mean±SEM, *P<0.05. **P<0.01 by two-tailed t-test. (C) On days 14 and 16 after CT26 inoculation, i.t. injection with PBS or 0.2 µg recombinant murine CCL19 at a volume of 100 µL. The tumor size was measured every 4 days and the mean±SEM was indicated. N.S., not significant. n=5. CCL19, chemokine (C-C motif) ligand 19; CP, cyclophosphamide; iMSC, immortalized MSC; i.p., intraperitoneal; i.t., intratumoral; MSC, mesenchymal stem/stromal cells; s.c., subcutaneously.
Fig 3: Generation of CCL19-expressing Fib and MSC. (A) Murine Fib and MSC were immortalized by transfection with SV40T-GFP vector. GFP expression in primary (p)Fib, immortalized (i)Fib, pMSC, and iMSC was examined by flow cytometry. (B) mRNA expression of iFib, iMSC, CCL19-expressing iFib (iFib/CCL19), and CCL19-expressing iMSC (iMSC/CCL19) was examined by RT-PCR. ß-Actin was used as a control. (C) The levels of CCL19 in the supernatants were determined by ELISA. Data are presented as the mean±SEM. **P<0.01 by Student’s t-test. (D) CT26 colon carcinoma cells (5×105) were inoculated subcutaneously into BALB/c mice with or without pMSC or iMSC. Tumor size was measured every 4 days. Data are presented as the mean±SEM. CCL19, chemokine (C-C motif) ligand 19; iFib, immortalized fibroblasts; iMSC, immortalized MSC; MSC, mesenchymal stem/stromal cells; pFib, primary fibroblasts; pMSC, primary MSC; RT, reverse transcription.
Fig 4: Antitumor effect induced by iMSC/CCL19 local therapy is T cell-dependent. (A) BALB/c nu/nu mice were inoculated s.c. with CT26 (5×105 cells) with or without iMSC or iMSC/CCL19. Thereafter, tumor size was measured every 4 days. n=5, respectively. (B) CT26 tumor-bearing mice received i.t. injection with iMSC/CCL19 (arrowheads) and i.p. injection with 100 µg of control IgG, anti-CD4 (depletion) or anti-CD8 (depletion) antibody (arrows). Tumor size on subsequent days after CT26 inoculation (left), the mean tumor size on day 30 (right). **P<0.01. *P<0.05 by Tukey-Kramer test (ANOVA). n=5, respectively. (C) iMSC or iMSC/CCL19 were injected i.t. on days 14 and 16 after tumor inoculation. Cryosections of the tumor were prepared 17 days after tumor inoculation and stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD11c antibodies. Representative images of inner (upper) and peripheral (lower) regions of tumor were shown. (D–G) Number of CD3, CD4, CD8, and CD11c-positive cells were shown. **P<0.01. *P<0.05 by Tukey-Kramer test (ANOVA). N.S., not significant. CCL19, chemokine (C-C motif) ligand 19; iMSC, immortalized MSC; i.p., intraperitoneal; i.t., intratumoral; MSC, mesenchymal stem/stromal cells; s.c., subcutaneously.
Fig 5: iMSC/CCL19 local therapy attracts F4/80- CD11c+ CCR7+ DC and increases IFN-?+ CD8+ T cells iMSC or iMSC/CCL19 were injected i.t. on days 14 and 16 after tumor inoculation. Tumor cells were collected 17 days after tumor inoculation, and tumor-infiltrating immune cells were analyzed by flow cytometry. (A) Representative plots (left) and the percentages of indicated CD11c+ CCR7+ among CD45+ F4/80cells are shown (right). *P<0.05 by Tukey-Kramer test (ANOVA). N.S., not significant. (B) Representative plots (left) and the percentages of CD4+ CD11c+ cells among CD45+ F4/80cells are shown (right). (C, D) CT26 tumor-bearing mice were received i.t. injection with iMSC/CCL19 (arrowheads) and i.p. injection with 100 µg of control IgG or anti-CD4 (depletion) antibody as described in figure 4b. (C) Represent plots (left) and the percentages of CD11c+ F4/80 cells gated on CD45+ cells were shown. (D) The percentages of IFN-?+ CD8+ T cells among CD3+ T cells are shown (represent plots: left, bar graph; right). **P<0.01 by Tukey-Kramer test (ANOVA). n=3. Splenocytes collected from tumor-bearing mice were analyzed as a control. CCL19, chemokine (C-C motif) ligand 19; IFN, interferon; IgG, immunoglobulin G; iMSC, immortalized MSC; i.p., intraperitoneal; i.t., intratumoral; MSC, mesenchymal stem/stromal cells.
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