Article Figures & Data
Figures
- Figure 1.
Characterization of the human UCB–MSCs. A, cultured human UCB–MSCs with a fibroblast-like morphology visualized by phase-contrast microscopy. B, immunophenotype profile of human UCB–MSCs. C, the ability of human UCB–MSCs to be differentiated into adipocytes, osteocytes, and chondrocytes was determined by Oil Red O, Alizarin Red S, and alcian blue staining, respectively.
- Figure 2.
UCB–MSCs can be successfully transduced to release functional IL15. UCB–MSCs were transduced with lentivirus carrying genes encoding GFP or GFP and IL15, and named MSC-GFP and MSC-IL15, respectively. A, to determine the efficiency of transduction, transduced cells were analyzed for GFP expression by flow cytometry. B, IL15 mRNA expression in MSC-IL15 was verified by semiquantitative RT-PCR. C, MSC-GFP and MSC-IL15 were cultured at 5 × 105 per mL for 24 hours. Supernatants were collected and IL15 concentration was measured by ELISA. D, membrane-bound IL15 on the surface of MSC-IL15 was detected by FACS. To test whether IL15 released by MSCs had any biological functions, splenocytes from naïve mice were incubated with the culture supernatants of MSC-IL15. E, growth of splenocytes was tested by MTT metabolization assays. The absorbance at 595 nm was as a measure for the relative number of viable cells. F, cytokine-induced activation of STAT5 and STAT3. Activated splenocytes were analyzed by FACS with phospho-STAT5 or STAT3-specific antibody staining. G, the IFNγ contents of the culture supernatants were measured by ELISA. Data are means ± SD of two independent experiments, each performed in triplicate. **, P < 0.01, versus control groups.
- Figure 3.
In vivo antitumor activity of MSC-IL15 in a synegenic mice model of Pan02 tumor. A, prophylactic effect of MSC-IL15. 1 × 106 Pan02 tumor cells were injected into C57BL/6 mice subcutaneously. Different numbers of MSC-IL15 or MSC-GFP were administered by i.v. injection at day 0 after Pan02 tumor inoculation. Tumor growth was recorded over time. B and C, therapeutic effect of MSC-IL15. 1 × 106 Pan02 tumor cells were injected into C57BL/6 mice subcutaneously. After 7 days, 1 × 106 MSC-IL15 or MSC-GFP were administered by i.v. injection. Tumor growth (B) and animal survival (C) was recorded over time. D, 1 × 106 Pan02 tumor cells were injected into C57BL/6 mice subcutaneously. After 7 days, 1 × 106 MSC-IL15, or 5 μg recombinant IL15 were administered by i.v. injection. Tumor growth was recorded over time. E, analysis of apoptotic cells in tumors from MSC-IL15–treated mice. Ten days after MSC-IL15 injection, tumors were harvested and used for H&E staining and TUNEL detection. F, analysis of lymphocyte infiltration in tumors from MSC-IL15–treated mice. Tumor sections were stained with PE-conjugated anti-CD3 or anti-NK1.1. Images are representative of treated mice. **, P < 0.01, versus control groups.
- Figure 4.
MSC-IL15 home to tumor tissue and secrete IL15 in vivo. A, the migration of MSCs and genetically modified MSCs in response to conditioned media from Pan02 cells was determined in vitro. Representative photomicrographs of stained filters show migrated UCB–MSCs. Magnification, ×100. B, in vivo MSC-IL15 migration and persistence assays, bioluminescent imaging of the profile of tumor to detect MSC-IL15 labeled with PKH26 in tumor tissues. PKH26-labeled MSC-IL15 (red) show that MSC-IL15 incorporated into tumor mass. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) for counterstaining. C, the level of IL15 from tumor and serum following therapy. An enzyme-linked immunosorbent assay for IL15 level was performed in triplicate with tumor homogenates or pooled serum samples from mice in different groups obtained on day 14 following therapy. **, P < 0.01, versus control groups.
- Figure 5.
CD8+ T cells and NK cells were responsible for MSC-IL15–mediated antitumor activity in Pan02 pancreatic tumor model. Naïve C57BL/6 mice were treated with anti–asialo-GM1 and/or anti-mouse CD8 antibody for 3 days. Then 1 × 106 Pan02 tumor cells were injected into C57BL/6 mice subcutaneously. Seven days later, 1 × 106 MSC-IL15 was administered by i.v. injection treatment. Tumor growth (A) and animal survival (B) were recorded over time. *, P < 0.05; **, P < 0.01, versus control groups.
- Figure 6.
Induction of tumor-specific long-term immunity by MSC-IL15 in Pan02 pancreatic tumor model. A and B, induction of tumor-specific CTLs after MSC-IL15 injection. Ten days after MSC-IL15 treatment, the splenocytes were isolated and restimulated in vitro for 5 days with the irradiated Pan02 cells and assayed for CTL activity against 51Cr-labeled Pan02 (A) and B16 cell targets (B). C, tumor-specific CTL activity of IL15-treated mice was also determined by CTL assay. D, ELISPOT assay. After 10 days of MSC-L or IL15 treatment, spleen cells were isolated and stimulated by Pan02 cells. The number of IFNγ+ cells was determined by ELISPOT assay. All experiments were repeated in triplicate. Columns, mean; bars, SE. E and F, rechallenge experiments. Tumor-free mice were rechallenged either with Pan02 or unrelated syngeneic B16 tumor cells. Tumor growth and survival were recorded over time. G and H, adoptive transfer experiments. Antigen-specific lymphocytes were generated from tumor-free mice of 100 days of posttumor challenge, and CD4+ and CD8+ T cells were purified by magnetic bead cell sorting. Stimulated cells were infused i.v. into naïve mice. On the next day, mice were challenged with Pan02 tumor cells. Control mice were infused with splenocytes from naïve mice. Tumor growth and survival were recorded over time. *, P < 0.05; **, P < 0.01, versus control groups.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF - 71K, The expression of immunologic markers on MSC surface by FACS analysis of CD14, CD11b, and CD16 expressions.
- Supplementary Figure 2 - PDF - 177K, The effect of MSCs on human and murine NK cells activation. Human and mouse NK cells were stimulated by recombinant human or murine IL-15 (5ug/ml) for 24 hours, in the presence or absence of MSCs. CD69 and CD107a expressions on NK cells were detected by FACS. In addition, the effect of MSC-IL-15 on CD69 and CD107a expressions of mouse NK cells was also detected.
- Supplementary Figure 3 - PDF - 113K, MSC-IL-15 suppresses Pan02 tumor growth via a cell dose-dependent manner. 1x106 Pan02 tumor cells were injected into C57BL/6 mice substancuously. After 7 days, various cell doses of MSC-IL-15 were administrated by i.v. injection. Tumor growth was recorded over time. **,P<0.01.
- Supplementary Figure 4 - PDF - 101K, Intratumor infiltration of gammadelta T cells were determined by flow cytometry at 10 days after MSC-IL-15 injection. **, P<0.01.
- Supplementary Figure 5 - PDF - 115K, (A) Intratumor infiltration of CD4+FoxP3+ regulatory T cells were determined by flow cytometry at 10 days after MSC-IL-15 injection. (B) mRNA levels of Foxp3 in tumor tissue were detected by qRT-PCR. (C) MSC-GFP with tumor cells was co-injected, and 7 days later, MSC-IL-15 was administrated. Tumor growth was recorded over time. *, P<0.05; **, P<0.01.
Volume 13, Issue 8
