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. 2013 Jun;62(6):1083-91.
doi: 10.1007/s00262-013-1407-9. Epub 2013 Apr 19.

Dendritic Cell Immunotherapy Combined With Gemcitabine Chemotherapy Enhances Survival in a Murine Model of Pancreatic Carcinoma

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Free PMC article

Dendritic Cell Immunotherapy Combined With Gemcitabine Chemotherapy Enhances Survival in a Murine Model of Pancreatic Carcinoma

Tomar Ghansah et al. Cancer Immunol Immunother. .
Free PMC article

Abstract

Pancreatic cancer is an extremely aggressive malignancy with a dismal prognosis. Cancer patients and tumor-bearing mice have multiple immunoregulatory subsets including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that may limit the effectiveness of anti-tumor immunotherapies for pancreatic cancer. It is possible that modulating these subsets will enhance anti-tumor immunity. The goal of this study was to explore depletion of immunoregulatory cells to enhance dendritic cell (DC)-based cancer immunotherapy in a murine model of pancreatic cancer. Flow cytometry results showed an increase in both Tregs and MDSC in untreated pancreatic cancer-bearing mice compared with control. Elimination of Tregs alone or in combination with DC-based vaccination had no effect on pancreatic tumor growth or survival. Gemcitabine (Gem) is a chemotherapeutic drug routinely used for the treatment for pancreatic cancer patients. Treatment with Gem led to a significant decrease in MDSC percentages in the spleens of tumor-bearing mice, but did not enhance overall survival. However, combination therapy with DC vaccination followed by Gem treatment led to a significant delay in tumor growth and improved survival in pancreatic cancer-bearing mice. Increased MDSC were measured in the peripheral blood of patients with pancreatic cancer. Treatment with Gem also led to a decrease of this population in pancreatic cancer patients, suggesting that combination therapy with DC-based cancer vaccination and Gem may lead to improved treatments for patients with pancreatic cancer.

Figures

Figure 1
Figure 1
Increased Tregs in the spleens of tumor-bearing mice. (A) CD4+CD25+Foxp3+ cells in the spleens (n=4) of naïve and Panc02 tumor-bearing mice were measured by flow cytometry. *indicates p<0.05. (B) CD4+CD25+ T cells were purified from the spleens of naïve or Panc02-bearing mice and co-cultured with OT-I T cells stimulated with OVA257-264 peptide. Proliferation was measured after 72 hours of co-culture. (C) Depletion of Tregs does not enhance DC immunotherapy. Mice were injected with Panc02 cells. Starting on day 3 and continuing every 3-4 days after, mice received NrIgG or PC-61 antibodies. Mice received DC injections intratumorally on days 3, 7, and 10. Tumor growth was measured.
Figure 2
Figure 2
Increased MDSC in the spleens and tumors of Panc02-tumor bearing mice. (A) Spleens (n=4 per group) were collected from naïve and Panc02 tumor-bearing mice on days 14, 20, 24 and 27 after Panc02 injection. (B) CD11b+Gr1+ MDSC percentages in the tumors of mice on days 20, 24, and 27 after injection of Panc02 tumor. CD11b+Gr1+ cells were measured by flow cytometry.
Figure 3
Figure 3
MDSC from Panc02-bearing mice suppress CD8+ and CD4+ T cell responses. CD11b+Gr1+ cells were purified from the spleens of Panc02-bearing mice. (A) Pmel T cells were cultured with gp10025-33 peptide alone, or at a 1:3 ratio with purified Gr1- or Gr1+ cells. Proliferation was measured after 72 hours. (B) OT-II T cells were cultured with DC pulsed with OVA323-339 peptide alone or at a 1:10 ratio with purified Gr1- or Gr1+ cells. Proliferation was measured after 72 hours. *indicates p<0.01.
Figure 4
Figure 4
Gemcitabine reduces MDSC in Panc02-bearing mice. On day 27 after Panc02 injection, (A) spleens and (B) tumors were collected from tumor-bearing and tumor-bearing mice treated with 120 mg/kg of Gemcitabine. CD11b+Gr1+ cells were measured in the splenocytes by flow cytometry. *indicates p<0.001, **indicates p<0.01
Figure 5
Figure 5
Delay of tumor growth and enhanced survival in mice treated with DC and Gem therapy. Mice were injected s.c. with Panc02 tumor cells (n=8 mice per group). Mice received 120 mg/kg Gem i.p. starting on day 3 and continuing every 3-4 days until day 42. DC vaccines were injected intratumorally on days 3, 7, and 10. Mice received control (PBS), Gem alone (Gem), DC alone (DC), or combination therapy with DC and Gem (DC+Gem). (A) Tumor size. (B) Percent survival. (C) Splenocytes were collected on day 21 and T cells were restimulated for 48 hours with media alone (CM), B16 cells, or Panc02 cells. Supernatants were collected and IFN-γ production was measured by ELISA. (D) Tumors were collected on day 21. The number of CD3+CD8+ T cells was calculated per milligram of tumor. (E) The percentage of CD3+CD8+ T cells producing IFN-gamma was measured. Data shows the mean ± SD (n=3). *indicates p<0.05.
Figure 5
Figure 5
Delay of tumor growth and enhanced survival in mice treated with DC and Gem therapy. Mice were injected s.c. with Panc02 tumor cells (n=8 mice per group). Mice received 120 mg/kg Gem i.p. starting on day 3 and continuing every 3-4 days until day 42. DC vaccines were injected intratumorally on days 3, 7, and 10. Mice received control (PBS), Gem alone (Gem), DC alone (DC), or combination therapy with DC and Gem (DC+Gem). (A) Tumor size. (B) Percent survival. (C) Splenocytes were collected on day 21 and T cells were restimulated for 48 hours with media alone (CM), B16 cells, or Panc02 cells. Supernatants were collected and IFN-γ production was measured by ELISA. (D) Tumors were collected on day 21. The number of CD3+CD8+ T cells was calculated per milligram of tumor. (E) The percentage of CD3+CD8+ T cells producing IFN-gamma was measured. Data shows the mean ± SD (n=3). *indicates p<0.05.
Figure 6
Figure 6
Gemcitabine therapy reduces MDSC in the peripheral blood of patients with pancreatic cancer. (A) Percentage of CD14-CD11b+CD33+ cells in the PBMC of healthy donors and pancreatic cancer patients as measured by flow cytometry. (B) Percentage of CD14-CD11b+CD33+ cells in the PBMC of pancreatic cancer patients prior to Gem therapy (pre) and after 1 cycle of 1000 mg/m2 Gem therapy (post-Gem) as measured by flow cytometry. *indicates p<0.05. **indicates p<0.01.

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