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Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma.

Zhao T, Ren H, Jia L, Chen J, Xin W, Yan F, Li J, Wang X, Gao S, Qian D, Huang C, Hao J - Oncotarget (2015)

Bottom Line: We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice.Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects.Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Department of Pancreatic Cancer, Tianjin, China.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is the worst prognoses among all the malignancies. Now, gemcitabine (Gem) is the first line chemotherapeutic drug for advanced pancreatic cancer. However, Gem is usually ineffective to the PDAC because of high degree of drug resistance. Hypoxia and immune suppressive milieu are the best-described hallmarks of PDAC; therefore, we investigated the impact of hypoxia inducible factor-1 (HIF-1) inhibitor, PX-478, in combination with Gem on the induction of immunogenic cell death (ICD). We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice. Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects. Pancreatic cell lines treated with Gem and PX-478 can induce an increase in eIF2α phosphorylation was correlated with down-regulation of HIF-1α and elicited exposure of CRT and release of HMGB1 and ATP. Only co-treated cells induced DC maturation/phagocytosis and IFN-γ secretion by cytotoxic T lymphocytes. Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization. We propose that inhibition HIF-1α elicits Gem-induced immune response and eliminates PDAC cells by inducing ICD.

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Comparison of treatment-induced immune response in both DCs and cytotoxic T cells(A and B) DC maturation. DCs were stimulated with conditioned supernatant of freeze/thawed, PX-478, Gem or Gem/PX-478 treated cell culture medium. The maturation markers for DCs, CD83 or CD80, were evaluated by flow cytometry and compared between freeze/thawed, PX-478, Gem and Gem/PX-478-treated groups. **P<0.01. Data were expressed as mean ± SD and each value represented the mean of three replicates. (C) Fluorescence microscopy analysis of phagocytosis. After 24 hours co-culture of immature DCs with killed tumor cells, the engulfment of tumor cells was verified by fluorescence microscopy. DCs were stained with DiO (green) and CFPAC-1 cells were stained with DiI (red). Double positive or yellow cells are fully phagocytized cells. (D) IFN-γ production. Immature DCs were pulsed with killed tumor cells for 24 hours and then co-cultured with autologous CD3+/CD8+ T lymphocytes. IFN-γ concentration in the supernatant was assessed after 10 days using the IFN-γ ELISA kit. A representative out of three independent experiments is depicted. ***P<0.001.
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Figure 6: Comparison of treatment-induced immune response in both DCs and cytotoxic T cells(A and B) DC maturation. DCs were stimulated with conditioned supernatant of freeze/thawed, PX-478, Gem or Gem/PX-478 treated cell culture medium. The maturation markers for DCs, CD83 or CD80, were evaluated by flow cytometry and compared between freeze/thawed, PX-478, Gem and Gem/PX-478-treated groups. **P<0.01. Data were expressed as mean ± SD and each value represented the mean of three replicates. (C) Fluorescence microscopy analysis of phagocytosis. After 24 hours co-culture of immature DCs with killed tumor cells, the engulfment of tumor cells was verified by fluorescence microscopy. DCs were stained with DiO (green) and CFPAC-1 cells were stained with DiI (red). Double positive or yellow cells are fully phagocytized cells. (D) IFN-γ production. Immature DCs were pulsed with killed tumor cells for 24 hours and then co-cultured with autologous CD3+/CD8+ T lymphocytes. IFN-γ concentration in the supernatant was assessed after 10 days using the IFN-γ ELISA kit. A representative out of three independent experiments is depicted. ***P<0.001.

Mentions: To test whether ICD markers in the conditioned medium could enhance immune response, human immature dendritic cells (iDCs) (treated with GM-CSF and IL-4 for 5 days) were incubated with conditioned supernatants from Gem or Gem/PX-478 treated cells for another 24 hours. Maturation of DCs was determined by expression of CD80 or CD83 using flow cytometry. Treatment with Gem/PX-478 significantly increased expression of both CD83 (Figure 6A) and CD80 (Figure 6B), indicating of maturation of DCs. To determine the phagocytosis single of ecto-CRT, iDCs (treated with GM-CSF and IL-4 for 5 days) were co-cultured with pancreatic cancer cells treated with saline, Gem, PX-478, or Gem/PX-478 for 24 hours. And then, fluorescence microscopy was used to evaluate the phagocytosis of DCs. DCs activated with Gem/PX-478-treated cells showed more phagocytosis activity compared with single treatment, as shown by double positive, or yellow coloured cells (Figure 6C). Most importantly, the co-culture Gem/PX-478 condition medium challenged DCs with autologous CD3+/CD8+ T cells significantly increased the secretion of IFN-γ by cytotoxic T-cells compared with control and those treated with single agent (Figure 6D). Taken together, these data confirmed that conditioned supernatants of Gem/PX478-treated PDAC cells can enhance immune response by inducing maturation and phagocytosis of DCs and up-regulate cytotoxic function of T cells.


Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma.

Zhao T, Ren H, Jia L, Chen J, Xin W, Yan F, Li J, Wang X, Gao S, Qian D, Huang C, Hao J - Oncotarget (2015)

Comparison of treatment-induced immune response in both DCs and cytotoxic T cells(A and B) DC maturation. DCs were stimulated with conditioned supernatant of freeze/thawed, PX-478, Gem or Gem/PX-478 treated cell culture medium. The maturation markers for DCs, CD83 or CD80, were evaluated by flow cytometry and compared between freeze/thawed, PX-478, Gem and Gem/PX-478-treated groups. **P<0.01. Data were expressed as mean ± SD and each value represented the mean of three replicates. (C) Fluorescence microscopy analysis of phagocytosis. After 24 hours co-culture of immature DCs with killed tumor cells, the engulfment of tumor cells was verified by fluorescence microscopy. DCs were stained with DiO (green) and CFPAC-1 cells were stained with DiI (red). Double positive or yellow cells are fully phagocytized cells. (D) IFN-γ production. Immature DCs were pulsed with killed tumor cells for 24 hours and then co-cultured with autologous CD3+/CD8+ T lymphocytes. IFN-γ concentration in the supernatant was assessed after 10 days using the IFN-γ ELISA kit. A representative out of three independent experiments is depicted. ***P<0.001.
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Related In: Results  -  Collection

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Figure 6: Comparison of treatment-induced immune response in both DCs and cytotoxic T cells(A and B) DC maturation. DCs were stimulated with conditioned supernatant of freeze/thawed, PX-478, Gem or Gem/PX-478 treated cell culture medium. The maturation markers for DCs, CD83 or CD80, were evaluated by flow cytometry and compared between freeze/thawed, PX-478, Gem and Gem/PX-478-treated groups. **P<0.01. Data were expressed as mean ± SD and each value represented the mean of three replicates. (C) Fluorescence microscopy analysis of phagocytosis. After 24 hours co-culture of immature DCs with killed tumor cells, the engulfment of tumor cells was verified by fluorescence microscopy. DCs were stained with DiO (green) and CFPAC-1 cells were stained with DiI (red). Double positive or yellow cells are fully phagocytized cells. (D) IFN-γ production. Immature DCs were pulsed with killed tumor cells for 24 hours and then co-cultured with autologous CD3+/CD8+ T lymphocytes. IFN-γ concentration in the supernatant was assessed after 10 days using the IFN-γ ELISA kit. A representative out of three independent experiments is depicted. ***P<0.001.
Mentions: To test whether ICD markers in the conditioned medium could enhance immune response, human immature dendritic cells (iDCs) (treated with GM-CSF and IL-4 for 5 days) were incubated with conditioned supernatants from Gem or Gem/PX-478 treated cells for another 24 hours. Maturation of DCs was determined by expression of CD80 or CD83 using flow cytometry. Treatment with Gem/PX-478 significantly increased expression of both CD83 (Figure 6A) and CD80 (Figure 6B), indicating of maturation of DCs. To determine the phagocytosis single of ecto-CRT, iDCs (treated with GM-CSF and IL-4 for 5 days) were co-cultured with pancreatic cancer cells treated with saline, Gem, PX-478, or Gem/PX-478 for 24 hours. And then, fluorescence microscopy was used to evaluate the phagocytosis of DCs. DCs activated with Gem/PX-478-treated cells showed more phagocytosis activity compared with single treatment, as shown by double positive, or yellow coloured cells (Figure 6C). Most importantly, the co-culture Gem/PX-478 condition medium challenged DCs with autologous CD3+/CD8+ T cells significantly increased the secretion of IFN-γ by cytotoxic T-cells compared with control and those treated with single agent (Figure 6D). Taken together, these data confirmed that conditioned supernatants of Gem/PX478-treated PDAC cells can enhance immune response by inducing maturation and phagocytosis of DCs and up-regulate cytotoxic function of T cells.

Bottom Line: We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice.Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects.Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Department of Pancreatic Cancer, Tianjin, China.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is the worst prognoses among all the malignancies. Now, gemcitabine (Gem) is the first line chemotherapeutic drug for advanced pancreatic cancer. However, Gem is usually ineffective to the PDAC because of high degree of drug resistance. Hypoxia and immune suppressive milieu are the best-described hallmarks of PDAC; therefore, we investigated the impact of hypoxia inducible factor-1 (HIF-1) inhibitor, PX-478, in combination with Gem on the induction of immunogenic cell death (ICD). We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice. Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects. Pancreatic cell lines treated with Gem and PX-478 can induce an increase in eIF2α phosphorylation was correlated with down-regulation of HIF-1α and elicited exposure of CRT and release of HMGB1 and ATP. Only co-treated cells induced DC maturation/phagocytosis and IFN-γ secretion by cytotoxic T lymphocytes. Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization. We propose that inhibition HIF-1α elicits Gem-induced immune response and eliminates PDAC cells by inducing ICD.

Show MeSH
Related in: MedlinePlus