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A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice.

Liechtenstein T, Perez-Janices N, Gato M, Caliendo F, Kochan G, Blanco-Luquin I, Van der Jeught K, Arce F, Guerrero-Setas D, Fernandez-Irigoyen J, Santamaria E, Breckpot K, Escors D - Oncotarget (2014)

Bottom Line: MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase.As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients.Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.

View Article: PubMed Central - PubMed

Affiliation: Division of infection and immunity, Rayne Institute, 5 University Street, London, UK. Immunomodulation group, Navarrabiomed-FMS, calle Irunlarrea 3, Pamplona, Navarra, Spain.

ABSTRACT
Myeloid-derived suppressor cells (MDSCs) exhibit potent immunosuppressive activities in cancer. MDSCs infiltrate tumors and strongly inhibit cancer-specific cytotoxic T cells. Their mechanism of differentiation and identification of MDSC-specific therapeutic targets are major areas of interest. We have devised a highly efficient and rapid method to produce very large numbers of melanoma-infiltrating MDSCs ex vivo without inducing tumors in mice. These MDSCs were used to study their differentiation, immunosuppressive activities and were compared to non-neoplastic counterparts and conventional dendritic cells using unbiased systems biology approaches. Differentially activated/deactivated pathways caused by cell type differences and by the melanoma tumor environment were identified. MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase. These studies uncovered more than 60 potential novel therapeutic targets. As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients. Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.

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Ex vivo-differentiated B16-MDSCs possess characteristics of tumor-infiltrating MDSCs and strongly suppress activated T cells(A) Top immunoblot shows iNOS expression in the indicated myeloid cell types. GADPH was detected as a loading control. The histogram below shows the same result but by flow cytometry after intracellular staining with an iNOS-specific antibody. (B) The column graph represents secreted bioactive TGF-β in the indicated myeloid cultures, after 7 days of differentiation. 24 hours before TGF-β assessment myeloid cells were plated in medium without CM. Bioactive TGF-β was quantified using a TGF-β-reporter cell line. Standard deviations are represented as error bars. CMB16 medium, B16 conditioning medium only. (C) The bar graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8 T cells as a titration of MDSC-T cell ratios. Changes in IFN-γ secretion measured during the same experiment are shown in the bar graph on the right. No, represents T cells incubated without MDSCs, three independent experiments. (D) Same as c but detecting the expression of the proliferation marker Ki67 using MDSC:T cell ratios as indicated in the figure. Standard deviations are represented as error bars. iDCs, immature DCs. no, no T cell-activatory beads. Relevant statistical comparisons are shown within the graph. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences, respectively.
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Figure 4: Ex vivo-differentiated B16-MDSCs possess characteristics of tumor-infiltrating MDSCs and strongly suppress activated T cells(A) Top immunoblot shows iNOS expression in the indicated myeloid cell types. GADPH was detected as a loading control. The histogram below shows the same result but by flow cytometry after intracellular staining with an iNOS-specific antibody. (B) The column graph represents secreted bioactive TGF-β in the indicated myeloid cultures, after 7 days of differentiation. 24 hours before TGF-β assessment myeloid cells were plated in medium without CM. Bioactive TGF-β was quantified using a TGF-β-reporter cell line. Standard deviations are represented as error bars. CMB16 medium, B16 conditioning medium only. (C) The bar graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8 T cells as a titration of MDSC-T cell ratios. Changes in IFN-γ secretion measured during the same experiment are shown in the bar graph on the right. No, represents T cells incubated without MDSCs, three independent experiments. (D) Same as c but detecting the expression of the proliferation marker Ki67 using MDSC:T cell ratios as indicated in the figure. Standard deviations are represented as error bars. iDCs, immature DCs. no, no T cell-activatory beads. Relevant statistical comparisons are shown within the graph. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences, respectively.

Mentions: In tumor-bearing hosts, spleen and tumor-infiltrating MDSCs differ in phenotype and suppressive activities. Splenic MDSCs are not representative of tumor-infiltrating MDSCs [12]. Interestingly, ex vivo-differentiated B16-MDSCs were phenotypically equivalent to in vivo B16 melanoma intra-tumor MDSCs on representative markers, which included CD86, MHC II, CD62L, arginase-1 and PD-L1 (Supplementary Fig. S1). To further demonstrate this point, we compared them to 293T-MDSC and immature conventional DC controls. iNOS and TGF-β expression were evaluated as they are signatures of tumor-infiltrating MDSCs. Only B16-MDSCs expressed high levels of iNOS as ascertained by immunoblot and flow cytometry (Fig. 4A). Bioactive TGF-β was quantified with a bioassay [20]. Accordingly, B16-MDSCs produced a higher amount of bioactive TGF-β than either DCs or 293T-MDSCs (Fig. 4B).


A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice.

Liechtenstein T, Perez-Janices N, Gato M, Caliendo F, Kochan G, Blanco-Luquin I, Van der Jeught K, Arce F, Guerrero-Setas D, Fernandez-Irigoyen J, Santamaria E, Breckpot K, Escors D - Oncotarget (2014)

Ex vivo-differentiated B16-MDSCs possess characteristics of tumor-infiltrating MDSCs and strongly suppress activated T cells(A) Top immunoblot shows iNOS expression in the indicated myeloid cell types. GADPH was detected as a loading control. The histogram below shows the same result but by flow cytometry after intracellular staining with an iNOS-specific antibody. (B) The column graph represents secreted bioactive TGF-β in the indicated myeloid cultures, after 7 days of differentiation. 24 hours before TGF-β assessment myeloid cells were plated in medium without CM. Bioactive TGF-β was quantified using a TGF-β-reporter cell line. Standard deviations are represented as error bars. CMB16 medium, B16 conditioning medium only. (C) The bar graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8 T cells as a titration of MDSC-T cell ratios. Changes in IFN-γ secretion measured during the same experiment are shown in the bar graph on the right. No, represents T cells incubated without MDSCs, three independent experiments. (D) Same as c but detecting the expression of the proliferation marker Ki67 using MDSC:T cell ratios as indicated in the figure. Standard deviations are represented as error bars. iDCs, immature DCs. no, no T cell-activatory beads. Relevant statistical comparisons are shown within the graph. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences, respectively.
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Figure 4: Ex vivo-differentiated B16-MDSCs possess characteristics of tumor-infiltrating MDSCs and strongly suppress activated T cells(A) Top immunoblot shows iNOS expression in the indicated myeloid cell types. GADPH was detected as a loading control. The histogram below shows the same result but by flow cytometry after intracellular staining with an iNOS-specific antibody. (B) The column graph represents secreted bioactive TGF-β in the indicated myeloid cultures, after 7 days of differentiation. 24 hours before TGF-β assessment myeloid cells were plated in medium without CM. Bioactive TGF-β was quantified using a TGF-β-reporter cell line. Standard deviations are represented as error bars. CMB16 medium, B16 conditioning medium only. (C) The bar graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8 T cells as a titration of MDSC-T cell ratios. Changes in IFN-γ secretion measured during the same experiment are shown in the bar graph on the right. No, represents T cells incubated without MDSCs, three independent experiments. (D) Same as c but detecting the expression of the proliferation marker Ki67 using MDSC:T cell ratios as indicated in the figure. Standard deviations are represented as error bars. iDCs, immature DCs. no, no T cell-activatory beads. Relevant statistical comparisons are shown within the graph. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences, respectively.
Mentions: In tumor-bearing hosts, spleen and tumor-infiltrating MDSCs differ in phenotype and suppressive activities. Splenic MDSCs are not representative of tumor-infiltrating MDSCs [12]. Interestingly, ex vivo-differentiated B16-MDSCs were phenotypically equivalent to in vivo B16 melanoma intra-tumor MDSCs on representative markers, which included CD86, MHC II, CD62L, arginase-1 and PD-L1 (Supplementary Fig. S1). To further demonstrate this point, we compared them to 293T-MDSC and immature conventional DC controls. iNOS and TGF-β expression were evaluated as they are signatures of tumor-infiltrating MDSCs. Only B16-MDSCs expressed high levels of iNOS as ascertained by immunoblot and flow cytometry (Fig. 4A). Bioactive TGF-β was quantified with a bioassay [20]. Accordingly, B16-MDSCs produced a higher amount of bioactive TGF-β than either DCs or 293T-MDSCs (Fig. 4B).

Bottom Line: MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase.As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients.Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.

View Article: PubMed Central - PubMed

Affiliation: Division of infection and immunity, Rayne Institute, 5 University Street, London, UK. Immunomodulation group, Navarrabiomed-FMS, calle Irunlarrea 3, Pamplona, Navarra, Spain.

ABSTRACT
Myeloid-derived suppressor cells (MDSCs) exhibit potent immunosuppressive activities in cancer. MDSCs infiltrate tumors and strongly inhibit cancer-specific cytotoxic T cells. Their mechanism of differentiation and identification of MDSC-specific therapeutic targets are major areas of interest. We have devised a highly efficient and rapid method to produce very large numbers of melanoma-infiltrating MDSCs ex vivo without inducing tumors in mice. These MDSCs were used to study their differentiation, immunosuppressive activities and were compared to non-neoplastic counterparts and conventional dendritic cells using unbiased systems biology approaches. Differentially activated/deactivated pathways caused by cell type differences and by the melanoma tumor environment were identified. MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase. These studies uncovered more than 60 potential novel therapeutic targets. As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients. Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.

Show MeSH
Related in: MedlinePlus