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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 monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage(A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM293T, as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant differences (P<0.001). Experiments were repeated at least twice.
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Figure 3: Ex vivo monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage(A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM293T, as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant differences (P<0.001). Experiments were repeated at least twice.

Mentions: The relationship between intra-tumor M-MDSCs and G-MDSCs is still uncertain, as G-MDSCs could be either immature recruited neutrophils, or the terminal differentiation stage of M-MDSCs [14, 19]. To test whether M-MDSCs were G-MDSC precursors, their proportion was monitored over time. G-MDSC numbers increased in both 293T-MDSC and B16-MDSC cultures until it comprised the main subset after 10 days (Fig. 3A). In addition, M-MDSCs (CD11b+ GR1+ Ly6Gneg) were sorted on day 5 and cultured for 3 days. M-MDSCs converted into G-MDSCs as ascertained by Ly6G up-regulation (Fig. 3B). Interestingly, increase in MDSC numbers stopped from day 5 onwards (Fig. 3C), coinciding with G-MDSC differentiation from M-MDSCs (Fig. 3A). It has been shown that G-MDSCs are terminally differentiated and their viability is compromised in vivo. This also held true in our ex vivo system. The viability of sorted M-MDSCs and G-MDSCs was assessed by flow cytometry, and G-MDSCs were found to be less viable than M-MDSCs (Fig. 3C).


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 monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage(A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM293T, as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant differences (P<0.001). Experiments were repeated at least twice.
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Related In: Results  -  Collection

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Figure 3: Ex vivo monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage(A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM293T, as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P<0.05), very significant (P<0.01) and highly significant differences (P<0.001). Experiments were repeated at least twice.
Mentions: The relationship between intra-tumor M-MDSCs and G-MDSCs is still uncertain, as G-MDSCs could be either immature recruited neutrophils, or the terminal differentiation stage of M-MDSCs [14, 19]. To test whether M-MDSCs were G-MDSC precursors, their proportion was monitored over time. G-MDSC numbers increased in both 293T-MDSC and B16-MDSC cultures until it comprised the main subset after 10 days (Fig. 3A). In addition, M-MDSCs (CD11b+ GR1+ Ly6Gneg) were sorted on day 5 and cultured for 3 days. M-MDSCs converted into G-MDSCs as ascertained by Ly6G up-regulation (Fig. 3B). Interestingly, increase in MDSC numbers stopped from day 5 onwards (Fig. 3C), coinciding with G-MDSC differentiation from M-MDSCs (Fig. 3A). It has been shown that G-MDSCs are terminally differentiated and their viability is compromised in vivo. This also held true in our ex vivo system. The viability of sorted M-MDSCs and G-MDSCs was assessed by flow cytometry, and G-MDSCs were found to be less viable than M-MDSCs (Fig. 3C).

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