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

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.Myeloid-derived suppressor cells (MDSCs) have been recognized as major contributors to tumor-induced immunosuppression. Tumor-infiltrating MDSCs strongly inhibit cytotoxic T cells, and their expansion favors tumor progression and metastasis [1, 2]. Counteracting MDSC activities strongly enhances anti-cancer treatments and prolongs survival. Specific MDSC elimination by chemotherapy significantly contributes to anti-tumor efficacy [3-5]. Interestingly, conventional dendritic cells (DCs) remain unaffected by some of these chemotherapy treatments and the mechanisms underlying selective MDSC susceptibility to these drugs are currently unknown. The availability of large numbers of tumor-infiltrating MDSCs would significantly improve research in their biology and functions, and facilitate anti-MDSC drug discovery.MDSCs in mice comprise a heterogeneous population of immature CD11b^high Gr-1⁺ myeloid cells [6]. However, their discrimination from other myeloid cells such as immature DCs, M2 macrophages, monocytes and neutrophils remains somewhat ambiguous. Nevertheless, mouse MDSCs are classified into monocytic (M) and granulocytic (G) subsets, which differ in Ly6C-Ly6G expression profiles. M-MDSCs are Ly6C^high Ly6G^−/low while G-MDSCs are Ly6C^int/low Ly6G^high. Both subsets suppress immune responses through several pathways, including L-arginine depletion through arginase-1 (arg-1) and inducible nitric oxide synthase (iNOS) activity, increased generation of reactive oxygen species (ROS) and production of immunosuppressive cytokines such as TGF-β [7, 8].For their study, MDSCs are isolated from the spleen or directly of tumors from a large number of tumor-bearing mice [9-11]. However, spleen MDSCs are phenotypically and functionally different from tumor-infiltrating MDSCs [11, 12]. Moreover, isolated intra-tumor MDSCs are usually contaminated with other myeloid cells, do not proliferate well ex vivo, lack plasticity of differentiation and are prone to apoptosis [9, 13, 14]. In addition, low MDSC numbers are obtained from within tumors [12, 15]. Ex vivo MDSC production systems have been developed, which rely on incubation of bone marrow (BM) cells with high concentrations of recombinant GM-CSF, alone or in combination with other cytokines, and sometimes supplemented with cancer cell-derived conditioning medium. Nevertheless, these methods achieve MDSC differentiation efficiencies of around 30%-40% of total cells [13, 14]. In practical terms, none of these methods have yet replaced the purification of MDSCs directly from tumors of cancer-bearing mice. Therefore, high-throughput and drug discovery studies with isolated intra-tumor MDSCs are certainly a challenge.