| Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSC). MDSCs inhibit anti-tumor T cell responses through a variety of mechanisms including nutrient depletion, production of reactive oxygen and nitrogen species, VEGF expression, and regulatory T cell expansion. In cancer patients and murine tumor models, MDSC accumulation correlates with increased stage and tumor burden, but the frequency and mechanisms of MDSC induction in human cancer remain poorly understood. This study examined the ability of a diverse set of human solid tumor cell lines to induce MDSC using in vitro tumor co-culture methods. Newly induced suppressor cells were characterized for morphology, phenotype, and gene expression. Of over 100 solid tumor cell lines examined, 45 generated canonical CD33+HLA-DRlow Lineage-MDSC, with high frequency of induction by cervical, ovarian, colorectal, renal cell, and head and neck squamous cell carcinoma cell lines. CD33+ MDSC could be induced by some cancer cell lines of all tumor types examined with the notable exception of breast cancer cell lines (0/9, inclusive of models with different hormone and HER2 mutation status). Upon further examination, breast cancer cell lines and other tumor types with infrequent CD33+ MDSC induction preferentially induced an undiscovered CD11b+CD33low HLA-DRlowLineage- MDSC subset. Gene and protein expression, neutralization, and cytokine induction experiments determined that the ability of tumor cell lines to induce CD33+ MDSC depended upon over-expression of IL-1beta, IL-6, TNFalpha, VEGF, and GM-CSF, while CD11b+ MDSC induction correlated with over-expression of FLT3L and TGFbeta. Both CD33+ and CD11b+ MDSC subsets appeared as immature myeloid cells by Wright-Giemsa staining and had significantly up-regulated expression of inducible nitric oxide synthase, TGFbeta, NADPH oxidase, VEGF, and arginase-1 genes compared with normal myeloid cells. Furthermore, increased expression of transcription factors HIF1alpha, STAT3, and C/EBPbeta distinguished suppressive from non-suppressive tumor cell line-educated myeloid cells. Interestingly, CD33+ and CD11b+ subsets showed differential expression of these transcription factors and therapeutic reversal of suppressive function coincided with decreasing STAT3 and HIF1alpha in CD33+ cells but with decreasing C/EBPbeta in CD11b+ MDSC. These studies demonstrate the universal nature of MDSC induction by human cancers and characterize two distinct populations of MDSC recognized by CD33+HLADR lowHIF1alpha+ and CD11b+HLA-DR lowC/EBPbeta+ biomarkers. The unraveling of these different subsets in human cancers should enable the development of novel diagnostic and therapeutic reagents for cancer immunotherapy. |
