Добірка наукової літератури з теми "PMN-MDSCs, gene signature, markers expression, cancer"

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent immune suppressive activity. MDSCs regulate anti-tumor immunity by suppressing T cell proliferation. MDSCs can be further classified into granulocytic MDSCs (G-MDSCs) and monocytic MDSC (M-MDSCs). However, there is a lack of MDSC-specific markers since MDSCs, normal neutrophil granulocytes, and monocytes are defined by the same flow cytometry markers (CD11b+Gr1+). We used a breast cancer mouse model (MMTV-PyMT) to define cellular and molecular properties of MDSCs in single cell resolution. To test the capacity of MDSCs to inhibit immune responses, CD11b+Gr1+ cells from PyMT and wild type (WT) mice were sorted by fluorescence-activated cell sorting (FACS) from bone marrow, lung, and spleen, and then subjected to a T cell activation assay in co-culture with T cells. We found that predominantly spleen-derived CD11b+Gr1+ cells from PyMT significantly suppressed CD4 and CD8 T cell proliferation, while CD11b+Gr1+cells from bone marrow and lung showed no effect on T cell proliferation. Thus, we focused on spleen-derived CD11b+Gr1+ cells and utilized single cell RNA sequencing (scRNAseq) to compare breast cancer derived MDSCs to the respective cell populations harvested from normal, and non-tumor bearing hosts. Our computational analysis of 14,646 single cell transcriptomes revealed a separate G-MDSCs cluster from normal neutrophils and a M-MDSCs cluster from normal monocytes and a distinct gene expression signature for the entire MDSCs population. Our studies provide crucial insights into the biology of MDSCs, which may ultimately form the basis for novel marker and therapeutic avenues to improve cancer immunotherapy.

BackgroundA potently immunosuppressive tumor microenvironment facilitates progression of glioblastoma (GBM). Immunotherapies have had variable success in improving the outcome of GBM patients, suggesting that there is a need to gain insight into the mechanisms of immunosuppression. Our findings indicated that proliferating monocytic MDSCs (mMDSCs) accumulate in tumors of male mice and patients, while female tumor-bearing mice had an increase in circulating granulocytic MDSC (gMDSC) frequency, and a high gMDSC gene signature correlated with worse outcome of female patients.MethodsTo investigate the basis and prognostic value of sex differences in MDSC profile, we analyzed the role of sex hormones, determined gene expression signatures of MDSCs and preclinically tested the therapeutic benefit of candidate drugs predicted to be effective against individual MDSC subsets.ResultsIn line with the differential MDSC accumulation pattern, targeting the systemic gMDSCs with the anti-Ly6G neutralizing antibody extended the lifespan of female mice without affecting males. These differences were not driven by sex steroids, as castration or ovariectomy failed to alter MDSC subset accumulation patterns in GBM-bearing mice. Drug-prediction algorithms using the differential MDSC gene expression profiles predicted IL-1 inhibitors are effective against gMDSCs. Correspondingly, IL-1β was highly expressed in female but not male gMDSCs. Single-cell sequencing revealed that circulating but not tumor-infiltrating gMDSCs were the primary source of IL-1β and that its neutralization provided a female-specific survival advantage by reducing circulating gMDSCs. This was accompanied by declines in tumor infiltration of microglia, microglia activation status and tumor cell proliferation. In vitro, IL-1β inhibition reduced viability and expression of activation markers by primary microglia.ConclusionsThese findings highlight a novel peripheral gMDSC-microglia IL-1β mediated communication axis in female GBM and indicate expression differences in MDSC subsets can be leveraged for improved immunotherapy efficacy in a sex-specific, precision medicine strategy.

Abstract Background: Tumor-associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs) promote an immunosuppressive milieu by inhibiting T-cell activation and recruitment, leading to resistance to immune checkpoint therapies. Interleukin 10 (IL10) and prostaglandin E2 (PGE2) promote tumor initiation and spread, in part, through TAM polarization and MDSC recruitment. C/EBPβ is a critical transcription factor that regulates the immunosuppressive state of TAMs and MDSCs and is activated by IL10 and PGE2 signaling. We developed a proprietary engineered exosome loaded with antisense oligonucleotides (ASOs) targeting C/EBPβ (exoASO-C/EBPβ), that selectively delivers ASO to TAMs and MDSCs, thereby inhibiting C/EBPβ expression and inducing reprogramming from an immunosuppressive (M2) to a pro-inflammatory (M1) phenotype. Results: exoASO-C/EBPβ demonstrated dose-dependent target gene knockdown (KD) in primary human M2 macrophages in vitro with a 2-fold higher potency (IC50) compared to free ASO. C/EBPβ KD resulted in profound changes in gene expression and cytokine secretion profile consistent with reprogramming to a M1 phenotype. exoASO-C/EBPβ induced a significant induction of pro-inflammatory cytokines, in M2 macrophages generated with IL10 or PGE2, both of which show prominent activation of the C/EBPβ pathway. Biodistribution in tumor-bearing mice (IT and IV) demonstrate selective uptake by MDSCs and TAMs. Intra-tumoral (IT) administration of exoASO-C/EBPβ in CT26 tumors resulted in a significantly higher KD of C/EBPβ in tumor-associated CD11b+ cells, as compared to CD11b- cells. Additionally, exoASO-C/EBPβ induced changes in Nos2, CD206 and Csf1r mRNA expression in CD11b+ cells, demonstrating in vivo M2 to M1 reprogramming. Furthermore, IT microinjections of exoASO-C/EBPβ in YUMM1.7 tumor-bearing mice resulted in a significant induction of pro-inflammatory M1 markers TNFα and iNOS, in contrast, free C/EBPβ ASO showed limited increase in M1 markers. exoASO-C/EBPβ administered IT as a single agent demonstrated significant anti-tumor activity in multiple tumor models including: the CT26-colorectal cancer (60% complete responses (CRs)), MB49-bladder cancer (70% CRs) and B16F10-melanoma (30% CRs). Combination treatment with anti-PD1 in all these models further increased the anti-tumor efficacy. Finally, we used a myeloid cell specific C/EBPβ/PGE2/IL10 gene signature to identify cancer indications where exoASO-C/EBPβ therapy might have the most therapeutic significance. Conclusion: exoASO-C/EBPβ is a novel therapeutic candidate that selectively targets and attenuates a critical transcription factor in immunosuppressive myeloid cells, resulting in reprogramming of TAMs and potent anti-tumor activity across multiple TAM-rich mouse models as a monotherapy. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, SuChul Jang, Kelvin Zhang, Yanyan Liu, William Dahlberg, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Exosome-mediated delivery of antisense oligonucleotides targeting C/EBPβ reprograms tumor-associated macrophages and induces potent single agent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3508.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is resistant to PD1/PDL1 blocking antibodies. The purpose of this study was to evaluate if immunotherapy with LOAd703, an oncolytic adenovirus armed with trimerized CD40L and 4-1BBL, can sensitize PDAC to mAbs targeting PD1/PDL1. The high content of myeloid-derived suppressor cells (MDCSs) in the tumor microenvironment may in part explain the limited anti-tumor T cell response in PDAC. We have previously shown that gemcitabine can reduce MDSCs in patients with PDAC, with the T cell proliferative capacity remaining intact. Further, LOAd703 has been demonstrated to efficiently and robustly kill PDAC cells and stimulate the maturation of dendritic cells (DCs), which, in turn, induces T cell activation. Patients with advanced PDAC, colorectal, biliary, and ovarian cancer (NCT03225989) were treated with intratumoral injections of LOAd703 combined with appropriate chemotherapy such as gemcitabine ± nab-paclitaxel for PDAC patients, or combined with gemcitabine if there was no standard of care. Blood sampling was performed for immune cell profiling and anti-adenovirus antibodies (flow cytometry, ELISA). Tumor biopsies were also analyzed for mRNA expression (NanoString; PanCancer Immune Profiling Panel). The study was approved by the ethical review board and the Swedish Medical Products Agency. The dose of LOAd703 was escalated (5 × 10e10 VP, 1 × 10e11 VP, 5 × 10e11VP) in separate cohorts of new patients and administered every other week. To date, blood samples from 23 patients have been analyzed by flow cytometry to profile immune cells. The mean percentages of monocytic- and granulocytic MDSCs, Tregs and M2-like myeloid cells were significantly decreased after treatment initiation. The effector memory (EM) and central memory (CM) CD8+ T cells were significantly increased, while naïve and CM cells were increased among CD4 T cells. Both CD4 and CD8 T cells expressing ICOS were present post-treatment but only CD8 T cells showed significant elevation of PD1. The NanoString data were analyzed using published mRNA immune signatures (bioinformatics). LOAd703 in combination with chemotherapy inflamed tumor lesions as shown by increased markers of the T cell inflamed signature (16 genes), T effector cell signature (19 genes), expanded immune signature (25 genes) and IFNg-related gene signature (12 genes) (week 1 versus week 13). Anti-adenovirus antibody titers increased in all patients after treatment initiation. The antibody levels did not correlate with the dose of virus, radiological response to treatment, nor overall survival. The results presented herein show that LOAd703 combined with chemotherapy enhances immune reactivity in patients with immune cold tumors as demonstrated by increases in EM and CM T cells in the peripheral blood, while suppressive immune cells were decreased. Further, the tumor milieu was inflamed post treatment as shown by mRNA profiling. Hence, LOAd703 may sensitize immune cold tumors to mAbs targeting PD1/PDL1. Citation Format: Jessica Wenthe, Emma Eriksson, Linda Sandin, Tanja Lövgren, Justyna Leja Jarblad, Hanna Dahlstrand, Ulla Olsson-Strömberg, Aglaia Schiza, Anders Sundin, Sandra Irenaeus, Eric Rowinsky, Gustav Ullenhag, Angelica Loskog. Inflaming advanced solid tumors including pancreatic cancer using LOAd703, a TMZ-CD40L/4-1BBL-armed oncolytic virus [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-018.

Abstract Most cancers express tumor antigens that can be recognized by T cells of the host. The fact that cancers become clinically relevant and grow nonetheless implies that immune escape must occur to allow cancer outgrowth. We have observed two major phenotypes of human melanoma metastases based on gene expression profiling and confirmatory assays. One subgroup of patients has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I IFN signature. In contrast, the other major subset lacks this phenotype and appears to display immune “exclusion”. Factors that influence the degree of spontaneous immune infiltration are being investigated, as sources of inter-patient heterogeneity. These include tumor cell-intrinsic oncogenic events, the composition of the gut microbiota, and polymorphisms in immune regulatory genes. We now know that each of these dimensions can be functionally important. The first tumor cell-intrinsic oncogenic pathway identified that mediates immune exclusion is the Wnt/β-catenin pathway. Tumors with active β-catenin fail to recruit Batf3-lineage dendritic cells into the tumor site. Regarding the commensal microbiota, mouse models identified commensal Bifidobacterium as one key component that augments spontaneous anti-tumor immunity and increases efficacy of anti-PD-L1 therapy in vivo. Similar analyses in human cancer patients revealed bacteria sequences enriched in anti-PD-1 responders, and also bacteria sequences enriched in non-responders. Fecal transfer into germ-free mice has confirmed a causal role for the gut microbiota in regulating immunotherapy efficacy. Recent experiments have revealed that one major mechanism by which gut microbes impact on distant ani-tumor immunity is through modulation of immune-regulatory myeloid cells, ie the M1/M2 ratio and MDSCs. Regarding germline variants, our first identified SNP connected to immune cell infiltration is in the PKCδ gene. Loss of function variants are associated with greater immune cell infiltration. PKCδ knockout hosts show improved immune-mediated tumor control and anti-PD-L1 efficacy, but with comparable T cell priming. However, activated T cell accumulation in the tumor microenvironment increases overtime, which is associated with a shift from M2 to M1. Myeloid cell-specific PKCδKO mice using LysM-Cre Tg mice recapitulate the phenotype. Thus, in each of these 3 instances, tumor and host factors impact on anti-tumor immunity by modulating myeloid cell participation—Batf3-DCs, MDSCs, and M1/M2 cells. Citation Format: Thomas F. Gajewski. Germline and microbiome variants impact immunotherapy efficacy through modulation of myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr PL04-03.

Abstract A potently immunosuppressive tumor microenvironment facilitates progression of glioblastoma (GBM). We previously demonstrated that myeloid-derived suppressor cell (MDSC) subsets promote tumorigenesis in a sex-specific manner, contributing to sexual dimorphism in GBM incidence and prognosis. Our findings indicated that proliferating monocytic MDSCs (mMDSCs) accumulate in tumors of male mice and patients, while female tumor-bearing mice had an increase in circulating granulocytic MDSC (gMDSC) frequency, and a high gMDSC gene signature correlated with worse outcome of female patients. However, the mechanisms underlying sexual dimorphism of MDSC heterogeneity remain understudied and can provide insights for improved immunotherapy response. Using syngeneic mouse glioma models and sequencing approaches, we show that expression of Y-chromosome-linked genes correlates with upregulation of multiple RNA transcription-related pathways specifically in male mMDSCs. Consistently, adoptive transfer of male mMDSCs but not gMDSCs worsened GBM outcome in male recipients, while the transfer of sex-matched mMDSCs did not impact survival of female mice. In contrast to this cell-intrinsic regulatory pathway, sex steroids had no impact on MDSC profile, as castration or ovariectomy failed to alter MDSC subset accumulation patterns in GBM-bearing mice. Correspondingly, IL-1β, which we had identified as a female-specific drug target, was highly expressed in female but not male gMDSCs. Single-cell sequencing revealed that circulating but not tumor-infiltrating gMDSCs were the primary source of IL-1β and that its neutralization provided a female-specific survival advantage by reducing circulating gMDSCs. This was accompanied by declines in tumor infiltration of microglia, microglia activation status and tumor cell proliferation. In vitro, IL-1β inhibition reduced viability and expression of activation markers by primary microglia. These findings highlight a peripheral gMDSC-microglia communication axis mediated by IL-1β signaling in females with GBM and indicate that expression differences in MDSC subsets represent opportunities for improved immunotherapy efficacy while accounting for sex as a biological variable.

BackgroundPatients with relapsed metastatic osteosarcoma have no effective treatments available to them,1 and immunotherapy thus far has not succeeded in improving outcomes.2–5 We aim to understand the immune architecture of the tumor microenvironment (TME) of osteosarcoma, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with osteosarcoma.Methods66 osteosarcoma tissue specimens were stained and analyzed by immunohistochemistry. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional multiparameter flow cytometry (MFC). Distinct regions from 16 pulmonary metastases (PMs) were microdissected, and RNA was extracted to perform comparative transcriptomic studies. Clinical follow-up (median 24 months) was available from resection.ResultsDigital image analysis of immunohistochemistry demonstrated significantly higher infiltrating immune cells in the PMs compared to primary bone tumors, concentrated at the tumor-normal lung ‘PM interface’ region, and elevated expression of multiple immune checkpoint molecules at the PM interface (figure 1). MFC confirmed the increased expression of the immune checkpoint molecules programmed cell death 1 (PD-1, p<0.01) and lymphocyte activation gene 3 (LAG-3, p<0.01), as well as the activation marker IFN-γ (p<0.05) in CD8+ TILs. Gene expression profiling provided further evidence for the presence of TILs with expression of activation markers and inhibitory immune checkpoint molecules at the PM interface compared to the PM interior (figure 2). A strong M2 macrophage signature was present in both regions. Further analysis revealed that genes related to neutrophil and myeloid cell chemotaxis and known to be associated with polymorphonuclear myeloid-derived suppressor cells were highly expressed at the PM interface, along with genes for multiple subsets of dendritic cells (figure 3). Expression of PD-L1, LAG-3, and CSF1R at the PM interface were associated with worse progression-free survival (PFS), while gene sets associated with productive T cell immune response were associated with improved PFS (figure 4).Abstract 506 Figure 1Immunohistochemistry of osteosarcoma pulmonary metastasesA. H&E with demarcation of tumor-normal lung interface (center green line) and area quantified as the ‘PM interface’ (outer green lines). Pulmonary metastases demonstrate a higher concentration of immune cells (CD3 p<0.001, CD8 p<0.001, CD163 p<0.01) and PD-1 (p<0.001)/PD-L1 (p<0.05) at the PM interface.B. H&E with demarcation of PM interface as above. Pulmonary metastases demonstrating increased staining of TIM-3 (p<0.01), LAG-3 (p<0.01) and IDO1 (p<0.0001) at the PM interface (no significant concentration of CSF1R at PM interface).Abstract 506 Figure 2Activated/exhausted lymphocyte signatures at PM interfaceA. Heatmap displaying significant genes that contribute to leading-edge of core enrichment subset via Gene Set Enrichment Analysis (GSEA) demonstrating higher expression of immune regulatory molecules at the PM interface compared to the PM interior. Expression levels were converted into heatmaps and colors quantitatively correspond to fold changes. FDR=GSEA false-discovery rate q-value.B. Heatmap illustrating coefficients of xCell analysis shows higher expression of markers of cytotoxicity and activation, as well as multiple checkpoint molecules, at the PM interface, with evidence that they are being contributed chiefly by T cells. Intensity represents xCell coefficient, which corresponds to the amount that a particular region (PM interior or PM interface) or cell population (T cells, B cells, or myeloid cells) contributes to the expression of a specific gene.Abstract 506 Figure 3Genes related to dendritic cells and MDSCs at PM interfaceA. By GSEA, genes associated with multiple subclasses of antigen-presenting dendritic cells are significantly upregulated at the PM interface (cDC1=conventional type 1 dendritic cell; cDC2=conventional type 2 dendritic cell; pDC=plasmacytoid dendritic cell; moDC=monocyte-derived dendritic cell). FDR=GSEA false-discovery rate q-value.B. Heatmap shows heightened expression of cytokines, chemokines and endothelin transcripts associated with development, recruitment and maintenance of PMNs and granulocytic MDSCs at the PM interface compared to the PM interior.Abstract 506 Figure 4Markers of immune TME at PM interface correlate with PFSA. Hazard ratios for immunohistochemistry markers at the PM interface as they relate to PFS. For absolute count biomarkers (CD3, CD8, Foxp3, PD-1, CD163, and LAG-3) the unit is per 100 cells, and for percentage biomarkers (PD-L1, CSF1R, TIM-3, and IDO1) the unit is per 1%.B. Hazard ratios for gene sets at the PM interface as they relate to PFS. NS=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001ConclusionsIn contrast to primary bone osteosarcoma ‘immune deserts,’ osteosarcoma PMs represent an ‘immune-excluded’ TME where immune cells are present but are halted at the PM interface. TILs can produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs that prevent TILs from penetrating into the PM interior, including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with PFS of patients, which could be potentially manipulated for successful immunotherapy.Ethics ApprovalThis study was approved by Johns Hopkins University’s Ethics Board, approval number FWA00005752.ReferencesMirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: Data from the surveillance, epidemiology, and end results program. Cancer 2009;115(7):1531–43.Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, et al. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): A multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol 2017;18(11):1493–501.Davis KL, Fox E, Merchant MS, Reid JM, Kudgus RA, Liu X, et al. Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): A multicentre, open-label, single-arm, phase 1–2 trial. Lancet Oncol 2020;21(4):541–50.D’Angelo SP, Mahoney MR, Van Tine BA, Atkins J, Milhem MM, Jahagirdar BN, et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma (alliance A091401): Two open-label, non-comparative, randomised, phase 2 trials. Lancet Oncol 2018;19(3):416–26.Paoluzzi L, Cacavio A, Ghesani M, Karambelkar A, Rapkiewicz A, Weber J, et al. Response to anti-PD1 therapy with nivolumab in metastatic sarcomas. Clin Sarcoma Res 2016;6:24.

Myeloid-derived suppressor cells (MDSCs) are innate immune cells that acquire the capacity to suppress adaptive immune responses during cancer. It remains elusive how MDSCs differ from their normal myeloid counterparts, which limits our ability to specifically detect and therapeutically target MDSCs during cancer. Here, we sought to determine the molecular features of breast cancer–associated MDSCs using the widely studied mouse model based on the mouse mammary tumor virus (MMTV) promoter–driven expression of the polyomavirus middle T oncoprotein (MMTV-PyMT). To identify MDSCs in an unbiased manner, we used single-cell RNA sequencing to compare MDSC-containing splenic myeloid cells from breast tumor–bearing mice with wild-type controls. Our computational analysis of 14,646 single-cell transcriptomes revealed that MDSCs emerge through an aberrant neutrophil maturation trajectory in the spleen that confers them an immunosuppressive cell state. We establish the MDSC-specific gene signature and identify CD84 as a surface marker for improved detection and enrichment of MDSCs in breast cancers.

Abstract Mutation in isocitrate dehydrogenase (mIDH) is the main genetic lesion that defines clinical glioma subtypes and prognosis. This gain of function mutation is associated with the production of the oncometabolite, R-2-hydroxyglutarate, that inhibits α-ketoglutarate dependent enzymes such as TET2 and the Jumonji-C domain containing demethylases. The resultant epigenetic modifications elicit profound effects on the tumor biology and on the glioma-infiltrating immune cells. Here, we report that in genetically engineered mouse glioma models(1), IDH1 mutation caused an expansion of tumor infiltration granulocytes. Upon phenotypic and functional characterization, we uncovered that granulocytes in mIDH1 glioma express low level of immunosuppressive molecules and did not inhibit T-cell function. Single-cell sequencing revealed that these granulocytes are heterogeneous and composed of three distinct populations; neutrophils, pre-neutrophils, and a small fraction of immunosuppressive PMN-MDSCs. Moreover, primary human gliomas showed a higher cellular fraction exhibiting the PMN-MDSCs gene signature in wtIDH1 tumors than the mIDH1 tumors. The mechanism by which mIDH1 mediates non-immune suppressive granulocytes expansion involves epigenetic reprogramming which leads to enhanced expression of granulocyte colony-stimulating factor (G-CSF) in stem-like cells. High G-CSF gene expression is correlated with favorable patient outcome solely in LGG-astrocytoma with mIDH1. Thus, G-CSF represents a potential therapeutic that can be harnessed to improve immunotherapeutic responses in wild type IDH1 glioma patients.

655 Background: Myeloid-derived suppressor cells (MDSC) are capable of inducing profound immune suppression of anti-tumor T cell responses. Here we examined the ability of intratumoral MDSC accumulation to alter the immune landscape of RCC and to identify the factors involved. Methods: Using flow cytometry, 41 clear cell RCC nephrectomy samples were assessed for their total MDSC infiltrate and the percentage of granulocytic (PMN-MDSC) or monocytic (M-MDSC) subsets. RNA isolated from these tumor samples and 4 non-tumor samples was also evaluated for expression levels of immune-related genes using a Nanostring PanCancer immune panel. A multiple linear regression model was used to identify which immune-related genes might be significantly associated with PMN-MDSC intratumoral accumulation. Log-Rank test was used for comparison of survival curves. Results: Genes encoding cytokines, cancer-testis antigens, interleukins, and T cell function were differentially expressed by clear cell RCC and the non-tumor samples. The transcription of 55 immune-related genes was significantly associated (FDR adjusted p < 0.05) with PMN-MDSC infiltration, including mediators of MDSC recruitment such as CXCL1, CXCL3, CXCL5, CXCR2, IL8, LIF, S100A8, CSF3R, and CCR3, and molecules governing MDSC expansion and function such as IL10, C/EBPB, CD44, COX2, and NFATC3. When a statistically significant 10 gene signature was analyzed in the context of patient survival using the TCGA database of clear cell RCC patients (n = 945), a strong association between the expression of these genes and reduced survival was observed. Moreover, this association seemed to be additive, with expression of 7-10 of these genes correlating with poorer survival when compared to patients expressing 3 or less (p < 0.0001). Conclusions: Consistent with previous studies defining the immunoregulatory role of MDSCs, these results suggest that MDSC accumulation can alter the inflammatory state of the tumor, and indicate that mediators associated with the function of PMN-MDSCs can have a negative impact on outcome. Mechanistic studies aimed at identifying the functionally relevant mediators are ongoing.

Recently, we demonstrated that neutrophil populations displaying polymorphonuclear myeloid derived suppressor cell (PMN-MDSC)-like features appear in abundance in both CD66b+ low-density neutrophils (LDNs) and normal-density neutrophils (NDNs) of healthy subjects receiving G-CSF for stem cell mobilization (GDs). We also uncovered that the mature, but not immature, fraction of PMN-MDSC populations present in GDs, similarly to what also shown by others in cancer patients, are those exerting the most potent immunosuppressive functions. Herein, by RNA-seq computational analysis, we report the identification and characterization of a distinct gene signature common to mature CD66b+CD11b+CD16+CD10+ LDNs/mNDNs from GDs (GD-mNDNs/mLDNs) and mature CD66b+CD11b+CD16+CD10+ PMN-MDSCs (mPMN-MDSCs) from head and neck (HNC) and non-small cell lung (NSCLC) cancer patients. We also show that this GD/NSCLC/HNC mPMN-MDSC gene signature derives, mostly from the maintenance of genes already expressed by normal immature neutrophil precursors, and in a minor, but more specific, quote, from genes selectively upregulated along the maturation of PMN-MDSC-committed cells. Furthermore, by searching for mRNAs included within the latter group of genes encoding surface proteins, we found CD84 expression markedly elevated on mPMN-MDSC populations, not only from GDs and HNC/NSCLC, but also from diffuse large B cell lymphoma (DLBCL), renal cell cancer (RCC), transitional cell carcinoma (TCC) and breast cancer (BC) patients. Overall, data prove that mLDNs and mNDNs from GDs represent excellent cellular models suitable either to define the molecular features, or to identify specific markers, that can be shared by mPMN-MDSC populations from different origin.