Polymorphonuclear Myeloid-derived Suppressor Cells Research Articles

Attempt to assess direct interactions between tumor burden, myeloid-derived suppressor cells and PD-1- and TIM-3-expressing T cells in multiple myeloma patients

The avoidance of immune surveillance by malignant plasma cells (PCs) in multiple myeloma (MM) is mediated by different mechanisms, among which an induction of T cell exhaustion and expansion of myeloid-derived suppressor cells (MDSCs) appear to play substantial roles, but it is still a lack of data on possible MDSC-mediated induction of T cell exhaustion. The aim of the present work was to evaluate possible relationship between frequencies of MM PCs, MDSCs and phenotypically exhausted PD-1+ and TIM-3+ T cells in bone marrow (BM) samples and peripheral blood (PB) of MM patients at various disease stages. Peripheral blood (n = 88) and BM samples (n = 56) were obtained from MM patients (newly diagnosed (n = 6), patients in remission (n = 71) and with progressive disease (n = 11)). Frequencies of T cells expressing checkpoint receptors PD-1 and TIM-3, polymorphonuclear MDSCs (PMN-MDSCs, Lin-CD14-HLA-DR- CD33+CD15+/CD66b+), monocyte MDSCs (M-MDSCs, CD14+HLA-DRlow/-), early MDSCs (E-MDSCs, Lin-HLA-DR-CD33+CD15-/CD66b-), and MM PCs (CD45dimCD38+CD138+CD56+CD19-CD117+CD27- CD81-) were assessed with flow cytometry. Circulating and BM-resident PD-1+/TIM-3+T cell subsets, BM E-MDSCs, as soon as MM PCs and serum beta2-microglobulin (B2-M) levels were gradually increased in patients at different stages. Despite that, there were no associations between the markers of tumor load and the studied cell subsets. In patients in remission, BM PMN-MDSCs negatively correlated with CD4+T cells, CD4+PD-1+ and CD8+TIM-3+T cell subsets; there were positive correlations between BM E-MDSCs and CD4+PD-1+TIM-3+ cells and PB M-MDSCs and CD8+PD-1+ and (as a trend) CD8+TIM-3+T cells. We found no associations for the samples of patients at diagnosis and with progression. We can conclude that a possible mutual influence of malignant PCs, MDSCs and PD-1+/TIM-3+T cells is nonlinear, especially during a manifest tumor growth at diagnosis and progression. The detected negative correlations between resident PMN- MDSCs and T cell subsets might be associated with MDSC suppressive function, affecting both predominantly activated PD-1+ cells and exhausted TIM-3+ subsets. The positive correlations between BM E-MDSCs and CD4+PD-1+TIM-3+ cell subset and circulating M-MDSCs and PD-1+ and TIM-3+ CD8+T cells might confirm an ability of MDSCs to induce T cell exhaustion.

A single-cell RNA sequencing atlas of circulating leukocytes from healthy and osteosarcoma affected dogs.

Translationally relevant animal models are essential for the successful translation of basic science findings into clinical medicine. While rodent models are widely accessible, there are numerous limitations that prevent the extrapolation of findings to human medicine. One approach to overcome these limitations is to use animal models that are genetically diverse and naturally develop disease. For example, pet dogs spontaneously develop diseases that recapitulate the natural progression seen in humans and live in similar environments alongside humans. Thus, dogs represent a useful animal model for many areas of research. Despite the value of the canine model, species specific reagent limitations have hampered in depth characterization of canine immune cells, which constrains the conclusions that can be drawn from canine immunotherapy studies. To address this need, we used single-cell RNA sequencing to characterize the heterogeneity of circulating leukocytes in healthy dogs (n = 7) and osteosarcoma (OS) affected dogs (n = 10). We present a cellular atlas of leukocytes in healthy dogs, then employ the dataset to investigate the impact of primary OS tumors on the transcriptome of circulating leukocytes. We identified 36 unique cell populations amongst dog circulating leukocytes, with a remarkable amount of heterogeneity in CD4 T cell subtypes. In our comparison of healthy dogs and dogs with OS, we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) myeloid-derived suppressor cells (MDSCs), as well as aberrations in gene expression within myeloid cells. Overall, this study provides a detailed atlas of canine leukocytes and investigates how the presence of osteosarcoma alters the transcriptional profiles of circulating immune cells.

P140Cap inhibits β-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of β-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of β-Catenin depends on its ability to localize in and stabilize the β-Catenin destruction complex, promoting enhanced β-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the β-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

Correlation between circulating myeloid-derived suppressor cells and serum miRNA21 in non-small cell lung cancer and its clinical significance

Objective: To investigate the effect and clinical significance of myeloid-derived suppressor cells (MDSCs) levels and serum miRNA-21 expression in advanced non-small cell lung cancer (NSCLC) patients. Methods: Advanced NSCLC patients treated at the First People’s Hospital of Tongxiang City, Zhejiang Province, between July 2021 and June 2022 were included in this study. MDSCs in the peripheral circulation were assessed by flow cytometry, and serum miRNA-21 expression was assessed by q-PCR. The effects of miRNA-21 on MDSC proliferation and ARG-1 and inducible nitric oxide synthase (iNOS) secretion from MDSCs were examined using in vitro cell experiments. In addition, the correlation between MDSC and CEA, CRP and Ki67 was analyzed. Results: Compared with those in the peripheral circulation of individuals in the control group, the expression levels of polymorphonuclear-MDSCs (PMN-MDSCs) and miRNA-21 in the peripheral circulation of NSCLC patients were significantly higher, and the two groups were significantly positively correlated. In in vitro cell experiments, miRNA-21 mimics promoted MDSC proliferation and increased ARG-1 and iNOS secretion and miRNA-21 inhibtor has an opposite result. In addition, PMN-MDSCs in the peripheral circulation of NSCLC patients were significantly positively correlated with carcinoembryonic antigen (CEA), C-reactive protein (CRP), and Ki67. Conclusion: The blood of advanced NSCLC patients contains high levels of MDSCs and miRNA-21, and the two may interact to impact on NSCLC.

Hepatocellular carcinoma (HCC) tumor microenvironment is more suppressive than colorectal cancer liver metastasis (CRLM) tumor microenvironment

Background and purposeWhile HCC is an inflammation-associated cancer, CRLM develops on permissive healthy liver microenvironment. To evaluate the immune aspects of these two different environments, peripheral blood-(PB), peritumoral-(PT) and tumoral tissues-(TT) from HCC and CRLM patients were evaluated.Methods40 HCC and 34 CRLM were enrolled and freshly TT, PT and PB were collected at the surgery. PB-, PT- and TT-derived CD4+CD25+ Tregs, M/PMN-MDSC and PB-derived CD4+CD25− T-effector cells (Teffs) were isolated and characterized. Tregs’ function was also evaluated in the presence of the CXCR4 inhibitor, peptide-R29, AMD3100 or anti-PD1. RNA was extracted from PB/PT/TT tissues and tested for FOXP3, CXCL12, CXCR4, CCL5, IL-15, CXCL5, Arg-1, N-cad, Vim, CXCL8, TGFβ and VEGF-A expression.ResultsIn HCC/CRLM-PB, higher number of functional Tregs, CD4+CD25hiFOXP3+ was detected, although PB-HCC Tregs exert a more suppressive function as compared to CRLM Tregs. In HCC/CRLM-TT, Tregs were highly represented with activated/ENTPD-1+Tregs prevalent in HCC. As compared to CRLM, HCC overexpressed CXCR4 and N-cadherin/vimentin in a contest rich in arginase and CCL5. Monocytic MDSCs were highly represented in HCC/CRLM, while high polymorphonuclear MDSCs were detected only in HCC. Interestingly, the function of CXCR4-PB-Tregs was impaired in HCC/CRLM by the CXCR4 inhibitor R29.ConclusionIn HCC and CRLM, peripheral blood, peritumoral and tumoral tissues Tregs are highly represented and functional. Nevertheless, HCC displays a more immunosuppressive TME due to Tregs, MDSCs, intrinsic tumor features (CXCR4, CCL5, arginase) and the contest in which it develops. As CXCR4 is overexpressed in HCC/CRLM tumor/TME cells, CXCR4 inhibitors may be considered for double hit therapy in liver cancer patients.

Disrupting CXCR2-mediated MDSC trafficking enhances antitumor efficacy of vaccinia virotherapy

Abstract Oncolytic virotherapy aims to potentiate antitumor responses via multiple mechanisms, including direct tumor lysis, increasing immune cell infiltration, modulating innate and adaptive immune interactions, and vascular modification. In an ideal situation, the oncolysis leads to release of tumor antigens and danger signals, to attract immune cells particularly dendritic cells (DC) into the tumor microenvironment (TME) and to recruit functional cytotoxic T lymphocytes (CTL). However, inflammation in the TME also has detrimental effects to promote cancer progression through reducing intratumoral T cell viability, promoting cancer cell proliferation and angiogenesis, and attracting immune suppressive cells. This complex balancing act dictates the outcome of oncolytic virotherapy and is yet fully understood. In this study, we demonstrated that in situ vaccinia virotherapy primarily recruited polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) into the TME where they exhibited strong suppression of T cell effector function in a ROS-dependent way. Single-cell RNA sequencing analysis confirmed the heightened suppressive activity of PMN-MDSC at the transcriptomic level and identified CXCR2 as a therapeutic target expressed on PMN-MDSC. Abrogating PMN-MDSC trafficking by CXCR2-specific small molecule inhibitor during in situ vaccinia virotherapy exhibited enhanced antitumor efficacy in three syngeneic cancer models, through manipulating MDSC and T cell populations in the TME, activating CTL and skewing suppressive TME into an antitumor environment. Our results warrant clinical development of CXCR2 inhibitor in combination with oncolytic virotherapy. Hong Kong Research Grant Council (TRS: T12-703/19-R and T11-709/21-N; CRF: C1134-20G and C7156-20G); Hong Kong Health and Medical Research Fund (HMRF 05162326), the Hong Kong Pneumoconiosis Compensation Fund Board (PCFB).

Tryptophan diet promotes myeloid-derived suppressor cell response via aryl hydrocarbon receptor in Sjogren’s syndrome

Abstract Myeloid-derived suppressor cells (MDSCs) are heterogeneous myeloid cell populations with immunosuppressive capacity, which contribute to immune regulation and tolerance induction. We previously reported impaired MDSC function in patients with primary Sjögren’s syndrome (pSS) and mice with experimental SS (ESS). In this study, we first found that aryl hydrocarbon receptor (AhR) was highly expressed by human and murine polymorphonuclear MDSCs (PMN-MDSCs). Indole-3-propionic acid (IPA), a natural AhR ligand produced from dietary tryptophan, significantly promoted PMN-MDSC differentiation and their suppressive function on CD4 +T cells. In contrast, feeding tryptophan-free diet resulted in decreased PMN-MDSC response, a phenotype that could be reversed by IPA supplementation. Notably, AhR expression was reduced in PMN-MDSCs during ESS development, while AhR antagonism resulted in exacerbated ESS pathology and dysregulated T effector cells, which could be phenocopied by tryptophan-free diet. Interferon regulatory factor 4 (IRF4), a repressive transcription factor, was upregulated in PMN-MDSCs during ESS progression. ChIP analysis revealed the binding capacity of IRF4 to the promoter region of AhR, while IRF4 deficiency markedly enhanced AhR-mediated PMN-MDSC responses. Furthermore, tryptophan-enriched diet or dietary supplementation of IPA markedly ameliorated salivary glandular pathology in ESS mice with restored MDSC immunosuppressive function. Together, our results identify a novel function of AhR in modulating PMN-MDSC response and demonstrate the therapeutic potential of targeting AhR for the treatment of pSS.

Depletion of Polymorphonuclear Myeloid-Derived Suppressor Cells Increases Efficacy of Listeria monocytogenes Cancer Vaccine

Abstract The tumor microenvironment is highly immunosuppressive, allowing cancer to evade the immune system by inducing the dysfunction, exhaustion, or subversion of CD8+ T-cell functions. Cancer immunotherapies using attenuated strains of Listeria monocytogenes (Listeria) engineered to express tumor antigens have been demonstrated to promote cancer antigen-specific CD8+ T-cells that infiltrate the tumor microenvironment and control mouse models of cancer. However, in human clinical trials, initial IV therapies utilizing Listeria did not demonstrate strong clinical efficacy, suggesting that the generation of tumor-specific CD8+ T-cells recognizing tumor antigens alone is insufficient to provide robust tumor control. Here, we demonstrate that intratumoral (IT) or intravenous (IV) inoculation of an attenuated form of Listeria(ΔActA) demonstrated no therapeutic benefit in the context of establish MC38 tumor-bearing mice while predominantly recruiting and infecting CD11b+Ly6G+CD14+ cells in the tumor microenvironment, markers associated with polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). In vitro analysis of PMN-MDSC infection by Listeria revealed increased immunosuppressive function, indicating a possible adverse outcome of Listeria treatment in vivo. Further, IV or IT injection of Listeria in the context of PMN-MDSC depletion increased the therapeutic efficacy of Listeria therapy and promoted a robust CD8+ T-cell mediated anti-tumor response. Collectively, our results demonstrate that depletion of PMN-MDSCs may be required to mediate the therapeutic efficacy of Listeria inoculation in the context of cancer immunotherapy and the production of an effective anti-tumor immune response. Supported by grants from NIH (DP2CA247830) & Cancer Research Coordinating Committee (CRCC) (C23CR5612)

Spleen tyrosine kinase inhibition restores neutrophil homeostasis in COVID-19

Abstract Spleen tyrosine kinase (SYK) is a novel therapeutic target that inhibits neutrophil and macrophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial. We demonstrated that SYK inhibition was associated with reduced neutrophil activation, increased circulation of mature neutrophils (CD10+CD33−), and decreased circulation of low-density granulocytes (LDG) and polymorphonuclear myeloid-derived suppressor cells (HLA-DR–CD33+CD11b−). Additionally, LDG transcriptional profiles were distinct in COVID-19 compared to during autoimmune conditions. Together, these data suggest that SYK inhibition may mitigate proinflammatory neutrophil cellular and soluble mediator responses thought to contribute to immunopathogenesis of severe COVID-19. This work has been funded in part by the Intramural Research Program of the NIH Clinical Center (NIHCC) including a Research Award for Staff Clinicians (FY2020/FY2021), NHLBI, National Institute of Allergy and Infectious Diseases (NIAID), and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAIMS)

Irradiation plus myeloid-derived suppressor cell-targeted therapy for overcoming treatment resistance in immunologically cold urothelial carcinoma

BackgroundRadiotherapy (RT) has recently been highlighted as a partner of immune checkpoint inhibitors. The advantages of RT include activation of lymphocytes while it potentially recruits immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). This study aimed to investigate the mechanism of overcoming treatment resistance in immunologically cold tumours by combining RT and MDSC-targeted therapy.MethodsThe abscopal effects of irradiation were evaluated using MB49 and cisplatin-resistant MB49R mouse bladder cancer cells, with a focus on the frequency of immune cells and programmed cell death-ligand 1 (PD-L1) expression in a xenograft model.ResultsMB49R was immunologically cold compared to parental MB49 as indicated by the fewer CD8+ T cells and lower PD-L1 expression. Polymorphonuclear MDSCs increased in both MB49 and MB49R abscopal tumours, whereas the infiltration of CD8+ T cells increased only in MB49 but not in MB49R tumours. Interestingly, PD-L1 expression was not elevated in abscopal tumours. Finally, blocking MDSC in combination with RT remarkably reduced the growth of both MB49 and MB49R abscopal tumours regardless of the changes in the frequency of infiltrating CD8+ T cells.ConclusionsThe combination of RT and MDSC-targeted therapy could overcome treatment resistance in immunologically cold tumours.

Abstract 66: Galectin-1 mediated chronic tumoral-STING activation promotes metastasisthrough MDSC recruitment

Abstract Tumor cells alter the immune microenvironment to facilitate metastatic progression. Galectin-1 (Gal1) is a known modulator of tumor growth and progression in multiple cancer types including head and neck cancer (HNC). However, its contribution to the regulation of metastasis is poorly understood. Through this study we report a previously unknown role for Gal1 in modulating the STING pathway to regulate metastasis. The cGAS-STING pathway has paradoxical roles in cancer depending on the kinetics and strength of its induction, with chronic STING activation being linked to immune suppression and tumor progression while acute activation being associated with immune activation. However, little is known about the pathways that promote chronic STING activation. Using HNC and lung cancer models, we show that Gal-1 enhances STING protein stability, leading to sustained NF-κΒ activation and heightened chemokine-driven recruitment of myeloid derived suppressor cells (MDSCs). We show that Gal1-STING connection fosters the establishment of pre-metastatic niche through polymorphonuclear MDSCs (PMN-MDSCs), which modify the local lung microenvironment to support metastatic spread. Notably, RNA sequencing of MDSCs isolated from pre-metastatic lungs indicate the role of PMN-MDSCs in remodeling collagen and the extracellular matrix in the pre-metastatic compartment. Our findings reveal an unexpected role of STING activation in metastatic progression of HNC and lung cancer models and establish Gal1 as an endogenous positive regulator of STING. Citation Format: Dhanya K. Nambiar, Vignesh Vignesh Viswanathan, Hongbin Cao, Weiruo Zhang, Li Guan, Manish Chamoli, Brittany Holmes, Christina Kong, Rachel Hildebrand, Amanda Koong, Rie Eyben, Amato Giaccia, Lingyin Li, Edgar Engleman, Quynh Thu Le. Galectin-1 mediated chronic tumoral-STING activation promotes metastasisthrough MDSC recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 66.

Abstract 5963: Germline variant positive melanoma patients exhibit an enhanced inflammatory microenvironment

Abstract Germline pathogenic variants (gPV) in cancer susceptibility genes are present in roughly 15% of all melanoma diagnoses. Prior studies note improvements in checkpoint immunotherapy responsiveness in patients with a gPV, which is associated with an increased neo-antigen load in gPV patients. We have also previously reported improvements in melanoma specific survival (MSS) in gPV patients vs non-gPV. No studies to date have examined the immune environment in gPV patients which may account for improvements in survival. In our study, melanoma patients with a personal or family history of multiple cancers were offered germline testing utilizing a commercial germline cancer sequencing panel. Enrolled patients also received a blood draw with subsequent flow cytometry to analyze the immune phenotype. In a cohort of 540 melanoma patients, 80 had a gPV. Multivariable linear regression indicated that significantly fewer polymorphonuclear myeloid derived suppressor cells (PMN-MDSC’s) were noted relative to non-gPV patients when adjusting for melanoma stage at germline testing [β = -.45, 95% CI (-.76, -.15), p = .0036]. This difference was most significant in cutaneous, later stage (IIB-IV) patients, primarily stage III. We also interrogated patterns in gene signature between gPV and non-gPV patients in the TCGA PanCancer cutaneous melanoma cohort. We identified reductions in CCL27 (p = .007) and CCL1 (p = .06), genes associated with MDSC recruitment, across all stages in gPV patients, as well as reductions in ARG1 (p= .05) and S100A7 (p = .08), genes associated with PMN-MDSC’s, in later stage gPV patients. Pathway differences were analyzed using gene set enrichment analysis (GSEA). Analysis of Hallmark gene sets identified significantly upregulated signatures in inflammatory response, TNF-α signaling, IFN-γ signaling, and IL-2 STAT5 signaling in late stage gPV patients. KEGG pathway analysis further identified NK cell mediated cytotoxicity and TCR signaling as significantly upregulated in late stage gPV patients. Notably, fatty acid metabolism pathways, which have been implicated in immunosuppression through MDSC suppressive function, were downregulated in gPV patients. These results suggest that gPV melanoma is associated with both a depletion of PMN-MDSC’s and an upregulation of inflammatory microenvironmental genes in later stages which may explain improved outcomes. Citation Format: Alan Shen, Roshan Lodha, Brandon Bungo, Michelle Arbesman, Nikhil Pramod, Jennifer Ko, Brian Gastman, C. Marcela Diaz, Timothy Chan, Joshua Arbesman, Pauline Funchain. Germline variant positive melanoma patients exhibit an enhanced inflammatory microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5963.

Abstract 68: Dissecting cellular and molecular alterations in pre-invasive to invasive lung cancer progression at single-cell resolution

Abstract Non-small cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide, largely due to diagnosis at advanced metastatic stages. Increased implementation of low-dose computed tomography (CT)-guided screening has led to the identification of non-solid lesions or ground-glass opacities (GGOs) that are not visible on plain chest radiography and can harbor very early-stage tumors. Growth of the lesions and/or development of a solid component can indicate more aggressive behavior. Using the low-dose CT reduced lung cancer mortality by >20%, underscoring the need for early detection and intervention. However, the cellular and molecular determinants that contribute to the malignant progression of those early preinvasive lesions remain poorly understood. To assess intratumoral heterogeneity and associated dynamic changes in the immune contexture during early lung carcinogenesis, we performed single-cell RNA-seq and Hyperion imaging on surgically resected freshly harvested tissues from 21 patients (matched solid and non-solid components of lesions and adjacent non-involved lungs), representing a spectrum from preneoplasia to invasive disease. Analysis of >300K cells identified cellular, transcriptomic, and metabolic alterations as well as tumor-stroma cross-talk pathways. Specifically, preinvasive to invasive disease progression was associated with significant changes in the immune landscape determined by an increase in exhausted CD4/CD8 T-cells, suppressive T-regs, polymorphonuclear myeloid-derived suppressor cells, and M2-like macrophages; and a decrease in cytotoxic CD8 T-cells, NK cells, and inflammatory cDC2 cells, suggesting that activation of immunosuppressive/anti-inflammatory microenvironment and suppression of immune activation microenvironment may enable tumor progression. Citation Format: Liron Yoffe, Alain Borczuk, Timothy E. McGraw, Olivier Elemento, Nasser K. Altorki, Vivek Mittal. Dissecting cellular and molecular alterations in pre-invasive to invasive lung cancer progression at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 68.

Abstract 2877: Profiling of myeloid-derived suppressor cells in Pten-deficient in prostate cancer progression

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature immune cells that expand during cancer and can induce potent immune suppression. These cells play critical roles in supporting tumor growth and progression and their induction has been associated with the inactivation of PTEN. To date, few therapies targeting these cells are available which is partly due to their increasingly complex nature and context-dependent functions. Moreover, there is no clear definition to identify these cells. Enhancing our understanding of this cell population will aid in developing more effective cancer therapies. Here, we used gene expression profiling, flow cytometry, and quantitative immunohistochemistry to characterize tumor-infiltrating and circulating MDSCs in prostate carcinogenesis and progression in transgenic mouse models of prostate cancer driven by the conditional inactivation of Pten and Trp53. Transcriptomic profiling revealed enrichment of various myeloid-derived cell signatures in the early stages of prostate cancer including MDSCs, macrophages, and neutrophils. Flow cytometric analysis confirmed the increased infiltration of CD11b+/Ly6G+ (polymorphonuclear MDSCs (pMDSCs)) and CD11b+/Ly6C+ (monocytic MDSCs (mMDSCs)) cells in prostate tumors. The temporal dynamics of tumor-infiltrating myeloid cells were further profiled and showed that pMDSC and mMDSC abundances were 1.6 and 5.7-fold higher, respectively, in older mice with late-stage locally invasive adenocarcinomas (AdCa) compared to younger mice with early-stage prostatic intraepithelial neoplasia (PIN). In both instances, pMDSCs comprised most of the leukocyte infiltrate, however, mMDSCs tended to be positively correlated with tumor burden in AdCa but not PIN. In peripheral blood, CD44+/CD62L+ immature neutrophils were associated with high tumor burden in aged mice. Treatments targeting the androgen receptor (AR) signaling further exacerbated pMDSC infiltration in tumors and was associated with resistance to second androgen receptor-targeted agent (ARTA) therapy in a model of castration resistant prostate cancer. Our findings indicate that MDSCs are a prominent population in Pten-null prostate cancer and are implicated with resistance to prostate cancer treatment. MDSCs constitute a potentially targetable population and these models serve as viable platforms to further investigate their biological activity and further evaluate novel MDSC-directed therapies. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Eri Banno, Shogo Adomi, Mitsuhisa Nishimoto, Takafumi Minami, Yasunori Mori, Kazutoshi Fujita, Masahiro Nozawa Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Profiling of myeloid-derived suppressor cells in Pten-deficient in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2877.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

CD115− monocytic myeloid-derived suppressor cells are precursors of OLFM4high polymorphonuclear myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) consist of monocytic (M-) MDSCs and polymorphonuclear (PMN-) MDSCs that contribute to an immunosuppressive environment in tumor-bearing hosts. However, research on the phenotypic and functional heterogeneity of MDSCs in tumor-bearing hosts and across different disease stage is limited. Here we subdivide M-MDSCs based on CD115 expression and report that CD115− M-MDSCs are functionally distinct from CD115+ M-MDSCs. CD115− M-MDSCs increased in bone marrow and blood as tumors progressed. Transcriptome analysis revealed that CD115− M-MDSCs expressed higher levels of neutrophil-related genes. Moreover, isolated CD115− M-MDSCs had higher potential to be differentiated into PMN-MDSCs compared with CD115+ M-MDSCs. Of note, CD115− M-MDSCs were able to differentiate into both olfactomedin 4 (OLFM4)hi and OLFM4lo PMN-MDSCs, whereas CD115+ M-MDSCs differentiated into a smaller proportion of OLFM4lo PMN-MDSCs. In vivo, M-MDSC to PMN-MDSC differentiation occurred most frequently in bone marrow while M-MDSCs preferentially differentiated into tumor-associated macrophages in the tumor mass. Our study reveals the presence of previously unrecognized subtypes of CD115− M-MDSCs in tumor-bearing hosts and demonstrates their cellular plasticity during tumorigenesis.

Persistent immunosuppressive effects of dibutyl phthalate exposure in adult male mice

Increased exposure to manmade chemicals may be linked to an increase in immune-related diseases in humans and immune system dysfunction in wildlife. Phthalates are a group of endocrine-disrupting chemicals (EDCs) suspected to influence the immune system. The aim of this study was to characterize the persistent effects on leukocytes in the blood and spleen, as well as plasma cytokine and growth factor levels, one week after the end of five weeks of oral treatment with dibutyl phthalate (DBP; 10 or 100 mg/kg/d) in adult male mice. Flow cytometry analysis of the blood revealed that DBP exposure decreased the total leukocyte count, classical monocyte and T helper (Th) populations, whereas it increased the non-classical monocyte population compared to the vehicle control (corn oil). Immunofluorescence analysis of the spleen showed increased CD11b+Ly6G+ (marker of polymorphonuclear myeloid-derived suppressor cells; PMN-MDSCs), and CD43+staining (marker of non-classical monocytes), whereas CD3+ (marker of total T cells) and CD4+ (marker of Th cells) staining decreased. To investigate the mechanisms of action, levels of plasma cytokines and chemokines were measured using multiplexed immunoassays and other key factors were analyzed using western blotting. The observed increase in M-CSF levels and the activation of STAT3 may promote PMN-MDSC expansion and activity. Increased ARG1, NOX2 (gp91phox), and protein nitrotyrosine levels, as well as GCN2 and phosphor-eIRFα, suggest that oxidative stress and lymphocyte arrest drive the lymphocyte suppression caused by PMN-MDSCs. The plasma levels of IL-21 (promotes the differentiation of Th cells) and MCP-1 (regulates migration and infiltration of monocytes/macrophages) also decreased. These findings show that adult DBP exposure can cause persistent immunosuppressive effects, which may increase susceptibility to infections, cancers, and immune diseases, and decrease vaccine efficacy.

Driver Mutations Dictate the Immunologic Landscape and Response to Checkpoint Immunotherapy of Glioblastoma.

The composition of the tumor immune microenvironment (TIME) is considered a key determinant of patients' response to immunotherapy. The mechanisms underlying TIME formation and development over time are poorly understood. Glioblastoma (GBM) is a lethal primary brain cancer for which there are no curative treatments. GBMs are immunologically heterogeneous and impervious to checkpoint blockade immunotherapies. Utilizing clinically relevant genetic mouse models of GBM, we identified distinct immune landscapes associated with expression of EGFR wild-type and mutant EGFRvIII cancer driver mutations. Over time, accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) was more pronounced in EGFRvIII-driven GBMs and was correlated with resistance to PD-1 and CTLA-4 combination checkpoint blockade immunotherapy. We determined that GBM-secreted CXCL1/2/3 and PMN-MDSC-expressed CXCR2 formed an axis regulating output of PMN-MDSCs from the bone marrow leading to systemic increase in these cells in the spleen and GBM tumor-draining lymph nodes. Pharmacologic targeting of this axis induced a systemic decrease in the numbers of PMN-MDSC, facilitated responses to PD-1 and CTLA-4 combination checkpoint blocking immunotherapy, and prolonged survival in mice bearing EGFRvIII-driven GBM. Our results uncover a relationship between cancer driver mutations, TIME composition, and sensitivity to checkpoint blockade in GBM and support the stratification of patients with GBM for checkpoint blockade therapy based on integrated genotypic and immunologic profiles.

PMN-MDSCs modulated by CCL20 from cancer cells promoted breast cancer cell stemness through CXCL2-CXCR2 pathway

Our previous studies have showed that C-C motif chemokine ligand 20 (CCL20) advanced tumor progression and enhanced the chemoresistance of cancer cells by positively regulating breast cancer stem cell (BCSC) self-renewal. However, it is unclear whether CCL20 affects breast cancer progression by remodeling the tumor microenvironment (TME). Here, we observed that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were remarkably enriched in TME of CCL20-overexpressing cancer cell orthotopic allograft tumors. Mechanistically, CCL20 activated the differentiation of granulocyte-monocyte progenitors (GMPs) via its receptor C-C motif chemokine receptor 6 (CCR6) leading to the PMN-MDSC expansion. PMN-MDSCs from CCL20-overexpressing cell orthotopic allograft tumors (CCL20-modulated PMN-MDSCs) secreted amounts of C-X-C motif chemokine ligand 2 (CXCL2) and increased ALDH+ BCSCs via activating CXCR2/NOTCH1/HEY1 signaling pathway. Furthermore, C-X-C motif chemokine receptor 2 (CXCR2) antagonist SB225002 enhanced the docetaxel (DTX) effects on tumor growth by decreasing BCSCs in CCL20high-expressing tumors. These findings elucidated how CCL20 modulated the TME to promote cancer development, indicating a new therapeutic strategy by interfering with the interaction between PMN-MDSCs and BCSCs in breast cancer, especially in CCL20high-expressing breast cancer.

Hepatocyte SGK1 activated by hepatic ischemia-reperfusion promotes the recurrence of liver metastasis via IL-6/STAT3

BackgroundLiver metastasis is the leading cause of death in patients with colorectal cancer (CRC). Surgical resection of the liver metastases increases the incidence of long-term survival in patients with colorectal liver metastasis (CRLM). However, many patients experience CRLM recurrence after the initial liver resection. As an unavoidable pathophysiological process in liver surgery, liver ischemia-reperfusion (IR) injury increases the risk of tumor recurrence and metastasis.MethodsColorectal liver metastasis (CRLM) mouse models and mouse liver partial warm ischemia models were constructed. The levels of lipid peroxidation were detected in cells or tissues. Western Blot, qPCR, elisa, immunofluorescence, immunohistochemistry, scanning electron microscope, flow cytometry analysis were conducted to evaluate the changes of multiple signaling pathways during CRLM recurrence under liver ischemia-reperfusion (IR) background, including SGK1/IL-6/STAT3, neutrophil extracellular traps (NETs) formation, polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration.ResultsHepatocyte serum/glucocorticoid regulated kinase 1 (SGK1) was activated in response to hepatic ischemia-reperfusion injury to pass hepatocyte STAT3 phosphorylation and serum amyloid A (SAA) hyperactivation signals in CRLM-IR mice, such regulation is dependent on SGK-activated IL-6 autocrine. Administration of the SGK1 inhibitor GSK-650394 further reduced ERK-related neutrophil extracellular traps (NETs) formation and polymorphonucler myeloid-derived suppressor cells (PMN-MDSC) infiltration compared with targeting hepatocyte SGK1 alone, thereby alleviating CRLM in the context of IR.ConclusionsOur study demonstrates that hepatocyte and immune cell SGK1 synergistically promote postoperative CRLM recurrence in response to hepatic IR stress, and identifies SGK1 as a translational target that may improve postoperative CRLM recurrence.