Bioinorganic hybrid bacteriophage for modulation of intestinal microbiota to remodel tumor-immune microenvironment against colorectal cancer.

Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells, which are expanded in most cancer patients. MDSCs suppress host immune responses, leading to cancer growth and progression. Several studies demonstrated that there was a relationship between levels of MDSCs and tumorigenesis in colorectal cancer (CRC) patients. MDSCs are now being investigated for their role as possible therapeutic targets in cancer treatment. This review summarizes available studies that investigated MDSC expansion in CRC patients, as well as their role in CRC tumorigenesis, prognosis, and targeting. Based on the available studies, there is a possible relationship between high levels of MDSCs and CRC progression. Additionally, targeting MDSCs in CRC patients selectively represents a significant challenge for the development of targeted treatments. Targeting of MDSCs could be exploited in different ways including MDSC depletion, inhibition of MDSC function and recruitment, and enhancing MDSC differentiation. Overall, MDSCs could be exploited as prognostic biomarkers and potential therapeutic targets in CRC.

BackgroundMyeloid-derived suppressor cells (MDSCs) accumulate in the blood and tumor microenvironment (TME) and suppress anti-tumor immune responses.1 Cancer cells express the granulocyte-macrophage colony-stimulating factor (GM-CSF), which drives MDSC differentiation and function.2 3 4 It is upregulated in several cancers, including mesothelioma, pancreatic and colorectal, and it is linked to higher levels of intra-tumoral MDSCs and poorer overall survival.2 4 5 In animal models, knockdown of GM-CSF in pancreatic epithelium or pancreatic mesenchymal stem cells inhibits tumorigenesis, reduces intra-tumor MDSCs and enhances CD8+ T cell accumulation.6 7 8 Therefore, targeting the GM-CSF receptor alpha (GM-CSFRα) on MDSCs is an attractive strategy to restore anti-tumor immunity. Mavrilimumab is a clinical stage fully human monoclonal antibody that blocks GM-CSFRα. It has demonstrated efficacy and acceptable safety profile in patients with rheumatoid arthritis, and it’s currently undergoing investigation in phase II studies in giant cell arteritis and in patients with severe COVID-19 pneumonia and hyper-inflammation (NCT03827018, NCT04397497, respectively). The present study investigates its potential as a therapeutic strategy to target MDSCs in the TME as an adjuvant to immunotherapy.MethodsCancer cell supernatants were collected when cells reached confluency. Human GM-CSF was measured by ELISA. Healthy donor CD14+ monocytes were incubated (± mavrilimumab) with cancer cell supernatants for either 3 or 6 days followed by phenotypic analysis (CD14, CD33, HLA-DR, CD11b, CD206, CD80, PD-L1, Arginase-1) by flow cytometry. On day 3, autologous CD3+ T cells were stimulated with CD3/CD28 and IL-2 and co-cultured with putative MDSCs for 5 days. T-cell proliferation was evaluated by measuring carboxyfluorescein succinimidyl ester (CFSE) dilution in CD4+ and CD8+ T cells by flow cytometry.ResultsGM-CSF is expressed in the supernatant of cancer cell lines (HCT116, SW-480, Panc-1, Capan-1). Human monocytes cultured with conditioned medium from colorectal carcinoma (SW-480) or pancreatic adenocarcinoma (Capan-1) show downregulation of HLA-DR, increased expression of PD-L1, Arg-1, CD206, and can suppress T-cell proliferation in-vitro. Similarly, peripheral blood monocytes purified from pancreatic cancer patients suppress T-cell proliferation ex-vivo. Notably, Mavrilimumab inhibits the polarization of healthy donor monocytes to M-MDSCs and restores T-cell proliferation.ConclusionsTargeting of GM-CSFRα with mavrilimumab may alleviate the pro-tumorigenic and immunosuppressive functions of MDSCs in the TME. Future clinical studies should evaluate whether targeting of the GM-CSFRα in combination with immune checkpoint inhibitors is a viable therapeutic option to bolster their efficacy.Ethics ApprovalThe study was approved by the Institute of Immunology and Immunotherapy, University of Birmingham, UK Ethics Board. Healthy volunteer human material was obtained from commercial sources and approved by Stemexpress Institutional Review Board (IRB).ReferencesLaw AMK, Valdes-Mora F, Gallego-Ortega D. Myeloid-Derived Suppressor Cells as a Therapeutic Target for Cancer. Cells 2020;9(3):561.Khanna S, Graef S, Mussai F, et al. Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients. Clin Cancer Res 2018;24(12):2859–2872.Dolcetti L, Peranzoni E, Ugel S, et al. Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur J Immunol 2010;40(1):22–35.Takeuchi S, Baghdadi M, Tsuchikawa T, et al. Chemotherapy-derived inflammatory responses accelerate the formation of immunosuppressive myeloid cells in the tissue microenvironment of human pancreatic cancer. Cancer Res 2015;75(13):2629–2640.Chen Y, Zhao Z, Chen Y, et al. An epithelial-to-mesenchymal transition-inducing potential of granulocyte macrophage colony-stimulating factor in colon cancer. Sci Rep 2017;7(1):8265.Bayne LJ, Beatty GL, Jhala N, et al. Tumor-derived granulocyte-macrophage colony-stimulating factor regulates myeloid inflammation and T cell immunity in pancreatic cancer. Cancer Cell 2012;21(6):822–835.Pylayeva-Gupta Y, Lee KE, Hajdu CH, Miller G, Bar-Sagi D. Oncogenic Kras-induced GM-CSF production promotes the development of pancreatic neoplasia. Cancer Cell 2012;21(6):836–847.Waghray M, Yalamanchili M, Dziubinski M, et al. GM-CSF mediates mesenchymal-epithelial cross-talk in pancreatic cancer. Cancer Discov 2016;6(8):886–899.

Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [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 5167.

Introduction: Immune checkpoint inhibitors (ICIs) are an effective strategy to engage the adaptive arm of the immune system in several solid cancers. While approved for metastatic triple negative breast cancer and breast cancer with microsatellite instability or mismatch repair deficiencies, ICIs are still under investigation in HER2+ breast cancer. In preclinical studies, the effects of ICIs are dampened by immunosuppressive cells, such as myeloid derived suppressor cells (MDSC), in the breast tumor microenvironment (TME). Previous studies in mice showed that addition of entinostat, a histone deacetylase inhibitor, to ICIs improved survival and reduced immunosuppression in an early HER2+ breast tumor model. Here, we evaluated the addition of entinostat to ICIs in a mouse model of metastatic HER2+ breast cancer. We hypothesize that the different immune cell composition within the TMEs of breast and lung metastases will affect mechanisms of response to this treatment combination. Methods: mmTV-NeuN transgenic mice (NeuN) were challenged with syngeneic NT2.5-LM cells that spontaneously metastasize to the lungs. Mice were treated with different combinations of entinostat, anti-CTLA-4, anti-PD-1, and anti-HER2 for 3 weeks for analysis of survival and metastatic tumor burden. For analyses of tumor-infiltrating immune cells in pulmonary metastases, flow cytometry of dissociated lungs was done 6 weeks after tumor injection. Results: Unlike the previously published model of early-stage HER2+ breast cancer, combinations of entinostat and ICIs did not improve survival in NT2.5-LM bearing mice. Anti-HER2 therapy was the only agent to improve survival, and its effect was hindered by the addition of entinostat and ICIs. The numbers of pulmonary metatastases among treatment groups were not significantly different. Although the combination of entinostat and ICIs increased the percentage of cytotoxic CD8 T cells in the lungs, it also increased the percentage of the more suppressive monocytic-MDSCs and decreased the percentage of less suppressive granulocytic-MDSCs. Whereas entinostat decreased suppressive activity of MDSCs from primary tumors, entinostat did not significantly change functional markers of MDSCs in the lung. Conclusion: Entinostat and ICIs did not reduce breast metastases in the lung, nor did their combination improve survival. Previous studies showed that entinostat and ICIs reduced MDSC suppressive function within the TME. Reduced suppression was not observed in lung metastases. Investigations are underway to define mechanisms responsible for the differential effect of entinostat on MDSC phenotype in primary tumors but not in the metastatic niche. Citation Format: Julie K. Jang, Christine Rafie, Sofi Castanon, Brian J. Christmas, Kayla A. Cruz, Skylar Woolman, Evanthia T. Roussos Torres. Breast pulmonary metastases and associated myeloid derived suppressor cells are resistant to the effects of entinostat with checkpoint inhibitor in a murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2730.

Elevated levels of circulating monocytic myeloid derived suppressor cells in splenectomised β-thalassaemia/HbE patients.

Myeloid Derived Suppressor Cells (MDSCs) Regulate Tumor Growth, Immune Response and Regulatory T Cell (Treg) Development in the Multiple Myeloma Bone Marrow Microenvironment

Inhibition Of Myeloid Derived Suppressor Cells (MDSC) In The Multiple Myeloma Bone Marrow Microenvironment

The abnormal differentiation of myeloid cells is the key element of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) are a major component of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. Recent data indicated that the fate of MDSC depends on the localization of these cells. In contrast to peripheral lymphoid organs, inside tumors, MDSC rapidly differentiate to tumor associated macrophages with potent immune suppressive activity. Hypoxia is intricate part of tumor microenvironment. We have found that hypoxia plays a major role in regulation of MDSC differentiation to macrophages in tumor site. Hypoxia caused dramatic reduction in phosphorylated STAT3 in MDSC. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. In our experiments spleen MDSC in tumor-bearing mice had much higher level of STAT3 phosphorylation than their counterparts in tumor site from the same mice. In cancer patients MDSC in tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. In vitro culture of spleen MDSC in hypoxia led to the suppression of STAT3 activation. Furthermore, mice with constitutively active STAT3 had shown the dramatic expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, suggesting that STAT3 is a downstream target for hypoxia driven myeloid cells differentiation in cancer. We found that CD45 protein tyrosine phosphatase was a molecule that regulated STAT3 activation in hypoxia. The activity but not expression of CD45 phosphatase was up-regulated in tumor MDSC as compared to MDSC in spleens. The exposure of spleen MDSC to hypoxia led to the significant increase in CD45 activity. The siRNA mediated silencing of CD45 abrogated the effect of hypoxia on the down-regulation of STAT3 activity in MDSC. This suggests that hypoxia driven up-regulation of CD45 phosphatase activity suppresses STAT3 activation, which in turn plays a key role in differentiation of myeloid cells in tumor site. These unexpected findings may provide rationale for re-evaluation of therapeutic strategies targeting myeloid cells in cancer. Citation Format: Vinit Kumar. Hypoxia determines the fate of myeloid cell differentiation by controlling STAT3 activation in tumor site. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2014-3616

Hydroxyphenylpyruvate Dioxygenase Is a Metabolic Immune Checkpoint for UTX-deficient Colorectal Cancer

β2-AR Signaling Enhances MDSC Survival through Metabolic Reprograming and Impairs Therapeutic Efficacy in Hematologic Malignancies

Immune checkpoint inhibitor response in mismatch repair-deficient colorectal cancer and other solid tumors: is it truly disease-agnostic?

The abnormal differentiation of myeloid cells is one of the key features of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) are a major component of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. Previous data indicated that the fate of MDSC depends on the localization of these cells. In contrast to peripheral lymphoid organs such as spleen, inside tumors, MDSC rapidly differentiate to tumor associated macrophages with potent immune suppressive activity. We have demonstrated that this phenomenon is mediated by dramatic down-regulation of STAT3 activity in MDSC at tumor site. In cancer patients, MDSC at tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. The mice with constitutively active STAT3 had shown the dramatic expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, emphasizing the crucial role of STAT3 in myeloid cell differentiation. Hypoxia is an intricate part of tumor microenvironment. We have found that hypoxia caused the down-regulation of STAT3 activity in MDSC. However, the effect of hypoxia on STAT3 activity was not observed in tumor cells. We further demonstrate that the up-regulation of CD45 tyrosine phosphatase activity in MDSC exposed to hypoxia in tumor site was responsible for down-regulation of STAT3 activity. Using KO model we have shown that MDSC devoid of CD45 failed to experience the effect of hypoxia on pSTAT3 down-regulation as well as their differentiation to macrophage at tumor site. Furthermore, the positive regulation of CD45 activity in hypoxia was mediated by the disruption of CD45 protein dimerization via sialylation. The treatment of sialidase abrogated the effect of hypoxia on STAT3 activity in MDSC and differentiation of MDSC to macrophages. This novel mechanism of regulation of myeloid cell differentiation by STAT3 may provide rationale for re-evaluation of therapeutic strategies in cancer. Citation Format: Vinit Kumar, Pingyan Cheng, Thomas Condamine, Dmitry Gabrilovich. Tumor microenvironment regulates the fate of myeloid cells by controlling STAT3 activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2343. doi:10.1158/1538-7445.AM2015-2343

582 Background: Although a causal relationship for inflammation and immunity of cancer is more widely accepted today, the precise cell mechanisms mediating this relationship have not been elucidated. Vascular endothelial growth factor (VEGF), previously known as vascular permeability factor. 45 kDa protein, belongs to a family of platelet-derived growth factors. VEGF, inflammation-related protein, could contribute to the accumulation of immunosuppressive cells (MDSC, Treg, TAM, and Tie-2-expressingmonocytes) in tumor-bearing hosts through direct or indirect mechanisms. Methods: We tested the serum levels of VEGF by ELISA in 106 patients including 43 with gastric and 63 with colorectal cancer, and they were increased in advanced stages of gastric and colorectal cancer. Production of IL-17, pro-inflammatory cytokine, with a stimulation of PHA was measured by ELISA and MDSC (myeloid-derived suppressor cells), one of major immunosuppressing cells, was measured by flow cytometry (CD11b+CD14-CD33+). Neutrophil to lymphocyte ratio (NLR) and C-reactive protein (CRP) were used as inflammatory markers. Production of IL-12 and SI (stimulation index) of blastogenic response of lymphocytes were used as markers of cell-mediated immune response. Results: The concentrations of VEGF were positively correlated with levels of MDSC, production of IL-17, NLR and CRP, and were inversely correlated with IL-12 production, SI and nutritional markers including prealbumin and retinol binding protein. The patients were both divided into two groups with a serum level of VEGF (330 pg/ml) and OS (overall survival) of patients with stages III and IV gastric or colorectal cancer were both significantly worse in patients with high levels of VEGF than in those with low VEGF although the differences were not significant in patients with stagesⅠand II. Conclusions: The results of the present study suggested that VEGF may have an impact on advancement and progression involving inflammation and serve as useful markers of the immune suppression involving MDSC, malnutrition and poor prognosis in patients with gastric and colorectal cancer.

Tumor microenvironment is characterized by a strong immunosuppression, where myeloidderived suppressor cells (MDSC) induced by chronic inflammation play a major role. Using ret transgenic mouse melanoma model, which mimics clinical situation in human melanoma, we demonstrated MDSC accumulation in melanoma lesions that correlated with reduced anti-tumor T cell reactivity and accelerated tumor progression. The accumulation and activation of MDSC could be mediated not only by soluble inflammatory factors but also by tumor-derived extracellular vesicles, converting immature myeloid cells into immunosuppressive MDSC. Targeting of MDSC migration and functions significantly prolonged survival of tumor-bearing mice associated with the restoration of T cell anti-tumor reactivity. In advanced melanoma patients resistant to immunotherapy with immune checkpoint inhibitors, MDSC targeting induced a beneficial therapeutic effect. We suggest that chronic inflammatory mediators and MDSC are of critical importance for melanoma pathogenesis and their neutralization should be included in combined melanoma immunotherapies to increase their efficiency.

e16005 Background: MDSC are a heterogeneous population of immunosuppressive cells with potentially predictive implications in UC pts receiving CI. We hypothesized that MDSC populations may change after CI exposure. Methods: Serial peripheral blood samples were collected from mUC pts treated with CI. MDSC were measured in fresh unfractionated whole blood (WB) and in peripheral blood mononuclear cells (PBMC). MDSC were identified by flow cytometry in WB and defined as LinloCD33+/HLADR- [(T)otal MDSC]. MDSC subsets were defined as (G)ranulocytic (CD15+CD14-), (M)onocytic (CD15-CD14+), (I)mmature (CD15-CD14-), or CD11b+. MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. The Wilcoxon signed rank and rank sum tests were used to assess changes in MDSC populations while on CI. Results: 17 pts treated with CI (9 atezolizumab [A], 8 pembrolizumab [P]) had ≥ 2 MDSC samples for analysis. Median age at diagnosis was 71 (46-81), 12 men, 29% never smokers; 53% / 29% / 18% ECOG PS 0/1/2 and 59% visceral metastasis at the time of 1st sample collection. 10 pts received CI as 1st line therapy (Tx) in metastatic setting; 7 pts received chemotherapy as 1st-line Tx for mUC (6 platinum-based, 1 docetaxel) and CI as 2nd-line Tx. In 16 pts with samples before 1stdose, there was a relative decrease (median 36.3%, range -59.7 to +21.2) in PBMC % I-MDSCs between 1st and 2nd samples (p=0.06). Interestingly, PBMC %M-MDSC and %I-MDSC tended to increase compared to baseline in pts treated with P, while they tended to decrease in pts treated with A (Table). Conclusions: In this cohort of pts with mUC treated with CIs,MDSC changes differed based on CI (anti-PDL1 or anti-PD1). Further study in larger cohort with various prior Tx lines and longer follow up as well as correlations with Tx response, toxicity and outcomes are ongoing. [Table: see text]