Fatty Acid Metabolism in Myeloid-Derived Suppressor Cells and Tumor-Associated Macrophages: Key Factor in Cancer Immune Evasion.

Identification of Novel Tumor-Microenvironment-Regulating Factor That Facilitates Tumor Immune Infiltration in Colon Cancer

The tumor microenvironment (TME) plays a pivotal role in the fate of cancer cells, and tumor-infiltrating immune cells have emerged as key players in shaping this complex milieu. Cancer is one of the leading causes of death in the world. The most common standard treatments for cancer are surgery, radiation therapy, and chemotherapeutic drugs. In the last decade, immunotherapy has had a potential effect on the treatment of cancer patients with poor prognoses. One of the immune therapeutic targeted approaches that shows anticancer efficacy is a type 2 diabetes medication, metformin. Beyond its glycemic control properties, studies have revealed intriguing immunomodulatory properties of metformin. Meanwhile, several studies focus on the impact of metformin on tumor-infiltrating immune cells in various tumor models. In several tumor models, metformin can modulate tumor-infiltrated effector immune cells, CD8+, CD4+ T cells, and natural killer (NK) cells, as well as suppressor immune cells, T regulatory cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). In this review, we discuss the role of metformin in modulating tumor-infiltrating immune cells in different preclinical models and clinical trials. Both preclinical and clinical studies suggest that metformin holds promise as adjunctive therapy in cancer treatment by modulating the immune response within the tumor microenvironment. Nonetheless, both the tumor type and the combined therapy have an impact on the specific targets of metformin in the TME. Further investigations are warranted to elucidate the precise mechanisms underlying the immunomodulatory effects of metformin and to optimize its clinical application in cancer patients.

Cancer immunotherapy is considered to be one of the biggest breakthroughs in cancer therapy in the last decade. However, the success of immunotherapy has so far been limited to a few solid malignancies including melanoma, renal cell carcinoma, non-small-cell lung cancer (NSCLC) and a few hematologic malignancies. In 2011, ipilimumab, a therapeutic monoclonal antibody that blocks CTLA-4, the bona fide immune checkpoint, was US FDA-approved for advanced melanoma [1]. Subsequently, other checkpoint inhibitors including anti-PD-1 and anti-PD-L1 blockade antibodies were also demonstrated to yield an objective response in approximately 20–30% of patients of these malignant diseases; and among the patients who had an objective response, many had a durable response [2–4]. One of anti-PD-1 antibodies (pembrolizumab) was most recently approved by US FDA for unresectable or metastatic melanoma. In addition, sipuleucel-T, a dendritic cell vaccine, has been shown to improve the overall survival of metastatic prostate cancer and subsequently gained FDA approval [5]. Nevertheless, significant objective response and durable responses were not seen in sipuleucel-T-treated pancreatic cancer patients. Melanoma, renal cell carcinoma and NSCLC were unique in their high infiltration of effector lymphocytes in tumor microenvironment (TME) [6]. By contrast, many other solid malignancies including pancreatic cancer are characterized by a highly immunosuppressive TME [7]. Immune tolerance mechanisms within the TME are a major obstacle to effective treatment of these cancers with immunotherapy. Pancreatic cancer and many other malignancies are thus considered ‘nonimmunogenic’ neoplasms. This notion has drastically slowed the development and application of immune-based therapies for these diseases.

Cancer immunotherapy includes promising strategies based on immune checkpoint blockade (e.g., anti-PD-1/PD-L1, anti-CTLA-4). A limitation of such therapies for solid tumors stems from other immune-suppressive mechanisms mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages. We hypothesize that molecules that target specific suppressive immune cells in the tumor microenvironment can reprogram the pro-tumor microenvironment towards antitumor immunity. Using our proteome-scale target/antitarget network-based lead optimization method (CANDESIGN), we designed and synthesized cell-specific nontoxic chemical libraries with modular functions for anticancer potency and immunomodulation. We used machine learning iteratively on experimental data to identify cell-specific target/anti-target networks (gene programs) as well as designed and synthesized compound libraries targeting cell-specific programs for desired cancer and immune cell function ex vivo. Specifically, we altered suppressive function of MDSCs by targeting upregulated genes in activated monocytic MDSCs in the tumor microenvironment compared to cells in spleen. We synthesized a potent nontoxic compound that specifically modulates activated MDSC function and corresponding changes in CD4+ and CD8+ T-cell activity in a mouse bladder cancer model. We observed a significant reduction in tumor mass following treatment by oral gavage. Interestingly, the bladder cancer cells used in our mouse model (as well as human and dog bladder cancer cells) are insensitive to our lead compound in vitro, suggesting in vivo antitumor activity via immunomodulation. Our lead decreased frequency of monocytic and granulocytic MDSCs in the ascites and the tumor. Moreover, a decreased frequency of MDSC expressing suppressive functional markers (e.g., Arg1, iNOS, PD-L1) and increased IFNγ+CD8+ T cells were observed in the tumor microenvironment after compound treatment. Finally, we performed a small clinical trial on pet dogs with bladder tumors treated with our compound tablets for 4-5 weeks that resulted in ~50% reduction in tumor volume. We conclude that designing cell-specific compounds perturbing the tumor microenvironment to combat immune suppression gives a selective advantage to the immune system to combat solid tumors via single and combination drug/cell therapies. Citation Format: Erin Kischuk, Joydeb Majumder, Jonathan A. Fine, Travis C. Lantz, Deepika Dhawan, Deborah W. Knapp, Timothy L. Ratliff, Gaurav Chopra. Cell-specific gene program-based small-molecule immunomodulators targeting solid-tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4709.

Immunomodulatory Effects of Histone Deacetylase 6 Inhibition in Suppressor Immune Cells in Multiple Myeloma

Glioblastoma (GBM) development and therapeutic resistance has been accompanying with the tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). TAMs are heterogeneous cell populations of immune regulatory myeloid-derived suppressor cells (MDSCs) and polarization of anti-tumor macrophages (M1) into pro-tumor macrophages (M2). We investigated the role of myeloid cell NF-κB signaling in orthotopic GBM model using immune deficient and immune competent hosts. Interestingly, conditional deletion of canonical NF-κB signaling (p65) with Lysm-Cre (p65 KO) in myeloid cells, significantly inhibited syngeneic GL261 tumor growth in immune-competent mice compared to control mice. We studied the TAMs recruitment to the tumor and their polarization under the influence of TME. P65 KO mice displayed decreasing trend of immune cell infiltration (CD45), which phenotyped as decreased F4/80+, CD68+, CD206+ (M2) and Gr1+CD11b+ (MDSCs) macrophages, compared to control mice. This was associated with the increased CD80+ (M1) macrophages, increasing trend of CD4+ and CD8+ cytotoxic T cells, and decreased CD44+ mesenchymal cancer stem cells (CSCs) populations in the TME. Cytokine array data indicated that loss of canonical NF-κB signaling within the TAMs was implicated in increased production of IFNγ, IGF1, MCP1, MIP1α, and TNFα cytokines. Co-culture of T cells with p65 KO or control MDSCs identified increased proliferation of T cells with p65 KO MDSCs compared to control MDSCs. Conversely, GBM patient-derived xenografts and U251 GBM cell line-derived tumors showed increasing trend of growth in immune-deficient mice, following the transplantation of p65 KO bone marrow (BM) compared to control BM. Pro-tumor macrophages and CSCs were increased and T cell populations were decreased in human tumors grown in immune deficient mice transplanted with p65 KO BM, compared with control BM. In addition, analysis of human data set revealed higher expression of p65 subunit of NF-κB complex in brain tumor stroma compared to the tumor cells. The study suggests that canonical NF-κB signaling in TAMs is required for the tumor-promoting macrophage polarization and GBM growth in immunocompetent host compared to immune deficient host. Therefore, targeting myeloid-specific NFκB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity. Citation Format: Bhagelu R. Achyut, Jennifer Bradford, Kartik Angara, Mohammad Rashid, Meenu Jain, Thaiz Borin, ASM Iskander, Roxan Ara, Ali Arbab. Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3968. doi:10.1158/1538-7445.AM2017-3968

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in cancer biology, particularly in the modulation of innate immune cells within the tumor microenvironment. These lncRNAs significantly influence the phenotype and function of immune cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), natural killer cells (NK), neutrophils, and γδT cells. Thus, lncRNAs emerge as pivotal molecules in cancer development due to their capacity to modulate the innate immune system. Understanding the intricate mechanisms by which lncRNAs influence tumor-associated immune cells can pave the way for novel therapeutic strategies to restore effective anti-tumor immunity. This review highlights the diverse roles of lncRNAs in regulating the differentiation, activation, and effector functions of innate immune cells within the complex tumor microenvironment.

Cancer progression is determined by cancer cells as well as various immune cells that make up the tumor microenvironment (TME). These immune cells consist of so-called effector cells such as natural killer cells and cytotoxic T lymphocytes, which fight cancer progression, and of immunosuppressive immune cells including regulatory T cells and immature myeloid cells, which aid tumor progression. Immature myeloid cells in the TME are further divided into several populations, amongst which are tumor-associated dendritic cells (TADC), tumor-associated macrophages (TAM), and myeloid-derived suppressor cells (MDSC). While TADC and TAM can be found in different activation states, MDSC can be further subdivided in two subsets: polymorphonuclear and monocytic MDSC. In recent years, MDSC received much attention as they are believed to exert a plethora of inhibitory mechanisms to create a tumor promoting TME. The recruitment, activation, and function of MDSC in the TME are largely determined by the transcription factor signal transducer and activator of transcription 3 (STAT3). Therefore, this review will focus on the role of this key signaling pathway during the MDSC life cycle.

Efficacy of immunotherapeutic approaches against glioma is limited by the immunosuppressive tumor microenvironment. Tumor derived TGF-β, IL-10 and Prostaglandin E2 along with the presence of regulatory T cells (Tregs) and tumor associated macrophages (TAMs) promote the immune escape in gliomas. Also, tumor derived factors induce the expansion of myeloid derived suppressor cells (MDSCs). MDSCs represent a heterogeneous population of myeloid cells at various stages of differentiation that have the potential to inhibit anti-tumor T cell responses. Herein we demonstrate the accumulation of MDSCs in GL26 brain tumor bearing mice. Absolute numbers of Ly-6G+ (Gr-1high) MDSCs showed a 200 fold increase within the tumor mass 28 days post-tumor implantation. In contrast, the numbers of Ly-6C+ (Gr-1low) MDSCs did not significantly change within the tumor microenvironment. While this massive influx of MDSCs was noted within intracranial tumors, the levels of Ly-6G+ or Ly-6C+ MDSCs did not increase in the tumor draining lymph nodes (dLNs), spleen, bone marrow or blood of intracranial tumor bearing mice. Mice bearing GL26 or B16-F0 tumors in the flank showed a ∼3 fold increased influx of Ly-6G+ MDSCs within the tumor mass, the spleen and circulating MDSCs. Ly-6G+ MDSCs isolated from the brain tumors and spleens of GL26 intracranial tumor bearing mice inhibited tumor antigen-specific CD8+ T cell proliferation and T cell proliferation mediated by CD3 ligation . On the other hand, Ly-6C+ MDSCs did not did not elicit inhibition of T cell proliferation. Preliminary experiments using tumor cells' conditioned media indicate that CXCR2 signaling mediates the migration of MDSCs in a transwell assay and suggest the possibility that it could mediate MDSCs' migration into the tumor microenvironment in vivo. Overall, our data shows that MDSCs accumulate within the glioma mass and inhibit tumor-specific T cell responses. Strategies that inhibit MDSC recruitment to the tumor microenvironment and/or block their activity may therefore enhance the T cell mediated tumor clearance and suppress glioma progression. Supported by National Institutes of Health/ National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grants RO1-NS074387 and RO1-NS054193 Citation Format: Neha Kamran, Hikmat Assi, Marianela Candolfi, Mariela Moreno, Youping Li, Pedro R. Lowenstein, Maria G. Castro. Glioma-infiltrating myeloid derived suppressor cells inhibit anti-tumor T cell responses. [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 1085. doi:10.1158/1538-7445.AM2014-1085

Background: Overexpression of sialic acids on glycans, called hypersialylation is a common alteration in cancer that drives immune evasion via interaction with Sialic acid-binding immunoglobulin-like lectin (Siglec) immunoregulatory receptors on tumor-infiltrating immune cells. Myeloid derived suppressor cells (MDSCs) generated by cancer-induced aberrant myelopoiesis are highly immunosuppressive cells inhibiting immune cells via direct interaction or secretion of suppressive cytokines. Targeting the complex suppressive tumor microenvironment (TME) remains a challenge. Methods: Siglec expression of murine and human MDSCs in healthy conditions and tumor setting was assessed by flow cytometry. Functional analysis of Siglec-E knockout on MDSCs in mice was evaluated using SigE-LysMCre mice and suppressive capacity was tested in vitro in combination with SiglecE blocking and cleavage of Sialoglycans using Sialidase. Results were confirmed in the human setting using an in vitro assay to generate MDSC-like cells including RNA-Sequencing and a MDSC suppression assay with cancer-derived MDSCs. Results: MDSCs express multiple inhibitory Siglec receptors in humans and mice. Knockout of SiglecE in the myeloid compartment in vivo resulted in prolonged survival and increased T cell infiltration compared to litter mates expressing Siglec-E. Of note, desialylation of MDSCs and Siglec-E blocking in vitro reduced MDSCs suppressive function against T cells. Similarly, reducing Sialoglycans by Sialidase in human MDSC generated in vitro resulted in increased suppressive capacity and downregulation of functional MDSC markers on RNA level. Findings were validated with lung cancer patient derived MDSCs. Conclusion: Our results provide first insights into the importance of the Siglec-Sialoglycans axis to modulate MDSCs in the TME. Targeting glycosylation of MDSCs with Sialidase reduces their suppressive capacity making it a powerful approach to improve cancer immunotherapy. Further studies are needed to reveal the underlying mechanisms. Citation Format: Ronja Wieboldt, Andreas Zingg, Emanuele Carlini, Anastasiya Börsch, Heinz Läubli, Natalia Rodrigues Manutano. Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor 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 1259.

L-Arginine metabolism plays a crucial role in determining the M1/M2 polarization of macrophages. The M1 macrophages express inducible nitric oxide synthase (iNOS), while the M2 macrophages express arginase 1 and metabolize arginine into nitric oxide and urea, respectively. The tumor microenvironment promotes M2 macrophage polarization and consequently switches the metabolic fate of arginine from nitric oxide towards urea production. Importantly, infiltration of M2 macrophages or tumor-associated macrophages (TAMs) has been correlated with poor prognosis of various cancer types. Melatonin is well reported to have antitumor and immunomodulatory properties. However, whether and how it impacts the polarization of TAMs has not been elucidated. Considering the crucial role of arginine metabolism in macrophage polarization, we were interested to know the fate of L-arginine in TAMs and whether it can be reinstated by melatonin or not. We used a murine model of Dalton's lymphoma and established an in vitro model of TAMs. For TAMs, we used the ascitic fluid of tumor-bearing hosts to activate the macrophages in the presence and absence of lipopolysaccharide (LPS). In these groups, L-arginine metabolism was evaluated, and then the effect of melatonin was assessed in these groups, wherein the metabolic fate of arginine as well as the expression of iNOS and arginase 1 were checked. Furthermore, in the in vivo system of the tumor-bearing host, the effect of melatonin was assessed. The in vitro model of TAMs showed a Th2 cytokine profile, reduced phagocytic activity, and increased wound healing ability. Upon investigating arginine metabolism, we observed high urea levels with increased activity and expression of arginase 1 in TAMs. Furthermore, we observed reduced levels of LPS-induced nitric oxide in TAMs; however, their iNOS expression was comparable. With melatonin treatment, urea level decreased significantly, while the reduction in nitric oxide level was not as significant as observed in its absence in TAMs. Also, melatonin significantly reduced arginase activity and expression at the transcriptional and translational levels, while iNOS expression was affected only at the translational level. This effect was further investigated in the in vivo system, wherein melatonin treatment reversed the metabolic fate of arginine, from urea towards nitric oxide, within the tumor microenvironment. This effect was further correlated with pro-apoptotic tumor cell death in the in vivo system. Our results reinforced the immunomodulatory role of melatonin and offered a strong prospect for activating the anti-tumor immune response in cancer conditions.

Special Issue on Immune Responses in Tumors and Non-Transformed Inflammatory Microenvironments

lipid metabolic, metabolic reprogramming, immunometabolic, regulatory T cells, tumor-associated macrophages, myeloid-derived suppressor cells "Cold tumors" are malignancies with poor immune infiltration and limited response to immunotherapy, largely shaped by an immunosuppressive tumor microenvironment (TME) (1-3).Lipid metabolic reprogramming has emerged as a central mechanism sustaining this suppression.Rapidly proliferating tumor cells deplete nutrients and release byproducts, generating hypoxia, acidosis, and scarcity, which force both tumor and immune cells to rewire their metabolism (4, 5).Under these stresses, not only tumor cells but also immune cells undergo "immunometabolic" reprogramming to adapt to the hostile environment (6, 7).Lipids serve as fuels, signaling mediators, and membrane components, and their altered metabolism profoundly affects immune regulation (8,9).This mini review highlights how lipid reprogramming supports key immunosuppressive populations in the TME-regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs)-and explores therapeutic strategies that target lipid metabolism to improve cancer immunotherapy. Functions of lipids and metabolic targetsLipids play three essential roles in cellular physiology: they serve as alternative energy sources through b-oxidation when glucose is scarce, act as precursors of signaling mediators such as PGE 2 and leukotrienes, and provide structural components of membranes that support proliferation and immune receptor function (2, 7).In the tumor microenvironment, lipid metabolism is frequently rewired to sustain growth and survival.This involves increased uptake via FATPs (fatty acid transport proteins), CD36 (cluster of differentiation 36), FABPs (fatty acid-binding proteins), and LDLR (low-density lipoprotein receptor) (10, 11), enhanced de novo synthesis of fatty acids and cholesterol from acetyl-CoA through FASN (fatty acid synthase) and ACC (acetyl-CoA carboxylase) (12-14), and elevated mitochondrial FAO (fatty acid oxidation) mediated by CPT1

<div>Abstract<p>Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. This FRβ<sup>+</sup> subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8<sup>+</sup> T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors.</p>Significance:<p>FRβ serves as both a means to identify and target MDSCs and TAMs within the tumor, allowing for delivery of immunomodulatory compounds to tumor myeloid cells in a variety of cancers.</p></div>

Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. This FRβ+ subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8+ T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors. SIGNIFICANCE: FRβ serves as both a means to identify and target MDSCs and TAMs within the tumor, allowing for delivery of immunomodulatory compounds to tumor myeloid cells in a variety of cancers.