Myeloid-derived Suppressor Cells Markers Research Articles

Targeting Myeloid-Derived Suppressor Cells via Dual-Antibody Fluorescent Nanodiamond Conjugate

Fluorescent nanodiamonds (FNDs) are carbon-based nanomaterials that emit bright, photostable fluorescence and exhibit a modifiable surface chemistry. Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive cell population known to expand in cancer patients and contribute to worse patient outcomes. To target MDSC, glycidol-coated FND were conjugated with antibodies against the murine MDSC markers, CD11b and GR1 (dual-Ab FND). In vitro, dual-Ab FND uptake by murine MDSC was significantly higher than IgG-coated FND (94.7% vs. 69.0%, p < 0.05). In vivo, intra-tumorally injected dual-Ab FND primarily localized to the tumor 2 and 24 h post-injection, as measured by in vivo fluorescence imaging and flow cytometry analysis of the spleen and tumor. Dual-Ab FND were preferentially taken up by intra-tumoral MDSC, representing 87.1% and 83.0% of FND+ cells in the tumor 2 and 24 h post-injection, respectively. Treatment of mice with anti-PD-L1 immunotherapy prior to intra-tumoral injection of dual-Ab FND did not significantly alter the uptake of FND by MDSC. These results demonstrate the ability of our novel dual-antibody conjugated FND to target MDSC and reveal a potential strategy for targeted delivery to other specific immune cell populations in future cancer research.

CSF1R Ligands Expressed by Murine Gliomas Promote M-MDSCs to Suppress CD8+ T Cells in a NOS-Dependent Manner.

Glioblastoma (GBM) is the most common malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a subset of myeloid cells, expressing monocytic (M)-MDSC markers and dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate the TME. This study evaluated the mechanism of CCR2+/CX3CR1+ M-MDSC differentiation and T cell suppressive function in murine glioma models. We determined that bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Glioma-secreted CSF1R ligands M-CSF and IL-34 were identified as key drivers of M-MDSC differentiation while adenosine and iNOS pathways were implicated in the M-MDSC suppression of T cells. Mining a human GBM spatial RNAseq database revealed a variety of different pathways that M-MDSCs utilize to exert their suppressive function that is driven by complex niches within the microenvironment. These data provide a more comprehensive understanding of the mechanism of M-MDSCs in glioblastoma.

Protein Signature Differentiating Neutrophils and Myeloid-Derived Suppressor Cells Determined Using a Human Isogenic Cell Line Model and Protein Profiling.

Myeloid-derived suppressor cells (MDSCs) play an essential role in suppressing the antitumor activity of T lymphocytes in solid tumors, thus representing an attractive therapeutic target to enhance the efficacy of immunotherapy. However, the differences in protein expression between MDSCs and their physiological counterparts, particularly polymorphonuclear neutrophils (PMNs), remain inadequately characterized, making the specific identification and targeting of MDSCs difficult. PMNs and PMN-MDSCs share markers such as CD11b+CD14-CD15+/CD66b+, and some MDSC-enriched markers are emerging, such as LOX-1 and CD84. More proteomics studies are needed to identify the signature and markers for MDSCs. Recently, we reported the induced differentiation of isogenic PMNs or MDSCs (referred to as iPMNs and iMDSCs, respectively) from the human promyelocytic cell line HL60. Here, we profiled the global proteomics and membrane proteomics of these cells with quantitative mass spectrometry, which identified a 41-protein signature ("cluster 6") that was upregulated in iMDSCs compared with HL60 and iPMN. We further integrated our cell line-based proteomics data with a published proteomics dataset of normal human primary monocytes and monocyte-derived MDSCs induced by cancer-associated fibroblasts. The analysis identified a 38-protein signature that exhibits an upregulated expression pattern in MDSCs compared with normal monocytes or PMNs. These signatures may provide a hypothesis-generating platform to identify protein biomarkers that phenotypically distinguish MDSCs from their healthy counterparts, as well as potential therapeutic targets that impair MDSCs without harming normal myeloid cells.

Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

ObjectivesEpithelial ovarian cancer (EOC) is considered to be a prevalent female malignancy with both high incidence and mortality. It is reported that RNA-binding protein 3 (RBMS3) executives a tumor suppressor function in different cancers. This investigation was designed to examine the expression of RBMS3 in epithelial ovarian cancer, the effects on EOC cells, and its connection to immune cells that infiltrate tumors in the EOC microenvironment. MethodsThe expression levels of RBMS3 in EOC tissues as well as their correlations with immune cell infiltration and clinical outcome were examined using bioinformatics approaches. Western blotting as well as immunohistochemistry were carried out to determine the protein levels in EOC tissues. In addition, qRT-PCR was employed to look at the expression of the mRNA. The role of RBMS3 in EOC cells was investigated, and an RBMS3 lentiviral vector was developed. The effects of RBMS3 on subcutaneous tumor development, the proliferation protein Ki-67, the tumor angiogenesis indicator CD31, and its function in controlling the tumor immune microenvironment were evaluated by in vivo tests. ResultsThere was a considerable decrease in RBMS3 expression in EOC tissues, which was linked to a poor prognosis for patients and the infiltration of multiple immune cell. Given immunohistochemical studies, tissues with increased RBMS3 expression had decreased markers of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages, whereas M1 macrophage markers were elevated. RBMS3 appears to suppress the capabilities of proliferating, invading, and migrating in EOC cells according to in vitro tests, whereas tumors overexpressing RBMS3 developed more slowly in syngeneic mouse models. The overexpression of RBMS3 led to a decline in the levels of Ki-67 protein and CD31. Additionally, it showed a negatively correlation with markers of regulatory T cell, myeloid-derived suppressor cell, and M2 macrophage but a positive correlation with markers of M1 macrophage. ConclusionsThe findings revealed that elevated RBMS3 expression plays a tumor suppressor role in EOC and was connected to patient survival in EOC. The studies conducted in vitro and in vivo demonstrated a link between RBMS3 expression and the infiltration of certain immune cells, indicating a function for RBMS3 in the immunosuppressive tumor microenvironment and its promising efficiency as a novel target for immunotherapy against EOC.

Abstract 7519: Elucidation of the mechanism of acquisition of immune checkpoint inhibitor resistance by chronic inflammation

Abstract Introduction: Eight molecules that are structurally similar to angiopoietin but do not bind to angiopoietin's specific receptor Tie2 were identified and named angiopoietin-like factors (ANGPTL). ANGPTL2, particularly abundantly expressed in adipose tissue, promotes damage repair through tissue remodeling, and contributes to tissue homeostasis; however, in severe obesity, ANGPTL2 is overexpressed, resulting in irreversible tissue remodeling, which induces chronic inflammation and is involved in systemic insulin resistance and diabetes development. On the other hand, it is unclear how systemic chronic inflammation controlled by ANGPTL2 is involved in cancer progression and acquisition of immune checkpoint inhibitor (ICI) resistance. Therefore, we aimed to elucidate the mechanisms in the acquisition of ICI resistance by systemic chronic inflammation from the perspective of ANGPTL2. Materials and Methods: 1. To induce chronic inflammation in adipose tissue, BALB/c mice were fed high-fat-diet (HFD) for 6 weeks. CT26 cells, a mouse colon cancer cell line, were subcutaneously implanted in normal diet (ND) and HFD mice and anti PD-1/PD-L1 antibody was administered intraperitoneally. Immune cell fractions in tumor tissue and bone marrow were analyzed by flow cytometry. 2. CT26 was subcutaneously implanted in WT and systemic ANGPTL2-deficient (Angptl2−/−) mice, and anti PD-1/PD-L1 antibody was administered intraperitoneally. Tumor size and survival time were analyzed. Bone marrow cells were isolated and the immune cell fractions were analyzed by flow cytometry, and the expression of inflammatory cytokines in adipose tissue were also analyzed. 3. Bone marrow cells were isolated from BALB/c mice, and myeloid-derived suppressor cells (MDSCs) were established in vitro by GM-CSF and IL-6. Recombinant ANGPTL2 protein was added, and MDSC differentiation and function were evaluated by flow cytometry and qRT-PCR. Results: 1. HFD mice had larger tumor size and poorer response to ICI treatment compared to ND mice. In tumor microenvironment, MDSCs were increased in HFD mice. HFD mice had significantly increased MDSCs, especially polymorphonuclear MDSCs (PMN-MDSCs) in bone marrow. 2. ICI-treated Angptl2−/− mice showed higher treatment efficacy and prolonged survival compared to WT mice. In bone marrow, MDSCs were reduced in Angptl2−/− mice under ICI treatment. In adipose tissue, the expression of chronic inflammatory markers was increased. 3. In vitro, addition of recombinant ANGPTL2 protein to MDSCs induced differentiation from mouse bone marrow cells did not significantly increase the cell ratio of MDSCs. No changes in the expression of MDSCs markers were observed in qRT-PCR. Conclusions: Chronic inflammation induced by ANGPTL2 promotes MDSCs differentiation and maturation in the bone marrow, which may suppress anti-tumor immunity and contribute to ICI resistance. Citation Format: Shinsei Yumoto, Haruki Horiguchi, Hideo Baba, Yuichi Oike. Elucidation of the mechanism of acquisition of immune checkpoint inhibitor resistance by chronic inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7519.

Flow cytometric detection of CD11b+ Gr-1+ cells in nontumor-bearing mice: A propolis-elicited model.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous myeloid lineage population whose conventional surface phenotype is CD11b+ Gr-1+. Due to their rarity and fragility, analyses using primary isolated MDSCs are extremely difficult. However, counting CD11b+ Gr-1+ cells in associated tissues such as tumors and inflammatory lesions provides critical information regarding MDSC involvement in immune disorders in the tissues. Specific MDSC markers have not been identified, limiting our ability to apply histochemical approaches during MDSCs research. However, profiling surface antigens using multi-colorimetric flow cytometry enables us to easily monitor the abundance of MDSCs in vivo. Monitoring of mouse MDSCs and their subpopulations using flow cytometry is well established. In this article, I exemplify a conventional method of monitoring CD11b+ Gr-1+ cells in mouse adipose tissue after administration of Brazilian propolis ethanol extract, which is a strong inducer of MDSCs.

Depletion of Gr1+ myeloid cells attenuates high-fat-diet-aggravated esophageal squamous cell carcinoma in mice.

Obesity is a leading cause of multiple cancers, but whether it promotes esophageal squamous cell carcinoma (ESCC) and its underlying cancer-promoting mechanism remains unclear. To evaluate the effect of a high-fat diet (HFD) on ESCC and explore the role and mechanism of myeloid-derived suppressor cells (MDSCs) in HFD-induced ESCC, C57BL/6J mice were treated with 4-nitroquinoline 1-oxide (4NQO) to induce ESCC and randomly assigned to an HFD or a normal-fat diet. An anti-Gr1 monoclonal antibody was used to deplete MDSCs in the context of experimental diets and ESCC induction. The expression of MDSC markers CD11b and Gr1 and immune checkpoints (ICs) PD1, TIM3, and VISTA in lesions were detected by immunohistochemistry. The correlation between myeloid cell markers (CD11b and CD33) and ICs and their relationship with ESCC patient prognosis were further analyzed using the The Cancer Genome Atlas dataset. The results showed that HFD accelerated esophageal carcinogenesis, induced MDSC expansion, and upregulated IC expression, whereas depletion of Gr1+ myeloid cells significantly suppressed tumor growth, decreased the number of MDSCs, and downregulated IC expression in HFD mice. PD1, TIM3, and VISTA expressions were positively correlated with myeloid cell marker expression in human ESCC. Moreover, the high expression of IC molecules was associated with poor survival in patients with ESCC. These data indicate that HFD promotes the initiation and development of ESCC. Gr1+ myeloid cell targeting significantly inhibited ESCC formation in HFD mice, which may be associated with IC downregulation.

Prognostic impact of LILRB4 expression on tumor-infiltrating cells in resected non-small cell lung cancer.

Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4/ILT3) is an up-and-coming molecule that promotes immune evasion. We have previously reported that LILRB4 facilitates myeloid-derived suppressor cells (MDSCs)-mediated tumor metastasis in mice. This study aimed to investigate the impact of the LILRB4 expression levels on tumor-infiltrating cells on the prognosis of non-small cell lung cancer (NSCLC) patients. We immunohistochemically evaluated the LILRB4 expression levels of completely resected 239 NSCLC specimens. Whether the blocking of LILRB4 on human PBMC-derived CD33+ MDSCs inhibited the migration ability of lung cancer cells was also examined using transwell migration assay. The LILRB4 high group, in which patients with a high LILRB4 expression level on tumor-infiltrating cells, showed a shorter overall survival (OS) (p = 0.013) and relapse-free survival (RFS) (p = 0.0017) compared to the LILRB4 low group. Multivariate analyses revealed that a high LILRB4 expression was an independent factor for postoperative recurrence, poor OS and RFS. Even in the cohort background aligned by propensity score matching, OS (p = 0.023) and RFS (p = 0.0046) in the LILRB4 high group were shorter than in the LILRB4 low group. Some of the LILRB4 positive cells were positive for MDSC markers, CD33 and CD14. Transwell migration assay demonstrated that blocking LILRB4 significantly inhibited the migration of human lung cancer cells cocultured with CD33+ MDSCs. Together, signals through LILRB4 on tumor-infiltrating cells, including MDSCs, play an essential role in promoting tumor evasion and cancer progression, impacting the recurrence and poor prognosis of patients with resected NSCLC.

Overcoming resistance to immunotherapy by targeting GPR84 in myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) were found to gradually accumulate in the orthotopic esophageal cancer mouse model during tumor progression. Although the roles of MDSCs in promoting tumor growth and inhibiting immune response have been extensively explored, currently, there are still no effective means for targeting MDSCs clinically. The deficiency of specific markers of MDSCs was responsible for the limited strategy to eliminating in clinic. This study identified that GPR84 was exclusively overexpressed on MDSCs. It was further found that GPR84 was prominently expressed on MDSCs in clinical samples and tumor mouse models, which drives the immunosuppression on CD8+T cells by inhibiting PD-L1 degradation in lysosomes. Furthermore, G-CSF and GM-CSF were found to induce GPR84 expression through the STAT3/C/EBPβ signaling pathway. In addition, GPR84+MDSCs and PD-L1+MDSCs were highly accumulated in anti-PD-1 therapy-resistant patients with esophageal cancer, and high GPR84 signature risk was verified as a negative factor for the overall survival of patients with anti-PD-1 treatment. Finally, GPR84 antagonism combined with an anti-PD-1 antibody enhanced the antitumor responses. Therefore, targeting GPR84 enhanced anti-PD-1 efficacy in esophageal cancer and other malignant tumors. This combination therapy has the potential for tumor therapy in clinics.

Abstract 1259: Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment

Abstract 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.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Myeloid-derived suppressor cells and plasmacytoid dendritic cells are associated with oncogenesis of oral squamous cell carcinoma.

Myeloid-derived suppressor cells (MDSCs) help establish the tumor microenvironment by suppressing T-cell response in tumor-bearing hosts. Plasmacytoid dendritic cells (pDCs) activate antigen-specific T cells, thereby, maximizing their antitumor effects. IDO1 is associated with both MDSCs and pDCs and plays a major role in the formation of the tumor-mediated immunosuppressive environment. We utilized immunohistochemistry to examine the involvement of IDO1 in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs, precancerous lesions). We examined the expression of MDSC markers, CD11b and CD33, as well as pDC markers, CD303 and IDO1, in 60 OSCC and 45 precancerous lesion specimens and analyzed their association with clinicopathological parameters. Expression of these biomarkers identifying MDSCs and pDCs was high in precancerous lesions in patients with severe dysplasia and OSCC. While detecting pDCs, high CD303 and IDO1 expression levels were frequently observed in moderately or poorly differentiated OSCCs. CD11b, CD33, and CD303 levels were significantly correlated with the mode of invasion; CD33 was correlated with OSCC invasion depth while the other three markers tended to be highly expressed in superficial cancer cases showing microinvasion. Expression levels of all four biomarkers were significantly associated with the cancerization of OPMDs to OSCCs. We show, for the first time, that the infiltration of MDSCs and pDCs is significantly associated with progression of premalignant lesions to OSCC. This suggests that these cells may act as prognostic biomarkers for premalignant lesion progression and that immunotherapeutic approaches that control each of these immunosuppressive cells may protect against progression to malignancy.

Regulation of DNA-PK activity promotes the progression of TNBC via enhancing the immunosuppressive function of myeloid-derived suppressor cells.

DNA-dependent protein kinase (DNA-PK) is engaged in DNA damage repair and is significantly expressed in triple negative breast cancer (TNBC). Inhibiting DNA-PK to reduce DNA damage repair provides a possibility of tumor treatment. NU7441, a DNA-PK inhibitor, can regulate the function and differentiation of CD4+ T cells and effectively enhance immunogenicity of monocyte-derived dendritic cells. However, the effect of NU7441 on the tumor progression activity of immunosuppressive myeloid-derived suppressor cells (MDSCs) in TNBC remains unclear. In this study, we found that NU7441 alone significantly increased tumor growth in 4T1 (a mouse TNBC cell line) tumor-bearing mice. Bioinformatics analysis showed that DNA-PK and functional markers of MDSCs (iNOS, Arg1, and IDO) tended to coexist in breast cancer patients. The mutations of these genes were significantly correlated with lower survival in breast cancer patients. Moreover, NU7441 significantly decreased the percentage of MDSCs in peripheral blood mononuclear cells (PBMCs), spleen and tumor, but enhanced the immunosuppressive function of splenic MDSCs. Furthermore, NU7441 increased MDSCs' DNA-PK and pDNA-PK protein levels in PBMCs and in the spleen and increased DNA-PK mRNA expression and expression of MDSCs functional markers in splenic MDSCs from tumor-bearing mice. NU7441 combined with gemcitabine reduced tumor volume, which may be because gemcitabine eliminated the remaining MDSCs with enhanced immunosuppressive ability. These findings highlight that the regulation of DNA-PK activity by NU7441 promotes TNBC progression via enhancing the immunosuppressive function of MDSCs. Moreover, NU7441 combined with gemcitabine offers an efficient therapeutic approach for TNBC and merits deeper investigation.

Abstract 2121: Gene expression and functional characterization of obesity-induced MDSC to uncover the link between obesity and cancer risk

Abstract Strong epidemiologic evidence suggests that excess body weight gain is a major risk of at least 13 different types of cancers. One of the mechanisms explaining the increased cancer incidence in obese patients is associated with the chronic low-grade inflammatory condition. Myeloid cells including macrophages and Myeloid-derived suppressor cells (MDSC) are increased in advanced adiposity. Previous studies in mouse models have shown that obesity-induced MDSC play a protective role on metabolic control; however, in the presence of malignant cells, MDSC facilitates tumor growth and metastasis. Our data show that compared to non-obese individuals, patients with severe obesity (body mass index [BMI] > 40) have a significant increase of circulating granulocytic cells displaying a gene signature resembling tumor-MDSC. Intriguingly, obesity-induced MDSC lacked the potent immunosuppressive function; and therefore, we called them obesity-induced G-MDSC-like cells (Ob-MDSC). The mechanisms underlying the regulation of MDSC phenotype in obesity and obesity-related cancer are unclear. We found that LDL, one altered obesity-related factor, and LPS differentially impact the expression of several genes including the immunosuppressive MDSC markers, arginase-1 and iNOS, when comparing mouse bone marrow-derived MDSC from high-fat fed mice with low-fat fed mice. This finding indicates that the responsiveness of MDSC may be regulated by their long-lasting exposure to the obese microenvironment. Therefore, we propose that long-term adaptation of MDSC is induced by dysfunctional metabolic factors in obesity that modulate their response to subsequent triggers, such as signals derived by the tumor microenvironment (TMA) in the context of obesity-associated cancer. Our data from obese mice with Diethylnitrosamine (DEN)-induced hepatocellular carcinoma and genetically altered hepatic lipid and lipoprotein metabolism show an increased infiltration of G-MDSC associated with enhanced tissue chemokine signaling pathway, ECM remodeling/metastasis, inflammation, and epigenetic and transcriptional regulation. Together our data suggest that chronic exposure to an obesity-related environment establishes a phenotype prone to become pro-tumor MDSC. The identification of key regulators of the gene expression driving the immunosuppressive phenotype of MDSC in obesity is critical for the discovery of therapeutical targets to reduce the risk of obesity-associated cancers. Citation Format: Maria D. Sanchez-Pino, Ramesh Thylur Puttalingaiah, Jone Garai, Jovanny Zabaleta, Randall L. Mynatt, William Richardson, Augusto Ochoa. Gene expression and functional characterization of obesity-induced MDSC to uncover the link between obesity and cancer risk [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 2121.

Upregulation of Atypical Cadherin FAT1 Promotes an Immunosuppressive Tumor Microenvironment via TGF-β.

FAT atypical cadherin 1 (FAT1) promotes glioblastoma (GBM) by promoting protumorigenic inflammatory cytokine expression in tumor cells. However, tumors also have an immunosuppressive microenvironment maintained by mediators such as transforming growth factor (TGF)-β cytokines. Here, we have studied the role of FAT1 in tumor immune suppression. Our preliminary TIMER2.0 analysis of The Cancer Genome Atlas (TCGA) database revealed an inverse correlation of FAT1 expression with infiltration of tumor-inhibiting immune cells (such as monocytes and T cells) and a positive correlation with tumor-promoting immune cells [such as myeloid-derived suppressor cells (MDSCs)] in various cancers. We have analyzed the role of FAT1 in modulating the expression of TGF-β1/2 in resected human gliomas, primary glioma cultures, and other cancer cell lines (U87MG, HepG2, Panc-1, and HeLa). Positive correlations of gene expression of FAT1 and TGF-β1/2 were observed in various cancers in TCGA, Glioma Longitudinal Analysis Consortium (GLASS), and Chinese Glioma Genome Atlas (CGGA) databases. Positive expression correlations of FAT1 were also found with TGF-β1/2 and Serpine1 (downstream target) in fresh-frozen GBM samples using q-PCR. siRNA-mediated FAT1 knockdown in cancer cell lines and in primary cultures led to decreased TGF-β1/2 expression/secretion as assessed by q-PCR, Western blotting, and ELISA. There was increased chemotaxis (transmigration) of THP-1 monocytes toward siFAT1-transfected tumor cell supernatant as a consequence of decreased TGF-β1/2 secretion. Reduced TGF-β1 expression was also observed in THP-1 cultured in conditioned media from FAT1-depleted glioma cells, thus contributing to immune suppression. In U87MG cells, decreased TGF-β1 upon FAT1 knockdown was mediated by miR-663a, a known modulator. FAT1 expression was also observed to correlate positively with the expression of surrogate markers of MDSCs [programmed death ligand-1 (PD-L1), PD-L2, and interleukin (IL)-10] in glioma tumors, suggesting a potential role of FAT1 in MDSC-mediated immunosuppression. Hence, our findings elaborate contributions of FAT1 to immune evasion, where FAT1 enables an immunosuppressive microenvironment in GBM and other cancers via TGF-β1/2.

GPX1-associated prognostic signature predicts poor survival in patients with acute myeloid leukemia and involves in immunosuppression

ObjectiveTreatment of acute myeloid leukemia (AML) remains a challenge. It is urgent to understand the microenvironment to improve therapy and prognosis. MethodsBioinformatics methods were used to analyze transcription expression profile of AML patient samples with complete clinical information from UCSC Xena TCGA-AML datasets and validate with GEO datasets. Western blot, qPCR, RNAi and CCK8 assay were used to assay the effect of GPX1 expression on AML cell viability and the expression of genes of interest. ResultsOur analyses revealed that highly expressed GPX1 in AML patients links to unfavorable prognosis. GPX1 expression was positively associated with not only fraction levels of myeloid-derived suppressor cells (MDSCs), monocytes and T cell exhaustion, the expression levels of MDSC markers, MDSC-promoting CCR2 and immune inhibitory checkpoints (TIM3/Gal-9, SIRPα and VISTA), but also negatively with low fraction levels of CD4+ and CD8+ T cells. Silencing GPX1 expression reduced AML cell viability and CCR2 expression. Moreover, GPX1-targetd kinases were PKC family, SRC family, SYK and PAK1, which promote AML progression and the resistance to therapy. Furthermore, Additionally, GPX1-associated prognostic signature (GPS) is an independent risk factor with high area under curve (AUC) values of receiver operating characteristic (ROC) curves. High risk group based on GPS enriched not only with endocytosis which transfers mitochondria to favor AML cell survival in response to chemotherapy, but also NOTCH, WNT and TLR signaling which promote therapy resistance. ConclusionOur results revealed the significant involvement of GPX1 in AML immunosuppression via and provided a prognostic signature for AML patients.

Abstract 2730: 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 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.

MIF inhibits myeloid derived suppressor cell mediated immunosuppression by promoting an inflammatory M1 phenotypic shift

Cancers of the oral cavity and pharynx contribute 3% of yearly cancer cases in the United States, primarily as head and neck squamous cell carcinoma (HNSCC). HNSCC presents with a five-year survival rate of 66.2%, with primary risk factors being alcohol abuse or human papillomavirus infection. Myeloid derived suppressor cells (MDSC) are a myeloid lineage cell population known to accumulate within HNSCC tumors and promote a pro-tumor microenvironment by suppressing the host immune response against tumor cells. Macrophage migration inhibitory factor (MIF) is a cytokine released by immune cells and mediates inflammatory and chemotactic effects upon interaction with its receptor, CD74, expressed by antigen presenting cells (APC), including MDSC. Given their role in promoting tumor growth, our group sought to elucidate the function of host versus tumor cell derived MIF on MDSC. Given the chemotactic effects of MIF upon APC, we hypothesized that knockdown of Mif gene expression would result in a better outcome and reduced tumor burden as a result of reduced MDSC accumulation within the primary tumor microenvironment. Using murine HNSCC cell lines LY2 and MOC2 in conjunction with Mif+/+, Mif-/- and myeloid specific MIF knockout mice (m-Mif-/-), our group has revealed an anti-immunosuppressive role for host-derived MIF through its interaction with MDSC, with tumor derived MIF being immunosuppressive in nature. After injection of the tumor cell lines into the buccal mucosa of age matched mice, we monitored tumor development until terminal sacrifice. At this point, we extracted tumor cells for flow cytometric analysis and isolated tumor associated Gr-1+ MDSC. Extracted MDSC were co-cultured with naïve T cells fluorescently labeled with CFSE, to enable quantification of suppression of T cell proliferation. Furthermore, we extracted isolated MDSC RNA for analysis by RT-qPCR. Unexpectedly, our initial finding ran contrary to our hypothesis, as we found that Mif-/- mice had a mean tumor burden of 108.3 mg compared to Mif+/+ mice who presented with a mean tumor of only 64 mg (p = 0.00624). Flow cytometry revealed increased CD11b+ F4/80+ macrophages in mice expressing MIF. Interestingly, a smaller proportion of these cells were identified as MDSC in Mif+/+ and m-Mif-/- compared to Mif-/-, determined by expression of MDSC markers Ly6G and Ly6C. Our group found that both Mif+/+ and m-Mif-/- demonstrated reduced suppression of T cell proliferation compared to Mif-/-. Gene expression analysis revealed in Mif-/- MDSC upregulation of immunosuppressive genes Arg1, Ptgs2, S100a8 and S100a9. Seeing that both Mif+/+ and m-Mif-/- demonstrate similar anti-tumor phenotypic and molecular responses to HNSCC, our group has concluded that in response to host derived, but not tumor derived, MIF, MDSC at the tumor site shift towards a more anti-tumor M1 phenotype and additionally that MIF derived from non-myeloid host sources within the tumor microenvironment are sufficient to stimulate this response.

The Endocannabinoid Anandamide Attenuates Acute Respiratory Distress Syndrome by Downregulating miRNA that Target Inflammatory Pathways.

Acute respiratory distress syndrome (ARDS) is defined as a type of respiratory failure that is caused by a variety of insults such as pneumonia, sepsis, trauma and certain viral infections. In this study, we investigated the effect of an endocannabinoid, anandamide (AEA), on ARDS induced in the mouse by Staphylococcus Enterotoxin B (SEB). Administration of a single intranasal dose of SEB in mice and treated with exogenous AEA at a dose of 40 mg/kg body weight led to the amelioration of ARDS in mice. Clinically, plethysmography results indicated that there was an improvement in lung function after AEA treatment accompanied by a decrease of inflammatory cell infiltrate. There was also a significant decrease in pro-inflammatory cytokines IL-2, TNF-α, and IFN-γ, and immune cells including CD4+ T cells, CD8+ T cells, Vβ8+ T cells, and NK+ T cells in the lungs. Concurrently, an increase in anti-inflammatory phenotypes such as CD11b + Gr1+ Myeloid-derived Suppressor Cells (MDSCs), CD4 + FOXP3 + Tregs, and CD4+IL10 + cells was observed in the lungs. Microarray data showed that AEA treatment in ARDS mice significantly altered numerous miRNA including downregulation of miRNA-23a-3p, which caused an upregulation of arginase (ARG1), which encodes for arginase, a marker for MDSCs, as well as TGF-β2, which induces Tregs. AEA also caused down-regulation of miRNA-34a-5p which led to induction of FoxP3, a master regulator of Tregs. Transfection of T cells using miRNA-23a-3p or miRNA-34a-5p mimics and inhibitors confirmed that these miRNAs targeted ARG1, TGFβ2 and FoxP3. In conclusion, the data obtained from this study suggests that endocannabinoids such as AEA can attenuate ARDS induced by SEB by suppressing inflammation through down-regulation of key miRNA that regulate immunosuppressive pathways involving the induction of MDSCs and Tregs.

IMMU-06. DISCERNING THE DIFFERENT BONE MARROW IMMUNE LANDSCAPES

Abstract Roughly 400,000 people have bone metastases in the U.S. with the vast majority of these occurring in the spine. The etiology of bone metastasis still remains to be fully elucidated. This study explored the differences in immune landscape between long bone and spine that may contribute to higher rates of bone metastasis to the spine. Spines and femurs from male C57BL6/J mice (N=10) were processed for flow cytometry and immunophenotyping using Mass Cytometry by Time-Of-Flight (CyTOF). The cells were analyzed with CyTOF using a 33-surface protein marker mouse antibody panel. Spines (N=3) and femurs (N=2) from patients were analyzed with CyTOF using the Maxpar Complete Human T cell Immuno-Oncology Panel Set. There are global differences in the immune cell composition between the long bone and spine microenvironment. Flow cytometry revealed slight increases in the CD45+ and Cd11b+ cell populations in the bone marrow of murine spines compared to murine long bone, which are markers for myeloid-derived suppressor cells (MDSCs). Using CyTOF, significant differences in the immune cell landscape between long bone and spine were observed. In the murine long bone, an increase in monocytes/macrophages, myeloid progenitors, granulocytic MDSCs, granulocytes, and mast cells was observed compared to the spine. In the murine spine an elevation of CD8a+ DC cells, classical monocytes, MDSCs, pDCs, memory T helper cells, and NK T cells was seen. Evaluation of human long bone and spine revealed similar trends with a predominance of myeloid progenitor cells and monocytes in the human vertebra compared to the human long bone marrow. Significant differences in the immune microenvironment exist between the spine and long bone marrow in both murine and human samples. This is the first report of significant differences in immune cell populations between different skeletal locations. However, the functional significance of these differences has yet to be determined.