M2 Tumor-associated Macrophages Research Articles

Construction of Hierarchically Biomimetic Iron Oxide Nanosystems for Macrophage Repolarization-Promoted Immune Checkpoint Blockade of Cancer Immunotherapy.

Cancer immunotherapy is developing as the mainstream strategy for treatment of cancer. However, the interaction between the programmed cell death protein-1 (PD-1) and the programmed death ligand 1 (PD-L1) restricts T cell proliferation, resulting in the immune escape of tumor cells. Recently, immune checkpoint inhibitor therapy has achieved clinical success in tumor treatment through blocking the PD-1/PD-L1 checkpoint pathway. However, the presence of M2 tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) will inhibit antitumor immune responses and facilitate tumor growth, which can weaken the effectiveness of immune checkpoint inhibitor therapy. The repolarization of M2 TAMs into M1 TAMs can induce the immune response to secrete proinflammatory factors and active T cells to attack tumor cells. Herein, hollow iron oxide (Fe3O4) nanoparticles (NPs) were prepared for reprogramming M2 TAMs into M1 TAMs. BMS-202, a small-molecule PD-1/PD-L1 inhibitor that has a lower price, higher stability, lower immunogenicity, and higher tumor penetration ability compared with antibodies, was loaded together with pH-sensitive NaHCO3 inside hollow Fe3O4 NPs, followed by wrapping with macrophage membranes. The formed biomimetic FBN@M could produce gaseous carbon dioxide (CO2) from NaHCO3 in response to the acidic TME, breaking up the macrophage membranes to release BMS-202. A series of in vitro and in vivo assessments revealed that FBN@M could reprogram M2 TAMs into M1 TAMs and block the PD-1/PD-L1 pathway, which eventually induced T cell activation and the secretion of TNF-α and IFN-γ to kill the tumor cells. FBN@M has shown a significant immunotherapeutic efficacy for tumor treatment.

Reactive oxygen species/glutathione dual sensitive nanoparticles with encapsulation of miR155 and curcumin for synergized cancer immunotherapy

Considerable attention has been directed towards exploring the potential efficacy of miR-155 in the realm of cancer immunotherapy. Elevated levels of miR-155 in dendritic cells (DCs) have been shown to enhance their maturation, migration, cytokine secretion, and their ability to promote T cell activation. In addition, overexpression of mir155 in M2 macrophages boost the polarization towards the M1 phenotype. Conversely, miR-155 has the propensity to induce the accumulation of immunosuppressive cells like regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor tissue. To account for this discrepancy, it is imperative to get help from a drug that could deal with immunosuppressive effect. Curcumin (CUR) exhibits the capacity to prompt Tregs converse into T helper 1 cells, fostering the polarization of M2 tumor-associated macrophage towards the M1 phenotype, and impeding the recruitment and aggregation of MDSCs within the tumor microenvironment. Nonetheless, CUR is known to exert an immunosuppressive impact on DCs by hindering the expression of maturation markers, cytokines, and chemokines, thereby prevent DCs response to immunostimulatory agents. Hence, a reactive oxygen species/glutathione dual responsive drug conveyance platform (CUR/miR155@DssD-Hb NPs) was devised to co-deliver CUR and miR155, with the aim of exploring their synergistic potential in bolstering a sustained and robust anti-tumor immune response. In vitro and in vivo results have suggested that CUR/miR155@DssD-Hb NPs can effectively inhibit the viability of 4T1 and B16F10 tumor cells, trigger the release of damage associated molecular patterns, stimulate DCs maturation, subsequent activation of CD8+ T cells, diminish immunosuppressive cell populations (MDSCs, Tregs, M2 TAMs and exhausted T cells), promote the formation of long-term immunity and lessen the formation of metastatic nodules in the lungs. In summary, the co-delivery system integrating CUR and miR155 (CUR/miR155@DssD-Hb NPs) demonstrates promise as a promising strategy for the immunotherapy of melanoma and triple negative breast cancer.Graphical abstract

Hypoxia exosome derived CEACAM5 promotes tumor-associated macrophages M2 polarization to accelerate pancreatic neuroendocrine tumors metastasis via MMP9.

Exosomes play significant roles in the communications between tumor cells and tumor microenvironment. However, the specific mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic neuroendocrine tumors (pNETs) are not well understood. This study aims to investigate these mechanisms and made several important discoveries. We found that hypoxic exosomes derived from pNETs cells can activate tumor-associated macrophages (TAM) to the M2 phenotype, in turn, the M2-polarized TAM, facilitate the migration and invasion of pNETs cells. Further investigation revealed that CEACAM5, a protein highly expressed in hypoxic pNETs cells, is enriched in hypoxic pNETs cell-derived exosomes. Hypoxic exosomal CEACAM5 was observed to induce M2 polarization of TAM through activation of the MAPK signaling pathway. Coculturing pNETs cells with TAM or treated with hypoxic exosomes enhanced the metastatic capacity of pNETs cells. In conclusion, these findings suggest that pNETs cells generate CEACAM5-rich exosomes in a hypoxic microenvironment, which in turn polarize TAM promote malignant invasion of pNETs cells. Targeting exosomal CEACAM5 could potentially serve as a diagnostic and therapeutic strategy for pNETs.

Integrated analysis of tumor-associated macrophages and M2 macrophages in CRC: unraveling molecular heterogeneity and developing a novel risk signature.

Emerging investigations have increasingly highlighted the critical role of tumor-associated macrophages (TAMs) and M2 macrophages in cancer development, progression, and metastasis, marking them as potential targets in various cancer types. The main objective of this research is to discover new biomarkers associated with TAM-M2 macrophages in colorectal cancer (CRC) and to dissect the molecular heterogeneity of CRC by combining single-cell RNA sequencing and bulk RNA-seq data. By utilizing weighted gene co-expression network analysis (WGCNA), we acquired TAM-M2-associated genes by intersecting TAM marker genes obtained from scRNA-seq data with module genes of M2 macrophages derived from bulk RNA-seq data. We employed least absolute shrinkage and selection operator (LASSO) Cox analysis to select predictive biomarkers from these TAM-M2-related genes. Quantitative polymerase chain reaction (qPCR) was employed to validate the mRNA expression levels of the genes identified in the screening. This led to the development of the TAM-M2-related signature (TAMM2RS). We also conducted functional and immune landscape analyses of different risk groups. The combination of scRNA-seq and bulk RNA-seq analyses yielded 377 TAM-M2-related genes. DAPK1, NAGK, and TRAF1 emerged as key prognostic genes in CRC, which were identified through LASSO Cox analysis. Utilizing these genes, we constructed and validated the TAMM2RS, demonstrating its effectiveness in predicting survival in CRC patients. Our research offers a thorough investigation into the molecular mechanisms associated with TAM-M2 macrophages in CRC and unveils potential therapeutic targets, offering new insights for treatment strategies in colorectal cancer.

Bioconjugated Antibody-Trojan Immune Converter Enhance Cancer Immunotherapy with Minimized Toxicity by Programmed Two-Step Immunomodulation of Myeloid Cells.

Current immune checkpoint blockade therapy (ICBT) predominantly targets T cells to harness the antitumor effects of adaptive immune system. However, the effectiveness of ICBT is reduced by immunosuppressive innate myeloid cells in tumor microenvironments (TMEs). Toll-like receptor 7/8 agonists (TLR7/8a) are often used to address this problem because they can reprogram myeloid-derived suppressor cells (MDSCs) and tumor-associated M2 macrophages, and boost dendritic cell (DC)-based T-cell generation; however, the systemic toxicity of TLR7/8a limits its clinical translation. Here, to address this limitation and utilize the effectiveness of TLR7/8a, this work suggests a programmed two-step activation strategy via Antibody-Trojan Immune Converter Conjugates (ATICC) that specifically targets myeloid cells by anti-SIRPα followed by reactivation of transiently inactivated Trojan TLR7/8a after antibody-mediated endocytosis. ATICC blocks the CD47-SIRPα ("don't eat me" signal), enhances phagocytosis, reprograms M2 macrophages and MDSCs,and increases cross-presentation by DCs, resulting in antigen-specific CD8+ T-cell generation in tumor-draining lymph nodes and TME while minimizing systemic toxicity. The local or systemic administration of ATICC improves ICBT responsiveness through reprogramming of the immunosuppressive TME, increased infiltration of antigen-specific CD8+ T cells,and antibody-dependent cellular phagocytosis. These results highlight the programmed and target immunomodulation via ATICC could enhance cancer immunotherapy with minimized systemic toxicities.

Codonopsis pilosula-derived glycopeptide dCP1 promotes the polarization of tumor-associated macrophage from M2-like to M1 phenotype

The majority of the immune cell population in the tumor microenvironment (TME) consists of tumor-associated macrophages (TAM), which are the main players in coordinating tumor-associated inflammation. TAM has a high plasticity and is divided into two main phenotypes, pro-inflammatory M1 type and anti-inflammatory M2 type, with tumor-suppressive and tumor-promoting functions, respectively. Considering the beneficial effects of M1 macrophages for anti-tumor and the high plasticity of macrophages, the conversion of M2 TAM to M1 TAM is feasible and positive for tumor treatment. This study sought to evaluate whether the glycopeptide derived from simulated digested Codonopsis pilosula extracts could regulate the polarization of M2-like TAM toward the M1 phenotype and the potential regulatory mechanisms. The results showed that after glycopeptide dCP1 treatment, the mRNA relative expression levels of some M2 phenotype marker genes in M2-like TAM in simulated TME were reduced, and the relative expression levels of M1 phenotype marker genes and inflammatory factor genes were increased. Analysis of RNA-Seq of M2-like TAM after glycopeptide dCP1 intervention showed that the gene sets such as glycolysis, which is associated with macrophage polarization in the M1 phenotype, were significantly up-regulated, whereas those of gene sets such as IL-6-JAK-STAT3 pathway, which is associated with polarization in the M2 phenotype, were significantly down-regulated. Moreover, PCA analysis and Pearson's correlation also indicated that M2-like TAM polarized toward the M1 phenotype at the transcriptional level after treatment with the glycopeptide dCP1. Lipid metabolomics was used to further explore the efficacy of the glycopeptide dCP1 in regulating the polarization of M2-like TAM to the M1 phenotype. It was found that the lipid metabolite profiles in dCP1-treated M2-like TAM showed M1 phenotype macrophage lipid metabolism profiles compared with blank M2-like TAM. Analysis of the key differential lipid metabolites revealed that the interconversion between phosphatidylcholine (PC) and diacylglycerol (DG) metabolites may be the central reaction of the glycopeptide dCP1 in regulating the conversion of M2-like TAM to the M1 phenotype. The above results suggest that the glycopeptide dCP1 has the efficacy to regulate the polarization of M2-like TAM to M1 phenotype in simulated TME.

Abstract PO3-26-08: Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination

Abstract More than half of patients with stage IV breast cancer will eventually develop liver metastasis. Due to their nature, liver metastases are hard to treat, and the five-year survival rate for stage IV breast cancer is very low (~11%). In the recent years, vital roles macrophages play in cancer progression and immune evasion have become increasingly clear, making them an appealing target for therapeutic intervention. As one of the most abundant cell populations recruited to the tumor microenvironment, tumor-associated macrophages (TAM) are typically immunosuppressive Arg1+ M2-like macrophages, which support cancer growth, metastasis progression, and drug resistance. Numerous studies, including from our laboratory, have shown that reversal of the macrophage phenotype to the classical inflammatory CD80+ M1-like macrophages is very promising for cancer eradication. In this study, we used CRISPR loaded into lipid nanoparticles (CRISPR-LNP) to modulate macrophage polarization in breast cancer metastasis in vivo. We hypothesize that CRISPR-mediated gene editing through lipid nanoparticle (LNP) delivery of CRISPR enzyme and target gRNA (CRISPR-LNP) is a highly effective and safe approach. Using our CRISPR-LNP platform, we screened multiple genes with significant roles in macrophage polarization and found that targeting the RICTOR gene alone enabled reprograming of pro-tumorigenic M2 TAMs to anti-tumorigenic M1 phenotype in vitro and in vivo. We administered the CRISPR-LNP targeting RICTOR (CRISPR-RIC-LNP) on a syngeneic liver metastasis mouse model via intravenous injection. Single dose studies were conducted to determine biodistribution, treatment efficacy, and shift in immune landscape of the tumor after treatment via immunofluorescence staining, Imaging Mass Cytometry, and single cell RNA sequencing. A spatial map was built using digital pathology analysis for investigation of the interactions amongst immune and tumor cells within the tumor immune microenvironment. In 4T1 breast cancer liver metastatic model, we observed that CRISPR-RIC-LNP treatment: (i) accumulated in the liver metastases lesions in vivo 24 hours after injection. ~0.5% of total injected dose, which corresponds to ~75% of fluorescence from the whole body at 24h time point was confined only in the metastatic regions; (ii) led to modest increase of macrophage number, and (iii) significant shift of macrophage phenotype from immunosuppressive, pro-tumorigenic Arg1+, CD206+ macrophages to inflammatory, anti-tumor CD80+ phenotype (from ~5 to ~9% of total cell in the tumor lesions) 24 hours after injection; (iv) reduced the cancer cell number by almost 80%; and (v) dramatically reduced exhausted T-cell and regulatory T-cell population, as determined by single cell RNA sequencing analysis. We plan to validate our preliminary finding with flow cytometry analysis of metastatic lesions, and evalute the efficacy of CRISPR-RIC-LNP as single and in combinatorial therapies in survival studies. These data showed that reprograming macrophages from cancer-supporting to cancer-eliminating phenotype is a promising tool to suppress cancer cell growth and increase the presence of cytotoxic CD8+ T-cells. We propose that this approach can be efficiently used as single therapy or as combination with immune checkpoint inhibitors to obtain synergistic effects and efficiently eradicate cancer cells. We will assess the safety, optimize the accumulation in TME, and devise methods to limit exposure to healthy cells. In the future, we also aim to expand scope of our candidate therapy as therapeutics for other types of breast cancer metastases. Citation Format: Fransisca Leonard, Tyler Galbraith, Nourhan Abdelfattah, Arturas Ziemys, Chihiro Hashimoto, Harlan Cook, Yitian Xu, Shu-Hsia Chen, Roberto Rosato, Kyuson Yun. Genetic modification of tumor associated macrophages using CRISPR lipid nanoparticle platform altered tumor microenvironment and induced cancer cell elimination [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-08.

The Biological Role of Macrophage in Lung and Its Implications in Lung Cancer Immunotherapy.

The lungs are the largest surface of the body and the most important organ in the respiratory system, which are constantly exposed to the external environment. Tissue Resident Macrophages in lung constitutes the important defense against external pathogens. Macrophages connects the innate and adaptive immune system, and also plays important roles in carcinogenesis and cancer immunotherapy. Lung cancer is the leading cause of cancer-related death worldwide, with an overall five-year survival rate of only 21%. Macrophages that infiltrate or aggregate in lung tumor microenvironment are defined as tumor-associated macrophages (TAMs). TAMs are the main components of immune cells in the lung tumor microenvironment. The differentiation and maturation process of TAMs can be roughly divided into two different types: classical activation pathway produces M1 tumor-associated macrophages, and bypass activation pathway produces M2 tumor-associated macrophages. Studies have found that TAMs are related to tumor invasion, metastasis, and treatment resistance, and show potential as a new target for tumor immunotherapy. Therefore, the biological function of macrophages in lung and the role of TAMs in the occurrence, development, and treatment of lung cancer are discussed in this paper.

S100P facilitates LUAD progression via PKA/c-Jun-mediated tumor-associated macrophage recruitment and polarization

S100P, a member of the S100 calcium-binding protein family, is closely associated with abnormal proliferation, invasion, and metastasis of various cancers. However, its role in the lung adenocarcinoma (LUAD) tumor microenvironment (TME) remains unclear. In this study, we observed specific expression of S100P on tumor cells in LUAD patients through tissue immunofluorescence analysis. Furthermore, this expression was strongly correlated with the recruitment and polarization of tumor-associated macrophages (TAMs). Bioinformatics analysis revealed that high S100P expression is associated with poorer overall survival in LUAD patients. Subsequently, a subcutaneous mouse model demonstrated that S100P promotes recruitment and polarization of TAMs towards the M2 type. Finally, in vitro studies on LUAD cells revealed that S100P enhances the secretion of chemokines and polarizing factors by activating the PKA/c-Jun pathway, which is implicated in TAM recruitment and polarization towards the M2 phenotype. Moreover, inhibition of c-Jun expression impedes the ability of TAMs to infiltrate and polarize towards the M2 phenotype. In conclusion, our study demonstrates that S100P facilitates LUAD cells growth by recruiting M2 TAMs through PKA/c-Jun signaling, resulting in the production of various cytokines. Considering these findings, S100P holds promise as an important diagnostic marker and potential therapeutic target for LUAD.

A Tandem-Locked Chemiluminescent Probe for Imaging of Tumor-Associated Macrophage Polarization.

Tumor-associated macrophages (TAMs) play a role in both pro- and anti-tumor functions; and the targeted polarization from M2 to M1 TAMs has become an effective therapy option. Although detection of M1 TAMs is imperative to assess cancer immunotherapeutic efficacy, existing optical probes suffer from shallow tissue penetration depth and poor specificity toward M1 TAMs. Herein, we report a tandem-locked NIR chemiluminescent (CL) probe for specific detection of M1 TAMs. Through a combined molecular engineering approach via both atomic alternation and introduction of electron-withdrawing groups, near-infrared (NIR) chemiluminophores are screened out to possess record-long emission (over 800 nm), record-high CL quantum yield (2.7 % einstein/mol), and prolonged half-life (7.7 h). Based on an ideal chemiluminophore, the tandem-locked probe (DPDGN) is developed to only activate CL signal in the presence of both tumour (γ-glutamyl transpeptidase) and M1 macrophage biomarkers (nitric oxide). Such a tandem-lock design ensures its high specificity towards M1 macrophages in the tumor microenvironment over those in normal tissues or peripheral blood. Thus, DPDGN permits noninvasive imaging and tracking of M1 TAM in the tumor of living mice during R837 treatment, showing a good correlation with ex vivo methods. This study not only reports a new molecular approach towards highly efficient chemiluminophores but also reveals the first tandem-locked CL probes for enhanced imaging specificity.

SPP1 represents a therapeutic target that promotes the progression of oesophageal squamous cell carcinoma by driving M2 macrophage infiltration.

Tumour-associated macrophages (TAMs) are an important component of the tumour microenvironment (TME). However, the crosstalk between oesophageal squamous cell carcinoma (ESCC) cells and TAMs remains largely unexplored. Clinical samples and the TCGA database were used to evaluate the relevance of SPP1 and TAM infiltration in ESCC. Mouse models were constructed to investigate the roles of macrophages educated by SPP1 in ESCC. Macrophage phenotypes were determined using qRT‒PCR and immunohistochemical staining. RNA sequencing was performed to elucidate the mechanism. Increasing expression of SPP1 correlated with M2-like TAM accumulation in ESCC, and they both predicted poor prognosis in the ESCC cohort. Knockdown of SPP1 significantly inhibited the infiltration of M2 TAMs in xenograft tumours. In vivo mouse model experiments showed that SPP1-mediated education of macrophages plays an essential role in the progression of ESCC. Mechanistically, SPP1 recruited macrophages and promoted M2 polarisation via CD44/PI3K/AKT signalling activation and then induced VEGFA and IL6 secretion to sustain ESCC progression. Finally, blockade of SPP1 with RNA aptamer significantly inhibited tumour growth and M2 TAM infiltration in xenograft mouse models. This study highlights SPP1-mediated crosstalk between ESCC cells and TAMs in ESCC. SPP1 could serve as a potential target in ESCC therapy.

The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

Abstract 731: pHLIP targeted intracellular delivery of calicheamicin

Abstract Calicheamicin is a potent, cell-cycle independent enediyne antibiotic that binds and cleaves DNA. Toxicity has led to its approved use in a targeted form as antibody-drug conjugates (ADC) for the treatment of liquid tumors. Mylotarg, one of the first such ADCs, was voluntarily withdrawn from the market due to safety concerns. However, Mylotarg was recently reapproved for acute myeloid leukemia based on new data that demonstrated efficacy with an acceptable safety profile. Besponsa, a related ADC, was approved for acute lymphocytic leukemia in 2017. In both agents, Mylotag and Besponsa, calicheamicin is conjugated to antibodies via an acid-labile hydrazone linker. We used a reduced calicheamicin to conjugate it to a single cysteine residue at the membrane-inserting end of a pH Low Insertion Peptide (pHLIP) via a disulfide bond to generate a linkerless cytoplasmic release. The cytoplasmic reduction of the disulfide releases the calicheamicin, which leads to its activation, DNA binding, and strand scission. pHLIP is a tumor-targeting agent now in clinical trials with imaging and therapeutic payloads. We studied the interaction of pHLIP-calicheamicin with liposomal and cellular membranes and demonstrated that the agent exhibits cytotoxic activity both in highly proliferative cancer cells and in non-proliferative immune cells, such as polarized M2 macrophages. In mice, treatment led to the growth inhibition of immuno-suppressive CT26 tumors with no signs of toxicity. Biodistribution studies confirmed tumor targeting with no accumulation of the agent in organs and tissues. The fluorescent version of the agent was observed within the tumor mass and tumor-stroma interface, where CD206+ M2 tumor-associated macrophages (M2-TAMs) reside. Treatment of tumors led to the depletion of M2-TAMs within the tumor core. Thus, pHLIP-calicheamicin could be developed into an effective therapeutic for the treatment of solid tumors with abundant immuno-suppressive M2-TAMs. Citation Format: Michael DuPont, Craig Klumpp, Marissa Iraca, Dana Allababidi, Hannah Visca, Donald Engelman, Oleg Andreev, Anna Moshnikova, Yana Reshetnyak. pHLIP targeted intracellular delivery of calicheamicin [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 731.

1-Ethoxycarbonyl-beta-carboline inhibits the M2 polarization of tumor-associated macrophages: A study based on network pharmacology and molecular docking analyses

AimThrough network pharmacology, molecular docking, molecular dynamics in combination with experimentation, we explored the mechanism whereby 1-ethoxycarbonyl-beta-carboline (EBC) regulates the M2 polarization of tumor-associated macrophages. MethodsNetwork pharmacology was adopted for analyzing the targets and signaling pathways related to the M2 polarization of EBC-macrophages, small molecular-protein docking was employed to analyze the possibility of EBC bonding to related protein, and molecular dynamics was introduced to analyze the binding energy between EBC and HDAC2. The M2 polarization of RAW264.7 macrophages was triggered in vitro by IL-4. After EBC intervention, the expressions of M1/M2 polarization-related cytokines were detected, and the mechanism of EBC action was explored in HDAC2-knockout RAW264.7 macrophages. A tumor-bearing mouse model was established in vitro to find the impact of EBC on tumor-associated M2 macrophages. ResultsAs revealed by the network pharmacology, molecular docking and molecular dynamics analyses, EBC was associated with 51 proteins, including HDAC2, NF-κB and HDAC4. Molecular docking and dynamics analyses suggested that HDAC2 was the main target of EBC. In vitro experiments discovered that EBC could hinder the M2 polarization of RAW264.7 macrophages, which exerted insignificant effect on the M1-associated cytokines, but could lower the levels of M2-associated cytokines. After knocking out HDAC2, EBC could not further inhibit the M2 polarization of macrophages. At the mouse level, EBC could hinder the tumor growth and the tissue levels of M2 macrophages, whose effect was associated with HDAC2. ConclusionOur study combining multiple methods finds that EBC inhibits the HDAC2-mediated M2 polarization of macrophages, thereby playing an anti-tumor role.

Abstract 5270: Androgen receptor blockade in macrophages primes NLRP3 inflammasome-mediated phagocytosis and tumor clearance in advanced prostate cancer

Abstract Immune-based therapies induce durable remissions in subsets of patients across multiple malignancies. However, there is limited efficacy of immunotherapy in metastatic castrate-resistant prostate cancer (mCRPC), manifested by an enrichment of immunosuppressive (M2) tumor-associated macrophages (TAM) in the tumor immune microenvironment (TME). Therefore, therapeutic strategies to overcome TAM-mediated immunosuppression are critically needed in mCRPC. Our single-cell RNA sequencing analysis in human tumors revealed that NLR family pyrin domain containing 3 (NLRP3), an innate immune sensing protein, is differentially expressed in TAM from metastatic PC patients treated with standard-of-care androgen deprivation therapy (ADT), relative to other tumor types and untreated primary PC. Furthermore, bulk RNA sequencing analysis in human mCRPC samples revealed an inverse relationship between NLRP3 expression and AR activity, with high NLRP3 expression associated with an M1 signature and a favorable clinical response to ICI in mCRPC. Based on these findings, we hypothesized that androgen axis blockade could enhance NLRP3 expression and potentiate innate immune tumor control in advanced PC. Critically, we discovered that blockade of TAM-intrinsic androgen receptor (AR) activity enhanced NLRP3 expression, but not inflammasome activity in the immunosuppressive (M2) TAM. In contrast, anti-tumor (M1) TAM exhibited high de novo NLRP3 expression, regardless of AR activity. The combination of AR blockade and NLRP3 agonism significantly enhanced phagocytosis of cancer cells by M2 TAM, whereas NLRP3 agonist treatment alone was sufficient to induce phagocytosis in M1 TAM. Following AR blockade/NLRP3 agonist combination treatment, all TAM acquired a distinct phenotype with high PD-L1 and CD86 expression, indicative of phagocytic TAM. Critically, NLRP3 agonism in combination with ADT resulted in significant tumor control in an aggressive c-myc driven advanced PC model, with 55% of mice achieving complete tumor clearance, which was abrogated by concurrent clodronate treatment (which systemically depletes phagocytic macrophages), thus demonstrating TAM-mediated phagocytosis enhancement as a major driver of the observed anti-tumor response. Collectively, our results identify NLRP3 as an AR-regulated “macrophage phagocytic checkpoint” that can be inducibly expressed and activated in TAM following ADT and NLRP3 agonist treatment, respectively, the combination resulting in TAM-mediated phagocytosis and tumor control. Citation Format: Kiranj Chaudagar, Srikrishnan Rameshbabu, Shenglin Mei, Taghreed Hirz, Ya-Mei Hu, Anna Argulian, Brian Labadie, Kunal Desai, Sam Grimaldo, Doga Kahramangil, Rishikesh Nair, Sabina DSouza, Dylan Zhou, Raymond Chen, Jordan Shafran, Mayme Loyd, Zheng Xia, David Sykes, Amy Moran, Akash Patnaik. Androgen receptor blockade in macrophages primes NLRP3 inflammasome-mediated phagocytosis and tumor clearance in advanced prostate cancer [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 5270.

Abstract 5283: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients and is characterized by a dense tumor microenvironment (TME) which impedes tumor entry of immune cells and delivery of therapeutics. Immunosuppressive myeloid populations of PDAC, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), are known to not only support PDAC development but also impede the anti-tumor immune response elicited by therapeutic agents. To improve prognosis while maintaining tolerability, this study aims to target polyamine metabolism that is upregulated in the immunosuppressive myeloid populations and cancer cells to disrupt their contribution to PDAC progression and the immunosuppressive TME. We previously demonstrated that polyamine blockade therapy (PBT), via blocking biosynthesis with difluoromethylornithine (DFMO) and transport with a novel inhibitor (Trimer44NMe), elicits an anti-tumor response and improves median survival in PDAC engrafted immunocompetent mice. In the current study, PBT was found to lead to a selective reduction of the dominant MDSC subtype in PDAC, polymorphonuclear (PMN) MDSCs. Upon co-culturing of MDSCs with CD8+ T cells, PBT dampened MDSC-mediated suppression of T cell activity. These findings were corroborated by tracking Arginase 1, a driver of MDSC-mediated immunosuppression, which exhibited a significant decrease in response to PBT at both the activity and gene expression level. Ex-vivo generated M2 macrophages and TAMs treated with PBT also demonstrated a shift from the M2 pro-tumor phenotype towards the anti-tumor M1 macrophage phenotype. In summary, these findings support that PBT is effective at reducing myeloid-mediated immunosuppression through subtype specific modulation and supports the use of PBT for treatment of PDAC in combination with other immunomodulatory therapeutics. Citation Format: Joseph A. Goode. Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer [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 5283.

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [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 1356.

APOC1 is a prognostic biomarker associated with M2 macrophages in ovarian cancer

BackgroundRecent studies have demonstrated that APOC1 is associated with cancer progression, exerting cancer-promoting and immune infiltration-promoting effects. Nevertheless, there is currently no report on the presence of APOC1 in ovarian cancer (OV).MethodIn this study, we conducted data analysis using the GEO and TCGA databases. We conducted a thorough bioinformatics analysis to investigate the function of APOC1 in OV, utilizing various platforms including cBioPortal, STRING, GeneMANIA, LinkedOmics, GSCALite, TIMER, and CellMarker. Additionally, we performed immunohistochemical staining on tissue microarrays and conducted in vitro cellular assays to validate our findings.ResultOur findings reveal that APOC1 expression is significantly upregulated in OV compared to normal tissues. Importantly, patients with high APOC1 levels show a significantly poorer prognosis. Furthermore, our study demonstrated that APOC1 exerted a crucial function in promoting the capacity of ovarian cancer cells to proliferate, migrate, and invade. Additionally, we have identified that genes co-expressed with APOC1 are primarily associated with adaptive immune responses. Notably, the levels of APOC1 in OV exhibit a correlation with the presence of M2 Tumor-associated Macrophages (TAMs).ConclusionAPOC1 emerges as a promising prognostic biomarker for OV and exhibits a significant association with M2 TAMs in OV.

Unveiling CXCR2 as a promising therapeutic target in renal cell carcinoma: exploring the immunotherapeutic paradigm shift through its inhibition by RCT001

BackgroundIn clear cell renal cell carcinoma (ccRCC), first-line treatment combines nivolumab (anti-PD-1) and ipilimumab (anti-CTLA4), yielding long-term remissions but with only a 40% success rate. Our study explored the potential of enhancing ccRCC treatment by concurrently using CXCR2 inhibitors alongside immunotherapies.MethodsWe analyzed ELR + CXCL levels and their correlation with patient survival during immunotherapy. RCT001, a unique CXCR2 inhibitor, was examined for its mechanism of action, particularly its effects on human primary macrophages. We tested the synergistic impact of RCT001 in combination with immunotherapies in both mouse models of ccRCC and human ccRCC in the presence of human PBMC.ResutsElevated ELR + CXCL cytokine levels were found to correlate with reduced overall survival during immunotherapy. RCT001, our optimized compound, acted as an inverse agonist, effectively inhibiting angiogenesis and reducing viability of primary ccRCC cells. It redirected M2-like macrophages without affecting M1-like macrophage polarization directed against the tumor. In mouse models, RCT001 enhanced the efficacy of anti-CTLA4 + anti-PD1 by inhibiting tumor-associated M2 macrophages and tumor-associated neutrophils. It also impacted the activation of CD4 T lymphocytes, reducing immune-tolerant lymphocytes while increasing activated natural killer and dendritic cells. Similar effectiveness was observed in human RCC tumors when RCT001 was combined with anti-PD-1 treatment.ConclusionsRCT001, by inhibiting CXCR2 through its unique mechanism, effectively suppresses ccRCC cell proliferation, angiogenesis, and M2 macrophage polarization. This optimization potentiates the efficacy of immunotherapy and holds promise for significantly improving the survival prospects of metastatic ccRCC patients.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels the molecular feature of M2 macrophages of head and neck squamous cell carcinoma.

The connection between head and neck squamous cell carcinoma (HNSC) and M2 tumour-associated macrophages is not yet fully understood. We gathered gene expression profiles and clinical data from HNSC patients in the TCGA database. Using Consensus Clustering, we categorized these patients into M2 macrophage-related clusters. We developed a M2 macrophage-related signature (MRS) through statistical analyses. Additionally, we assessed gene expression in HNSC cells using single-cell sequencing data (GSE139324). We identified three distinct M2 macrophage-related clusters in HNSC, each with different prognostic outcomes and immune characteristics. Patients with different MRS profiles exhibited variations in immune infiltration, genetic mutations and prognosis. FCGR2A may play a role in creating an immunosuppressive tumour microenvironment and could potentially serve as a therapeutic target for HNSC. Our study demonstrated that M2 macrophage-related genes significantly impact the development and progression of HNSC. The M2 macrophage-related model offered a more comprehensive assessment of HNSC patient prognosis, genetic mutations and immune features. FCGR2A was implicated in immunosuppressive microenvironments and may hold promise for the development of novel immunotherapeutic strategies for HNSC.