Tumor-associated Macrophages Research Articles

A nanotheranostics with hypoxia-switchable fluorescence and photothermal effect for hypoxia imaging-guided immunosuppressive tumor microenvironment modulation

Modulating the immunosuppressive tumor immune microenvironment (TIME) is considered a promising strategy for cancer treatment. However, effectively modulating the immunosuppressive TIME within hypoxic zones remains a significant challenge. In this work, we developed a hypoxia-responsive amphiphilic drug carrier using boron-dipyrromethene (BODIPY) dye-modified chitosan (CsB), and then fabricated a hypoxia-targeted nanotheranostic system, named CsBPNs, through self-assembly of CsB and pexidartinib (5-((5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl), PLX3397), an immunotherapeutic drug targeting tumor-associated macrophages (TAMs), for synergistic photothermal/immunotherapy and hypoxia imaging. CsBPNs demonstrated uniform size, good stability, and hypoxia-switchable fluorescence and photothermal effects, enabling deep penetration and hypoxia imaging capacities in three-dimensional tumor cell spheres and tumor tissues. In vitro and in vivo experiments showed that CsBPNs under laser irradiation promoted TAMs repolarization, reversed the immunosuppressive TIME, and enhanced the therapeutic outcome of PLX3397 in solid tumors by facilitating deep delivery into hypoxic regions and synergistic photothermal therapy. This work provides a new strategy for detecting and modulating the immunosuppressive TIME in hypoxic zones, potentially enabling more precise and effective photo-immunotherapy in the future.

Blocking CX3CR1+ Tumor-associated Macrophages Enhances the Efficacy of Anti-PD-1 Therapy in Hepatocellular Carcinoma.

The efficacy of immune checkpoint inhibitors (ICI) in the treatment of hepatocellular carcinoma (HCC) remains limited, highlighting the need for further investigation into the underlying mechanisms. Accumulating evidence indicates that tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) are implicated in immune evasion and treatment resistance. This study aimed to explore the contribution of TAMs in the HCC TME. Our findings reveal the critical involvement of CX3C motif chemokine receptor 1 (CX3CR1)-positive TAMs in inducing T cell exhaustion through interleukin-27 (IL-27) secretion, providing valuable insights into the mechanisms underlying the suboptimal efficacy of anti-PD-1 therapy in HCC. Moreover, we identified prostaglandin E2 (PGE2), released by immune-attacked tumor cells, as a key regulator of CX3CR1+ TAM phenotype transition. To augment the therapeutic response to current anti-PD-1 therapy, we propose an innovative treatment strategy that incorporates targeting CX3CR1+ TAMs in addition to anti-PD-1 therapy. In conclusion, our study contributes to the understanding of TAMs' role in cancer immunotherapy and highlights potential clinical implications for HCC treatment. The combination of targeting CX3CR1+ TAMs with anti-PD-1 therapy holds promise for enhancing the efficacy of immunotherapeutic interventions in HCC patients.

Proteomic profiling of oleamide-mediated polarization in a primary human monocyte-derived tumor-associated macrophages (TAMs) model: a functional analysis.

Tumor-associated macrophages (TAMs) play a critical function in the development of tumors and are associated with protumor M2 phenotypes. Shifting TAMs towards antitumor M1 phenotypes holds promise for tumor immunotherapy. Oleamide, a primary fatty acid amide, has emerged as a potent anticancer and immunomodulatory compound. However, the regulatory effects of oleamide on TAM phenotypes remain unclear. We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to study the influence of oleamide on primary human monocyte-derived TAM phenotypes, and we investigated the protein expression profiles based on mass spectrometry to analyze the effect of oleamide on macrophage polarization. Moreover, the advantageous binding scores between oleamide and these target candidate proteins are examined using molecular docking. Our study revealed that oleamide effectively suppressed the M2-like TAM phenotype by reducing interleukin (IL)-10 production and downregulating M2-like markers, including vascular endothelial growth factor A (VEGFA), MYC proto-oncogene, bHLH transcription factor (c-Myc), and mannose receptor C-type 1 (CD206). Moreover, the conditioned medium derived from oleamide-treated TAMs induces apoptosis of MDA-MB-231 breast cancer cells. Proteomic analysis identified 20 candidate up- and down-regulation proteins targeted by oleamide, showing modulation activity associated with the promotion of the M1-like phenotype. Furthermore, molecular docking demonstrated favorable binding scores between oleamide and these candidate proteins. Collectively, our findings suggest that oleamide exerts a potent antitumor effect by promoting the antitumor M1-like TAM phenotype. These novel insights provide valuable resources for further investigations into oleamide and macrophage polarization which inhibit the progression of breast cancer, which may provide insight into immunotherapeutic approaches for cancer.

A pancreatic cancer organoid incorporating macrophages reveals the correlation between the diversity of tumor-associated macrophages and cancer cell survival

Pancreatic ductal adenocarcinoma (PDAC) is a progressive cancer with a poor prognosis. It contains a complex tumor microenvironment (TME) that includes various stromal cell types. Comprehending cellular communications within the TME is difficult due to a lack of research models that can recapitulate human PDAC-TME. Previously, we recapitulated, in part, the PDAC-TME containing a diversity of cancer-associated fibroblasts (CAFs) in vitro. This was done by establishing a PDAC organoid by co-culturing patient-derived cancer cells with human induced pluripotent stem cell (hiPSC)-derived mesenchymal and endothelial cells, which was designated the fused pancreatic cancer organoid (FPCO). We further incorporated macrophages derived from the THP-1 cell line, which are the source of tumor-associated macrophages (TAMs), a major TME component, into FPCO, which was designated M0-FPCO. Bulk RNA sequencing (RNAseq) analysis revealed that macrophages in M0-FPCO (FPCO-Mac) lost their pro-inflammatory features but acquired pro-angiogenic features. Consistently, the formation of an endothelial cell network was enhanced in M0-FPCO. Single-cell RNA-seq (scRNA-seq) analysis revealed that M0-FPCO contained five TAM subpopulations similar to the corresponding TAM in human PDAC tissue in the integrated analysis, including SPP1+-TAM, which has been correlated with tumor angiogenesis and cell proliferation. Focusing on PDAC cells, we found that they could survive longer within the organoid in the presence of TAM. Consistent with the prolonged proliferation and survival of PDAC cells, PDAC subclusters were characterized by proliferative features, such as increased M0-FPCO. Therefore, by establishing a PDAC organoid with macrophages, we recapitulated the diversity of TAMs and identified the role of TAM in endothelial network formation as well as in the modulation of PDAC cell properties. SignificancePDAC organoids, including macrophages using hiPSC, showed that PDAC-TAM has angiogenic features and contributes to PDAC cell survival.

Lipid-laden macrophages recycle myelin to feed glioblastoma.

Tumor-associated microglia and macrophages (TAMs) make up the largest immune cell population in the glioblastoma (GBM) tumor microenvironment (TME). Given the heterogeneity and plasticity of TAMs in the GBM TME, understanding the context-dependent cancer cell-TAM symbiotic interaction is crucial for understanding GBM biology and developing effective therapies. In a recent issue of Cell, Kloosterman and colleagues identified a subpopulation of GPNMBhigh lipid-laden microglia and macrophages (LLMs) in GBM. Mesenchymal-like (MES-like) GBM cells help to generate the LLM phenotype. Reciprocally, LLMs are epigenetically rewired to recycle myelin and transfer the lipid from myelin to cancer cells, fueling MES-like GBM progression in an LXR/ABCA1-dependent manner. Together, leveraging LLMs opens new therapeutic possibilities for rewiring the metabolism-mediated tumor-TAM interaction during GBM progression.

Emerging immunologic approaches as cancer anti-angiogenic therapies.

Targeting tumor angiogenesis, the formation of new blood vessels supporting cancer growth and spread, has been an intense focus for therapy development. However, benefits from anti-angiogenic drugs like bevacizumab have been limited by resistance stemming from activation of compensatory pathways. Recent immunotherapy advances have sparked interest in novel immunologic approaches that can induce more durable vascular pruning and overcome limitations of existing angiogenesis inhibitors. This review comprehensively examines these emerging strategies, including modulating tumor-associated macrophages, therapeutic cancer vaccines, engineered nanobodies and T cells, anti-angiogenic cytokines/chemokines, and immunomodulatory drugs like thalidomide analogs. For each approach, the molecular mechanisms, preclinical/clinical data, and potential advantages over conventional drugs are discussed. Innovative therapeutic platforms like nanoparticle delivery systems are explored. Moreover, the importance of combining agents with distinct mechanisms to prevent resistance is evaluated. As tumors hijack angiogenesis for growth, harnessing the immune system's specificity to disrupt this process represents a promising anti-cancer strategy covered by this review.

A Review on Single-Cell analysis of Macrophages in Cancer

Cancer is characterized by uncontrolled cell growth and spread into surrounding tissue. According to the North American Association of Central Cancer Registries (NAACCR) and the National Center for Health Statistics, there were 1,918,030 cancer cases and a total of 609,360 deaths estimated in the US in 2022. Although the direct cause of cancer is not fully understood, it is recognized that the immune system plays a major role in the development and progression of tumors. Investigating tumor-associated macrophages (TAMs) is crucial for understanding the immunosuppressive role that they may play in the tumor microenvironment, which makes them a target for immunotherapies to combat cancer. However, angiogenesis, tumor initiation and invasion promoting signals prevent gaining deeper insight into the intricate molecular mechanisms involved in TAMs and the tumor microenvironment. While researchers in the past have used population samples to analyze cells, recent efforts have incorporated single-cell technologies to study the complex mixture of cells in a tumor, monitor the effects of immunotherapies and more. With the revolutionary advent of the first single-cell RNA sequencing (scRNA-seq), researchers have since been able to gain insight into single-cell data with unprecedented accuracy and develop new immunotherapies through different single-cell technologies, such as polymethylsiloxane (PDMS) and Multiplexed Ion Beam Imaging (MIBI) multiplexing. In this review, we underline the benefits of adopting single-cell technologies to identify distinct cell types in complex environments (i.e., tumors) and highlight the potential application of these technologies along with various multiplexing techniques to immunotherapies.

Inhalation of Bioorthogonal Gene‐Editable Spiky‐Pollen Reprograms Tumor‐Associated Macrophages for Lung Cancer Immunotherapy

AbstractAs a major component of tumor infiltration, tumor‐associated macrophages (TAMs) primarily promote the M2 phenotype of tumors in the tumor microenvironment. Additionally, the overexpression of anti‐phagocytic molecules in TAMs facilitates tumor cell evasion of TAMs’ phagocytic arrest. Therefore, there is an urgent need to develop a reliable strategy to reprogram TAMs for more effective anti‐tumor outcomes. In this study, innovative inhalable bioorthogonal gene‐editable microparticles (SPR) have been engineered that reprogram TAMs for lung cancer therapy based on spike‐covered sunflower sporopollenin exine capsules (SECs). These microparticles utilize SECs as delivery vehicles, with reduced palladium (Pd) nanoparticles on their surface carrying CRISPR/Cas9 lipid nanoparticles (LNP‐RNPs) that specifically knock down the anti‐phagocytic SIRPα gene on TAMs. The SPR microparticles employ a tripartite strategy to reprogram the TAMs: the physical stimulation through pollen spikes, the production of a Toll‐like receptor 7 agonist (imiquimod, IMD) via Pd‐mediated bioorthogonal catalysis, and the employment of CRISPR/Cas9 gene editing. In an orthotopic mouse model, the inhalation of SPR microparticles not only reprograms macrophages efficiently but also bolsters their tumor‐fighting abilities. Notably, the cured mice show no signs of recurrence even upon re‐exposure to the tumor, indicating a long‐lasting anti‐tumor effect.

Natural plant-derived polysaccharides targeting macrophage polarization: a promising strategy for cancer immunotherapy.

Tumor associated macrophages (TAMs) are the predominant innate immune cells in the tumor microenvironment (TME). Cytokines induce the differentiation of macrophages into distinct types of TAMs, primarily characterized by two phenotypes: M1-polarized and M2-polarized. Cancer growth is suppressed by M1-polarized macrophages and promoted by M2-polarized macrophages. The regulation of macrophage M1 polarization has emerged as a promising strategy for cancer immunotherapy. Polysaccharides are important bioactive substances found in numerous plants, manifesting a wide range of noteworthy biological actions, such as immunomodulation, anti-tumor effects, antioxidant capabilities, and antiviral functions. In recent years, there has been a significant increase in interest regarding the immunomodulatory and anti-tumor properties of polysaccharides derived from plants. The regulatory impact of polysaccharides on the immune system is mainly associated with the natural immune response, especially with the regulation of macrophages. This review provides a thorough analysis of the regulatory effects and mechanisms of plant polysaccharides on TAMs. Additionally, an analysis of potential opportunities for clinical translation of plant polysaccharides as immune adjuvants is presented. These insights have greatly advanced the research of plant polysaccharides for immunotherapy in tumor-related applications.

Integrated analyses reveal IDO1 as a prognostic biomarker coexpressed with PD-1 on tumor-associated macrophages in esophageal squamous cell carcinoma.

Inhibition of indolamine-2,3-dioxygenase 1 (IDO1) has been proposed as a promising strategy for cancer immunotherapy; however, it has failed in clinical trials. Macrophages in the tumor microenvironment (TME) contribute to immune escape and serve as potential therapeutic targets. This study investigated the expression pattern of IDO1 in TME and its impact on prognosis and therapeutic response of patients with esophageal squamous cell carcinoma (ESCC). RNA sequencing data from 95 patients with ESCC from The Cancer Genome Atlas (TCGA) database were used to explore the prognostic value of IDO1. Bioinformatics tools were used to estimate scores for stromal and immune cells in tumour tissues, abundance of eight immune cell types in TME, and sensitivity of chemotherapeutic drugs and immune checkpoint (IC) blockage. The results were validated using digitalized immunohistochemistry and multiplexed immunofluorescence in ESCC tissue samples obtained from our clinical center. TCGA and validation data suggested that high expression of IDO1 was associated with poor patient survival, and IDO1 was an independent prognostic factor. IDO1 expression positively correlated with macrophages in TME and PDCD1 within diverse IC genes. Single-cell RNA sequencing data analysis and multiplexed immunofluorescence verified the coexpression of IDO1 and PD-1 in tumor-associated macrophages (TAMs). Patients with high IDO1 expression showed increased sensitivity to various chemotherapeutic drugs, while were more likely to resist IC blockage. This study identifies IDO1 as an independent prognostic indicator of OS in patients with ESCC, reveals a compelling connection of IDO1, PD-1, and TAMs, and explores the sensitivity of patients with high IDO1 expression to chemotherapeutic drugs and their resistance to IC blockade. These findings open new avenues for potential targets in ESCC immunotherapy.

ICAT-Mediated Crosstalk Between Cervical Cancer Cells and Macrophages Promotes M2-Like Macrophage Polarization to Reinforce Tumor Malignant Behaviors.

Inhibitor of β-catenin and T-cell factor (ICAT) is a classical inhibitor of the Wnt signaling pathway. Nonetheless, our previous work found that ICAT is overexpressed in cervical cancer (CC), resulting in the augmentation of migration and invasion capabilities of CC cells. It remains unclear what molecular mechanism underlies this phenomenon. The interaction between cancer cells and the tumor microenvironment (TME) promotes the outgrowth and metastasis of tumors. Tumor-associated macrophages (TAMs) are a major constituent of the TME and have a significant impact on the advancement of CC. Consequently, our inquiry pertains to the potential of ICAT to facilitate tumor development through its modulation of the cervical TME. In this study, we first verified that ICAT regulated the secretion of cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in CC cells, leading to M2-like macrophage polarization and enhancement of the migration and invasion of CC cells. Furthermore, the system of co-culturing human umbilical vein endothelial cells (HUVECs) with macrophages revealed that depending on the CC cells' overexpression or inhibition of ICAT, the vascular tube formation by HUVECs can be either increased or decreased. Overall, our study indicates that ICAT stimulates M2-like polarization of TAMs via upregulating IL-10 and TGF-β, which results in increased neovascularization, tumor metastasis, and immunosuppression in CC. In upcoming times, inhibiting crosstalk between CC cells and TAMs may be a possible strategy for CC therapy.

Blockade of CD73 potentiates radiotherapy antitumor immunity and abscopal effects via STING pathway

Radiotherapy (RT) is a crucial treatment for colorectal cancer (CRC) patients, but it often fails to induce systemic antitumor immunity. CD73, an immunomodulatory factor, is upregulated after RT and associated with poor prognosis in CRC patients. This study aims to elucidate the mechanisms driving RT-induced CD73 upregulation in CRC and investigate how combining RT with CD73 blockade stimulates immune responses and induces abscopal effects. Findings revealed that RT-induced CD73 upregulation is mediated by the ataxia telangiectasia and Rad3-related (ATR) pathway and correlated with RT tolerance, as demonstrated through flow cytometry, immunofluorescence, and Western Blotting. Using flow cytometry and multicolor immunofluorescence, experiments demonstrated that in CRC subcutaneous tumor models, combination therapy reduces the infiltration of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs) while increasing dendritic cells (DCs) and CD8 + T cells, resulting in superior antitumor responses. Additionally, results from flow cytometry, Western Blot, and RNA sequencing demonstrated that combination therapy enhances the antigen-presenting ability of DCs and activates tumor antigen-specific CD8 + T cells, improving their function and delaying their depletion. The activation of the cGAS-STING and IFN-I pathways is crucial for this effect. In summary, the integration of RT with CD73 blockade effectively reverses the immunosuppressive TME and invigorates CD8 + T cell-driven, specific antitumor immune responses. These insights shed fresh light on the mechanisms governing the synergistic modulation of immunity by RT and CD73 blockade in CRC, offering promising avenues for the advancement of therapeutic strategies against CRC.

Abstract B036: Hypoxic cancer cells condition immunosuppressive macrophages via secreted non-protein mediators in pancreatic ductal adenocarcinoma

Abstract INTRODUCTION: Hypoxia is a defining feature of pancreatic ductal adenocarcinoma (PDAC) that contributes to the immunosuppressive microenvironment defined by increased tumor-associated macrophages (TAMs). However, there is an unmet need to define mechanisms by which hypoxia drives the immunosuppressive macrophage phenotype. We hypothesize that hypoxic cancer cells secrete non-protein metabolites to induce immunosuppressive phenotypes in macrophages. METHODS: We generated conditioned media (CM) by culturing the pancreatic adenocarcinoma cell line KPC under normoxia (21% O2) or hypoxia (0.5% O2) conditions for 48 hours. We treated bone marrow-derived macrophages (BMDMs) or the macrophage line RAW264.7 with CM. We assessed immunosuppressive gene expression using RNAseq and qRT-PCR. Proteins in CM were depleted using Sera-Mag Magnetic Carboxylate Modified Beads or denatured by boiling at 100°C for 15 minutes. We identified changes in metabolites between hypoxic and normoxic CM using HPLC. RESULTS: Compared to BMDMs treated with normoxic CM, we observed that BMDMs treated with hypoxic CM induced multiple immunosuppressive proteins (Arg1), cytokines (Ccl22, Cxcl3) and interleukins (Il1b, Il1a, Il6). Similarly, GSEA analysis demonstrated that hypoxic CM induced TNFa signaling via NfkB and IL6-JAK-STAT3 signaling. Consistent with RNA-seq analysis, qRT-PCR measured Arg1 expression 53 times higher in hypoxic CM-treated BMDMs compared to nomoxic CM (p=0.0286). Compared to normoxic CM, hypoxic CM stimulated macrophage migration, a marker for macrophage infiltration, as measured by transwell assays (p<0.0001). An increase in Arg1 expression in RAW264.7 cells was still observed when treated with protein depleted or boiled hypoxic CM, excluding proteins as soluble factor(s) mediating immunosuppression. To identify non-protein mediators of immune suppression, mass spectrometry quantified lipids and metabolites enriched in HCM. Metabolomic analysis identified 40 metabolites increased in hypoxic CM including the monounsaturated or polyunsaturated fatty acids linoelaidic acid, DGLA, and arachidonic acid. Similarly, lipidomic analysis revealed that multiple PC (phosphatidylcholine) and LPC (lysophosphatidylcholine) lipid species were enriched in HCM. CONCLUSION: We observe that hypoxic tumor cells induce immunosuppressive changes in macrophages likely via secreted non-protein metabolites. The hypoxic metabolome of PDAC tumor cells may be a key driver of the accumulation of immunosuppressive tumor associated macrophages. We are currently characterizing the role of individual lipids or metabolites in macrophage polarization. Citation Format: Matthew T Cribb, Sahar Fattani, Ayeisha Colon-Ortiz, Dadi Jiang, Michael Spiotto, Albert Koong. Hypoxic cancer cells condition immunosuppressive macrophages via secreted non-protein mediators in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B036.

Abstract B078: A comprehensive spatial atlas of neoadjuvant chemoradiation therapy in resected pancreatic cancer identifies cellular & microenvironmental determinants of persister populations

Abstract Background The molecular pathways involved in the response to radiation therapy (RT) in pancreatic ductal adenocarcinoma (PDAC) remain poorly understood. Despite extensive molecular stratification efforts, improvements in molecularly informed RT-based therapeutic strategies in the neoadjuvant setting have been limited. This study aims to elucidate the adaptive mechanisms and interactions within PDAC to identify new combinatorial therapeutic targets with RT. MethodsWe constructed a high-resolution molecular landscape of the cellular subtypes and spatial communities that constitute PDAC. Single-cell RNA sequencing (scRNA- seq) was performed on 16 PDAC biopsies of matched pre- and post-treatment samples from a Phase I/II dose-escalation clinical trial of pancreatic stereotactic body radiation therapy (SBRT). Additionally, Visium spatial transcriptomics (ST) was conducted on biopsies from primary PDAC patients collected at the time of surgery. These patients received either no neoadjuvant therapy (n = 8) or neoadjuvant chemoradiation (chemoRT) (n = 20). Cell type profiles were determined from scRNA-seq to compare changes in cell type proportions and signatures before and after RT. From these profiles, we employed robust cell type decomposition to analyze cell type signatures in our ST dataset. ResultsChemoRT induced several significant effects compared to controls within the tumor microenvironment, including an increase in the proportion of myofibroblasts, specifically senescent cancer-associated fibroblasts (CAFs), with signatures consistent with immune cell dysfunction. We observed an increase in CD8 and CD4 effector memory cells with RT; however, this occurred alongside a rise in immunosuppressive alternatively activated tumor-associated macrophages. Additionally, RT upregulated pathways related to HIF, cytokine production, B cell proliferation, and negative regulation of lymphocytes. Patients with increased infiltration of NK, memory B, and CD8 effector memory cells demonstrated improved overall survival outcomes. Inference of copy number variations enabled us to identify cancer subclones as either responsive or resistant in matched pre and post RT biopsies. Resistance was characterized by augmented hypoxia, KRAS activation, epithelial- mesenchymal transition, and interferon-gamma response signaling. Conversely, responders exhibited increased oxidative phosphorylation signaling and pathway enrichment associated with cell proliferation and cell cycle progression. Interestingly, RT induced a decrease in cancer cells characterized as harboring a chemoresistant basal-like molecular subtype. ConclusionThrough both longitudinal and therapy stratified tumor biopsies, we were able to observe significant selection pressures on stromal and cancer cells following RT. This includes upregulation of innate and adaptive immune pathways with a compensatory immunosuppressive response driven by myeloid cells and CAFs. This may provide opportunities for development of clinical trials to therapeutically target specific cellular phenotypes and their interactions. Citation Format: Vincent Bernard, Galia Jacobson, Kimal Rajapakshe, Jimin Min, Guangsheng Pei, Daniel Lin, Ayush Suresh, Dadi Jiang, Ching-Wei Tzeng, Manoop S Bhutani, Linghua Wang, Paola A Guerrero, Ethan B Ludmir, Albert C Koong, Anirban Maitra, Cullen M Taniguchi, Eugene J Koay. A comprehensive spatial atlas of neoadjuvant chemoradiation therapy in resected pancreatic cancer identifies cellular & microenvironmental determinants of persister populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B078.

Abstract B070: TGFB2 mRNA levels prognostically interact with Interferon-alpha receptor activation of IRF9 and IFI27, and makers for tumor-associated macrophages impacting overall survival in PDAC

Abstract Background: The treatment of pancreatic ductal adenocarcinoma (PDAC) is an unmet challenge, with the median overall survival rate remaining less than a year, even with the use of FOLFIRINOX-based therapies. Methods: In this study, we analyzed archived macrophage- associated mRNA expression using datasets deposited in the UCSC Xena web platform to compare normal pancreatic tissue and PDAC tumor samples. We also utilized TCGA datasets deposited in cBioportal (https://www.cbioportal.org/study/summary?id=paad_tcga_pan_can_atlas) and KMplotter (http://kmplot.com/analysis/index.php?p=service&cancer=pancancer_rnaseq) to relate mRNA expression levels to overall survival outcomes. The prognostic impact of TGFB2 mRNA expression levels was determined by comparing patients expressing high versus low levels of TGFB2 (50th percentile cut-off) in low macrophage TME. Results: The TGFB2 gene exhibited low mRNA expression levels in normal tissue, with less than one TPM, while, in tumor tissue, TGFB2 expression levels exhibited a 7.9-fold increase in mRNA expression relative to normal tissue (P < 0.0001). Interferon Alpha Inducible Protein 27 (IFI27), the downstream product of Interferon regulatory factor 9 (IRF9) and Signal transducer and activator of transcription 1 (STAT1) transcriptional activation by Interferon alpha/beta receptor 1 (IFNAR1) exhibited a significant 66.3-fold increase in expression in tumor tissue compared to normal tissue (P < 0.0001). The statistical contrast between the expression of IRF9 mRNA also displayed a significant (P<0.0001) 4.2-fold increase in mRNA expression levels. These increased levels of mRNA expression were found to be prognostically significant, whereby patients with high expression levels of either TGFB2, IRF9, or IFI27 showed median OS times ranging from 16 to 20 months (P<0.01 compared to 72 months for patients with low levels of expression for both TGFB2 and either IRF9 or IFI27). In TME with low macrophage levels, patients with high levels of TGFB2 mRNA exhibited significantly shorter OS outcomes than patients with low TGFB2 mRNA levels (Median OS of 72.2 versus 15.3 months, P<0.0001). Furthermore, expression of TGFB2 mRNA and TAM markers exhibited TGFB2 prognostic impact independent of 17 TAM markers suggested by multivariate Cox regression models. Conclusions: The results of our study suggest that a combination of pharmacological tools can be used in treating PDAC patients, targeting both TGFB2 and the components of the type-I interferon signaling pathway to improve OS outcomes. TGFB2 expression impacts OS independent of TAM markers, but the expression of TAM markers impacts the prognostic effect of TGFB2 mRNA expression. Citation Format: Sanjive Qazi, Wen-Han Chang, Cynthia Lee, Vuong Trieu. TGFB2 mRNA levels prognostically interact with Interferon-alpha receptor activation of IRF9 and IFI27, and makers for tumor-associated macrophages impacting overall survival in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B070.

Abstract C002: A pilot study to assess the safety, tolerability, and tumor responses with lenvatinib plus pembrolizumab in patients with advanced pancreatic ductal adenocarcinoma in second-line or later of treatment

Abstract Introduction Lenvatinib plus PD-1 inhibition substantially decreases tumor-associated macrophages and increases infiltration of CD8+ T-cells in mouse tumor models, enhancing anti- tumor activity, thus providing the background rationale to explore the clinical response and the safety of the combination of lenvatinib plus pembrolizumab in patients with pancreatic cancer. Methods Patients with metastatic pancreatic adenocarcinoma with disease progression on ≥ 1 line of systemic therapy were eligible for screening. Eligible patients received therapy on a 42 day cycle, receiving fixed doses of 400mg of pembrolizumab on day 1 and 20mg of lenvatinib daily. Blood and tumor tissue were collected to evaluate molecular determinants of response and changes in the tumor’s T-cell composition. Results Fifteen patients received treatment on protocol, with a median of 1 cycle of treatment. Of these 15 patients, 5 were female and 10 were male; 12 enrolled at a performance status of 0 and 3 at a 1; 10 had one prior line of therapy in the metastatic setting and 5 had 2 lines. Currently, 1 patient remains on protocol after 3 cycles of therapy; 3 other patients who had disease progression on protocol remain alive at the time of this submission and are on other treatments. While no tumor responses were observed on therapy, 7 patients had stable disease and remained on treatment beyond 1 cycle of therapy. In 3 of these 7 patients, CA 19-9 trend was consistent with cancer control (1 additional patient was a nonproducer). The disease control rate was 47% and the median progression free survival was 10 weeks [95% CI, 6.3 – 11.7]. Median overall survival was 19.8 weeks [95% CI, 13.5 – 26.1]. All 14 patients who discontinued therapy did so due to disease progression; none stopped therapy due to treatment related toxicity. Four patients had either lenvatinib dose delays or reductions per protocol due to toxicity; no patients had dose delays/modifications to the pembrolizumab. No unexpected adverse events were experienced. The most frequently occurring toxicities of any grade were fatigue (8), hypertension (7), proteinuria (6), nausea (4), hypothyroidism (4), mucositis (4), and hyponatremia (4). There was one occurrence of each of the following serious adverse events, with noted CTCAE grade (Gr) and attribution to therapy: sepsis (Gr3, unrelated), immune mediated endocrinopathy (Gr3, possibly related), hypotension (Gr3, possibly related), fatigue (Gr3, unrelated), lung infection (Gr3, probably related), enterocolitis (Gr3, probably related), fever (Gr3, possibly related). There were no grade 4 or 5 events. Conclusions The combination of pembrolizumab with lenvatinib demonstrated pancreatic cancer control which, while modest, is appreciated in this later line of therapy where few treatment options exist. These benefits were achieved while being a safe and manageable treatment for patients. Blood and tissue data will offer further insights into tumor responses, and combination of this therapy with chemotherapy and in an earlier line of treatment is being explored. Citation Format: Brandon G Smaglo, Ryan Sun, Shubham Pant, Michael S Lee, Dan Zhao, Phat H Le, Milind Javle, Jason A Willis, Christine Parseghian, Manoop Bhutani, Saman Chishty, Stacy Diao, Michael W Leung, Ryan W Huey, Maria Pia Morelli, Michael J Overman, Robert A Wolff. A pilot study to assess the safety, tolerability, and tumor responses with lenvatinib plus pembrolizumab in patients with advanced pancreatic ductal adenocarcinoma in second-line or later of treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C002.

Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

Glioma is a common primary malignant brain tumor with low survival rate. Immunotherapy with immune checkpoints inhibitors (ICI) can be a choice for glioma management, and extracellular vesicles (EVs) are recognized as a potential drug delivery system for various disease management due to their enhanced barrier permeation ability and immunomodulatory effect. The aim of this study is to develop ICI-loaded EVs (ICI/EV) that have sufficient efficacy in managing glioma. Calcium phosphate particles (CaP) were used to stimulate the secretion of EVs from murine macrophage cells. CaP conditioning of cells showed an enhanced amount of EVs secretion and macrophage polarization toward a proinflammatory phenotype. The CaP-induced EVs were shown to polarize macrophages into proinflammatory phenotype in vitro, as correlated with the conditioning method. ICI/EVs were successfully prepared with high loading efficiency using the sonication method. The EVs can be distributed throughout the entire brain upon intranasal administration and facilitate ICIs distribution into glioma lesion. Combinatory treatment with ICI/EVs showed benefit in glioma-bearing mice by reducing their tumor volume and prolonging their survival. Cytotoxic T cell infiltration, polarization of tumor-associated macrophage, and lower tumor proliferation were observed in ICI/EVs-treated mice. The developed ICI/EVs showed promise in immunotherapeutic management of glioma.

Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma

BackgroundMacrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies.MethodsIn this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT.ResultsMacrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME.ConclusionsIn summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

Unconventional p65/p52 NF-κB module regulates key tumor microenvironment-related genes in breast tumor-associated macrophages (TAMs)

The complex heterogeneity of tumor microenvironment (TME) of triple-negative breast cancer (TNBC) presents a significant obstacle to cytotoxic immune response and successful treatment, building up one of the most hostile oncological phenotypes. Among the most abundant TME components, tumor-associated macrophages (TAMs) have pivotal pro-tumoral functions, involving discordant roles for the nuclear factor kappa-B (NF-κB) transcription factors and directing to higher levels of pathway complexity.In both resting macrophages and TAMs, we recently revealed the existence of the uncharacterized NF-κB p65/p52 dimer. In the present study, we demonstrated its enhanced active nuclear localization in TAMs and validated selected immune target genes as directly regulated by dimer binding on DNA sequences.We demonstrated by ChIP-qPCR that p65/p52 enrichment on HSPG2 and CSF-1 regulatory regions is strictly dependent on macrophage polarization and tumor environment.Our data provide novel mechanisms of transcriptional regulation in TAMs, orchestrated by the varied and dynamic nature of NF-κB combinations, which needs to be considered when targeting this pathway in cancer therapies.Our results offer p65/p52, together with identified regulatory regions on genes impacting macrophage behavior and tumor biology, as novel molecular targets for TNBC, aimed at modulating TAMs functions towards anti-tumoral phenotypes and thus improving cancer treatment outcomes.

Towards imaging the immune state of cancer by PET: Targeting legumain with 11C-labeled P1-Asn peptidomimetics carrying a cyano-warhead

PurposeM2-type tumor-associated macrophages (TAM) residing in the tumor microenvironment (TME) have been linked to tumor invasiveness, metastasis and poor prognosis. M2 TAMs suppress T cell activation, silencing the recognition of the cancer by the immune system. Targeting TAMs in anti-cancer therapy may support the immune system and immune-checkpoint inhibitor therapies to fight the cancer cells. We aimed to develop a PET tracer for the imaging of M2 TAM infiltration of cancer, using activated legumain as the imaging target. Basic proceduresTwo P1-mimicking inhibitors with a cyano-warhead were labeled with carbon-11 and evaluated in vitro and in vivo with a CT26 tumor mouse model. Target expression and activity were quantified from RT-qPCR and in vitro substrate conversion, respectively. The co-localization of legumain and TAMs was assessed by fluorescence microscopy. The two tracers were evaluated by PET with subsequent biodistribution analysis with the dissected tissues. Parent-to-total radioactivity in plasma was determined at several time points after i.v. tracer injection, using reverse phase radio-UPLC. Main findingsLegumain displayed a target density of 40.7 ± 19.1 pmol per mg total protein in tumor lysate (n = 4) with high substrate conversion and colocalization with M2 macrophages in the tumor periphery. [11C]1 and [11C]2 were synthesized with >95 % radiochemical purity and 12.9–382.2 GBq/μmol molar activity at the end of synthesis. We observed heterogeneous tumor accumulation in in vitro autoradiography and PET for both tracers. However, excess unlabeled 1 or 2 did not compete with tracer accumulation. Both [11C]1 and [11C]2 were rapidly metabolized to a polar radiometabolite in vivo. Principal conclusionsThe legumain tracers [11C]1 and [11C]2, synthesized with high radiochemical purity and molar activity, accumulate in the legumain-positive CT26 tumor in vivo. However, the lack of competition by excess compound questions their specificity. Both tracers are rapidly metabolized in vivo, requiring structural modifications towards more stable tracers for further investigations.