Macrophage Tumor Research Articles

Imaging tumor and ascites-associated macrophages in a mouse model of metastatic ovarian cancer

BackgroundTumor-Associated Macrophages (TAMs) play a critical role in the pathogenesis and progression of ovarian cancer, a lethal gynecologic malignancy. [124I]iodo-DPA-713 is a PET radiotracer that is selectively trapped within reactive macrophages. We have employed this radioligand here as well as a fluorescent analog to image TAMs associated with primary tumors, secondary pulmonary metastases and gastrointestinal tract-associated macrophages, associated with ascites accumulation in a syngeneic mouse model of metastatic ovarian cancer. Intact female C57BL/6 mice were engrafted with ID8-Defb29-VEGF tumor pieces. One month after engraftment, the mice were selected for positive bioluminescence to show primary and secondary tumor burden and were then scanned by PET/MRI with [124I]iodo-DPA-713, observing a 24 h uptake time. PET data were overlayed with T2-weighted MRI data to facilitate PET uptake tissue identity. Additionally, mice were imaged ex vivo using Near IR Fluorescence (NIRF), capturing the uptake and sequestration of DPA-713-IRDye800CW, a fluorescent analog of the radioligand used here. Additionally, cell culture uptake of DPA-713-IRDye680LT in ID8-DEFb29-VEGF, IOSE hTERT and RAW264.7 cells was conducted to measure tracer uptake in ovarian cancer cells, ovarian epithelial cells and macrophage.ResultsPET/MRI data show an intense ring of radiotracer uptake surrounding primary tumors. PET uptake is also associated with lung metastases, but not healthy lung. Mice displaying ascites also display PET uptake along the gastrointestinal tract while sham-operated mice show minimal gastrointestinal uptake. All mice show specific kidney uptake. Mice imaged by NIRF confirmed TAMs uptake mostly at the rim of primary tumors while 1 mm secondary tumors in the lungs displayed robust, homogeneous uptake of the radio- and fluorescent analog. Ex vivo biodistribution of [124I]iodo-DPA-713 showed that contralateral ovaries in middle-stage disease had the highest probe uptake with tissues sampled in mid- and late-stage disease showing increasing uptake.Conclusion[124I]iodo-DPA-713 and DPA-713-IRDye800CW sensitively identify and locate TAMs in a syngeneic mouse model of metastatic ovarian cancer.

The role of NLRP3 and NLRP12 inflammasomes in glioblastoma.

Glioblastoma (GBM) is the deadliest malignant brain tumor, with a survival of less than 14 months after diagnosis. The highly invasive nature of GBM makes total surgical resection challenging, leading to tumor recurrence and declined survival. The heterocellular composition of the GBM reprograms its microenvironment, favoring tumor growth, proliferation, and migration. The innate immune cells in the GBM tumor microenvironment, including microglia, astrocytes, and macrophages, express pattern recognition receptors such as NLRs (Nucleotide-binding domain and leucine-rich repeat-containing) that sense pathogen- and damage-associated molecular patterns initiating inflammation. Upon activation, NLRP3 promotes inflammation by NLRP3 inflammasome formation. Auto-proteolytic cleavage and activation of Caspase-1 within the inflammasome leads to caspase-1-mediated cleavage, activation, and conversion of pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, leading to pyroptosis. In contrast, NLRP12 downregulates inflammatory responses in microglia and macrophages by regulating the NF-κB pathway. NLRP3 and NLRP12 have been implicated in the disease pathophysiology of several cancers with cell-context-dependent, pro- or anti-tumorigenic roles. In this review, we discuss the current literature on the mechanistic roles of NLRP3 and NLRP12 in GBM and the gaps in the scientific literature in the context of GBM pathophysiology with potential for targeted therapeutics.

MiR-122/NEGR1 axis contributes colorectal cancer liver metastasis by PI3K/AKT pathway and macrophage modulation

Colorectal cancer (CRC) is a prevalent malignant tumor in the gastrointestinal tract, with around 50% of patients experiencing distant metastases, predominantly to the liver. Colorectal cancer liver metastasis (CRLM) is a leading cause of CRC-related death, and effective treatments remain limited. This study aims to identify new targets for predicting and treating CRLM. Bioinformatics analysis highlighted the miR-122/NEGR1 axis as crucial in CRLM. In vitro assays (Colony formation, Wound healing, Transwell) explored the impact of this axis on CRC cell proliferation, invasion, and migration. Dual-Luciferase Reporter Gene Assay and RNA-pulldown confirmed miR-122/NEGR1 interaction. In vivo, CRLM model mice were used to investigate the axis’s effects on tumor metastasis and macrophage polarization. Immunofluorescence (IF), Quantitative Real-time PCR (qRT-PCR), Enzyme-linked Immunosorbent Assay (ELISA), and Western Blot (WB) analyzed macrophage polarization markers and cytokine/protein/RNA expression. Results showed increased miR-122 and decreased NEGR1 in liver metastases compared to primary tumors. The miR-122/NEGR1 axis enhanced CRC cell proliferation, migration, invasion, and affected the PI3K/AKT pathway. Furthermore, reduced NEGR1 promoted M2 macrophage polarization and accelerated liver metastasis in CRLM model mice. In conclusion, the miR-122/NEGR1 axis drives CRC progression and liver metastasis through the PI3K/AKT pathway and M2 macrophage polarization, representing a potential target for the therapy of CRLM.

Fatty Acids Derived from Royal Jelly Exert Anti-Inflammatory and Antibacterial Activities in the Treatment of Pseudomonas aeruginosa-Induced Acute Pneumonia.

Pseudomonas aeruginosa, an opportunistic pathogen, commonly causes hospital-acquired pneumonia. Royal jelly fatty acids (RJFAs), a mixture of various fatty acids extracted from royal jelly, exhibit antibacterial and anti-inflammatory properties in treating many infectious diseases. Nevertheless, the therapeutic mechanisms of RJFAs in treatment of acute P. aeruginosa pulmonary infection are still unclear. Herein, we initially extracted the fatty acids from royal jelly and characterized their chemical constituents using headspace gas chromatography-mass spectrometry. Furthermore, we examined the antibacterial effect of RJFAs in vitro and explored its therapeutic effect and molecular mechanisms in treating acute P. aeruginosa pulmonary infection invivo. The in vitro antibacterial studies revealed that RJFAs significantly inhibited P. aeruginosa growth. Moreover, the invivo studies showed that the RJFAs effectively mitigated the lung damage and inflammation induced by P. aeruginosa through impairing neutrophil infiltration, reducing the bacterial load in lung and diminishing the production of proinflammatory cytokines, including tumor necrosis factor (TNF-α), interleukin (IL-1β), IL-6, and macrophage inflammatory protein-2 (MIP-2). In addition, the mice treated with RJFAs exhibited reduced phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), c-Jun, and nuclear factor-kappa B (NF-κB) p65 in the lung tissues in comparison with that of the mice without drug treatment. These findings demonstrated that RJFAs exhibited significant antibacterial and anti-inflammatory effects in treating the P. aeruginosa-induced acute pneumonia, and the anti-inflammatory effects were exerted through suppressing the mitogen-activated protein kinase/activator protein-1 (MAPK/AP-1) pathway and NF-κB activation, suggesting a promising therapeutic potential of RJFAs against acute bacterial pneumonia.

Subcellular Proteomic Mapping of Lysine Lactylation.

Protein lactylation is a novel post-translational modification (PTM) involved in many important physiological processes such as macrophage polarization, immune regulation, and tumor cell growth. However, traditional methodologies for studying lactylation have predominantly relied on peptide enrichment from whole-cell lysates, which tend to favor the detection of high-abundance peptides, thus limiting the identification of low-abundance lactylated peptides. To address this limitation, here, we employed subcellular fractionation to separate proteins and map lactylated peptides from each isolated subcellular fraction using a model cell line. In brief, we identified 1,217 lysine lactylation (Kla) sites on 553 proteins across four subcellular fractions. Subsequent pathway enrichment analysis revealed that Kla proteins participate in distinct pathways depending on the subcellular contexts. In addition, this subcellular fractionation method enabled the discovery of 36 previously unreported Kla proteins and 223 novel Kla sites, many of which are present in low abundance. Notably, several proteins contain multiple newly identified Kla sites, exemplified by the transcriptional regulator ATRX. Furthermore, our results indicate the possibility of PTM crosstalk between Kla and other PTMs such as ubiquitination and sumoylation. In conclusion, subcellular fractionation facilitates the identification of Kla proteins that have been previously uncovered and could be overlooked by affinity enrichment of whole-cell lysates.

Oncolytic virus encoding 4-1BBL and IL15 enhances the efficacy of tumor-infiltrating lymphocyte adoptive therapy in HCC.

Previous studies have found that oncolytic virus (OVs) can improve the efficacy of TIL adoptive therapy in oral cancer, colon cancer, and pancreatic cancer. However, the curative effect in hepatocellular carcinoma (HCC) is still unclear. Therefore, this study aims to explore the therapeutic effect and mechanism of OVs encoding 4-1BBL and IL15 (OV-4-1BBL/IL15) combined with TIL adoptive therapy on HCC. In this study, the role and immunological mechanism of armed OVs combined with TILs were evaluated by flow cytometry and ELISA in patient-derived xenograft and syngeneic mouse tumor models. Co-culturing with TILs can up-regulate the expression of antigen-presenting cell (APC) markers on the surface of OV-infected primary HCC cells, and promote the specific activation ability and tumor-killing ability of TILs. OV-4-1BBL/IL15 combined with TIL adoptive therapy could induce tumor volume reduction and anti-tumor immune memory in patient-derived xenograft and syngeneic mouse tumor models. Furthermore, OV combined with TIL adoptive therapy can endow tumor cells with aAPC characteristics, activate T cells at the same time, and reprogram tumor macrophages into anti-tumor phenotype. OV-4-1BBL/IL15 can stimulate the anti-tumor potential of TIL therapy in HCC, and possess broad clinical application prospects.

Abstract C036: Pharmacological modulation of glutamine metabolism alters macrophage activity and tumor immune responses

Abstract As a critical component of innate and adaptive immunity, macrophages play an important role in shaping the tumor microenvironment (TME), which affects tumor initiation, progression, and response to treatment. Tumor-associated macrophages can display anti-tumor or pro-tumor activity, a cell fate attributed to macrophage polarization that depends on metabolomic regulation. Targeting macrophage metabolism could be a promising approach for cancer treatment by converting a pro-tumor TME into an anti-tumor TME. To examine in vivo mechanisms behind glutamine metabolism on macrophages, we treated immunocompetent murine ovarian cancer models with a clinical-grade glutamine prodrug, JHU083, and performed single-cell RNA sequencing (scRNAseq). In addition to the significant anti-tumor efficacy of JHU083, we found that JHU083 specifically suppressed the M2-like tumor macrophages while sparing M1-like macrophages. To better understand the molecular mechanism of how JHU083 causes macrophage polarization shift, we cultured an M1-like macrophage cell line with DON (6-Diazo-5-oxo-L-norleucine), the active component of JHU083, and performed bulk RNA sequencing. Using bulk RNAseq and scRNAseq transcriptome analyses on M1-like macrophages, glutamine inhibitor treatment significantly enhanced MHC class II genes, including CD74, H2-Aa, H2-Eb1, H2-Ea, H2-Ab1, and TNF family genes including Tnfsf12, and Tnfrsf14 which may promote T cell trafficking and cytotoxicity through reversing immunosuppressive tumor microenvironment. Moreover, DON-treated bone marrow-derived M1 macrophages promoted CD4+ T cell expansion. On the other hand, DON treatment significantly suppressed bone marrow-derived M2 macrophages. In conclusion, our findings suggest that glutamine metabolism reprogramming can promote anti-tumor functions in tumor macrophages. Therefore, targeting glutamine metabolism to regulate macrophage function and polarization can be a promising direction for future cancer therapy. Citation Format: Jiawei Fan, Sumeng Qi, Ie-Ming Shih, Tian-Li Wang. Pharmacological modulation of glutamine metabolism alters macrophage activity and tumor immune responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C036.

Abstract C045: The extracellular matrix glycoprotein periostin supports chemotherapy resistance by modulating macrophage recruitment and phagocytosis in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancer cases and is notoriously aggressive, often developing resistance to standard chemotherapy thus leading to a high rate of metastatic relapse. Evidence suggests that this resistance is linked to changes in the extracellular matrix (ECM) composition, particularly an increase in periostin (POSTN) expression, which is also associated with poor prognosis in breast cancer. Our recent publication showed that the ECM alone can influence macrophage phenotypes. Furthermore, another study from our lab has demonstrated that POSTN mediates resistance to anti-angiogenic therapy by recruiting macrophages in pancreatic neuroendocrine tumors. Notably, POSTN deletion reduced monocyte/macrophage recruitment and enhanced cytotoxic CD8+ T-cell infiltration. Based on these findings, we hypothesized that paclitaxel (PTX), a widely used chemotherapeutic drug for the management of TNBC, may stimulate POSTN production by fibroblasts leading to the recruitment of pro-tumoral macrophages that facilitate tumor growth resulting into therapy resistance. In this study, we showed that PTX induces increased POSTN expression in mouse models of TNBC through immunohistochemistry, and in both 2D and 3D in vitro models via interactions between TNBC cell lines and mammary fibroblasts, as demonstrated by immunofluorescence. An in vitro migration assay confirmed that PTX-induced POSTN enhances the recruitment of human monocytes and macrophages, which display a mixed immunosuppressive and inflammatory phenotype, as revealed by flow cytometry. Not only did these recruited macrophages exhibit a reduced phagocytic capacity against labelled TNBC cells in an in vitro phagocytosis assay, but we also found that POSTN plays a crucial role in inducing SIRPα expression, that may diminish macrophage phagocytic potential. Increased sialylation and Siglec-1 expression are known to be linked to poor prognosis and polarization towards a more immunosuppressive macrophage phenotype in TNBC. Interestingly in our model, we observed that higher concentrations of recombinant human POSTN induce increased Siglec-1 expression on macrophages, a receptor that interacts with sialic acids in the tumor ECM. Further work using a decellularized tissue model that preserves only the ECM from patient-derived tumor biopsies pre- and post-chemo, aims to further explore the correlation between PTX-induced POSTN, ECM sialylation, and Siglec-1 expression on macrophages. Additionally, we have developed POSTN-knockout murine cancer cells and fibroblasts using CRISPR/Cas9, allowing us to investigate how POSTN influences macrophage phenotypes and chemotherapy responses in vivo. Understanding the mechanisms behind chemotherapy resistance in TNBC is crucial for developing effective treatments for this aggressive cancer subtype. By elucidating the intricate interplay between POSTN, macrophages, and chemotherapy response, we aim to identify new therapeutic strategies to improve outcomes for TNBC patients. Citation Format: Ludovica Tarantola, Ioanna Keklikoglou, Oliver M. Pearce. The extracellular matrix glycoprotein periostin supports chemotherapy resistance by modulating macrophage recruitment and phagocytosis in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C045.

Abstract C006: Tumor-associated macrophages drive prostate cancer progression via IL-1β signaling

Abstract Inflammation is linked to prostate cancer progression. Inflammatory cell infiltrates are commonly observed in prostate biopsies, and inflammation-induced lesions (proliferative inflammatory atrophy, PIA) are precursors of prostate cancer. However, the mechanism by which inflammation impacts prostate cancer progression is poorly understood. Here, we investigated the significance of inflammation on prostate cancer progression using a cMyc-driven prostate adenocarcinoma mouse model (Hi-Myc). We observed a robust tumor-associated macrophage (TAM) infiltrate early during progression of Hi-Myc tumors. Depleting TAMs led to a decrease in both tumor weight and invasive area, demonstrating the functional importance of TAMs in tumor maintenance. To elucidate the molecular basis of how TAMs influence tumor progression, we collected Hi-Myc tumors throughout cancer development from the precursor stage of prostatic intraepithelial neoplasia to prostate adenocarcinoma and performed single-cell RNA sequencing (scRNA-seq). Our study revealed that a gene signature of strong IL-1β signaling activation was observed in TAMs from Hi-Myc tumors, but not in macrophages from wild-type prostates. Importantly, IL-1β neutralization led to delayed tumor progression with reduced tumor weight and invasive area. Furthermore, blocking IL-1β signaling decreased TAM infiltration, suggesting a positive feedback loop created by TAMs. To understand the effect of IL-1β on cancer cell invasion, we used a protease-dependent fluorescent probe to investigate the activity of major extracellular matrix degraders and found that IL-1β neutralization impairs MMP activity, likely through loss of expression by tumor cells and macrophages. To further investigate the targets of IL-1β signaling, we analyzed Il1r1 expression levels across all cell types and found the highest levels in cancer-associated fibroblasts (CAFs). Moreover, CAFs expressed elevated levels of the myeloid chemokines Ccl2, Csf1, Cxcl1, Cxcl2 as well as Il6 compared to fibroblasts from wild-type prostates. In vitro studies of wild-type prostate fibroblasts treated with recombinant IL-1β confirmed that IL-1β directly upregulates expression of these inflammatory cytokines and chemokines. In addition, IL-1β directly promotes proliferation of tumor-derived prostate cancer organoids in vitro. Overall, our study suggests that TAMs and CAFs cooperatively drive pro-tumorigenic IL-1β signaling in prostate cancer, demonstrating a direct mechanistic link between inflammation and prostate cancer progression. Citation Format: Young Sun Lee, Jimmy L. Zhao, Max Land, Joseph Chan, Perianne Smith, Roshan Sharma, Sanjay Kottapalli, Linda Fong, Zhenghao Chen, Cathy Wang, Jesse Kirkpatrick, Ava Soleimany, Samir Zaidi, Kayla Lawrence, Amanda Kulick, Teng Han, Zhen Sun, Philip Watson, Anuradha Gopalan, Ojasvi Chaudhary, Tianhao Xu, Ignas Masilionis, Ronan Chaligne, Dana Rathkopf, Michael Morris, Sangeeta Bhatia, Michael Haffner, Dana Pe'er, Charles Sawyers. Tumor-associated macrophages drive prostate cancer progression via IL-1β signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C006.

Unveiling the role of Pleckstrin-2 in tumor progression and immune modulation: insights from a comprehensive pan-cancer analysis with focus on lung cancer

Cancer remains a leading cause of mortality globally, highlighting the need for novel biomarkers to enhance prognosis and therapeutic strategies. Pleckstrin-2 (PLEK2), a member of the pleckstrin family, has been implicated in processes critical to tumor progression, but its role across cancers remains underexplored. This study systematically examined the expression patterns, prognostic relevance, and functional impact of PLEK2 across multiple cancer types. Using data from The Cancer Genome Atlas (TCGA), Genotype Tissue Expression Project (GTEx), and the Human Protein Atlas, we analyzed PLEK2 expression in both cancerous and normal tissues, revealing significant overexpression of PLEK2 in various cancers at the mRNA and protein levels. Single-cell RNA sequencing further indicated predominant expression of PLEK2 in tumor cells and macrophages within the tumor microenvironment. Survival analysis demonstrated that elevated PLEK2 expression correlated with poor prognosis in specific cancers, though its impact varied across cancer types. Functional assays showed that PLEK2 knockdown inhibited proliferation and migration in human cancer cell lines. In vivo studies using a Lewis lung carcinoma (LLC) model confirmed that PLEK2 knockdown suppressed tumor growth and enhanced the efficacy of PD-1 immunotherapy. Mechanistically, PLEK2 knockdown was associated with reduced AKT pathway activation, diminished tumor-associated macrophage infiltration, and increased CD8 T cell presence. Compounds like Navitoclax were also identified as potential PLEK2 inhibitors. In conclusion, PLEK2 played a multifaceted role in cancer progression and immune response modulation. Targeting PLEK2 might suppress tumor growth and overcome immunotherapy resistance, offering a promising biomarker and therapeutic target to improve cancer treatment outcomes.

Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy.

Achieving selective and durable inhibition of programmed death ligand 1 (PD-L1) in tumors for T cell activation remains a major challenge in immune checkpoint blockade therapy. We herein presented a set of clickable inhibitors for spatially confined PD-L1 degradation and radioimmunotherapy of cancer. Using metabolic glycan engineering click bioorthogonal chemistry, PD-L1 expressed on tumor cell membranes was labeled with highly active azide groups. This enables covalently binding of the clickable inhibitor with PD-L1 and subsequent PD-L1 degradation. A pH-activatable nanoparticle responding to extracellular acidic pH of tumor was subsequently used to deliver the clickable PD-L1 inhibitor into extracellular tumor microenvironment for depleting PD-L1 on the surface of tumor cell and macrophage membranes in vivo. We further demonstrated that a combination of the clickable PD-L1 inhibitor with radiotherapy (RT) eradicated the established tumor by inhibiting RT-up-regulated PD-L1 in the tumor tissue. Therefore, selective PD-L1 blockade in tumors via the clickable PD-L1 inhibitor offers a versatile approach to promote cancer immunotherapy.

Pancreatic cancer-derived exosomal miR-510 promotes macrophage M2 polarization and facilitates cancer cell aggressive phenotypes.

Extensive tumor microenvironment (TME) and tumor-associated macrophages (TAMs) contribute to the initiation and progression of pancreatic cancer (PC). Cancer cell-derived exosomal miRNAs that stimulate macrophage M2 polarization might play an important role in the process. In the current study, we observed significant upregulation of miR-510 in PC cell lines in comparison to normal HPDE cell line, with PANC-1 exhibiting the highest and MIA PaCa-2 the lowest miR-510 levels. Functional assays demonstrated that miR-510 overexpression enhanced, while its inhibition reduced, PC cell viability, migration, invasion, and EMT. In vivo, miR-510 mimics promoted tumor growth and macrophage M2 polarization, whereas miR-510 inhibition had the opposite effect. Exosomes from PANC-1 and MIA PaCa-2 cells, characterized by nanoparticle tracking analysis and TEM, contained significantly higher miR-510 levels than those from HPDE cells. Macrophages incubated with conditioned media from these PC cells showed increased M2 polarization markers, a process inhibited by the exosome inhibitor GW4869. The delivery of miR-510 via PC cell-derived exosomes facilitated macrophage M2 polarization and regulated the STAT signaling pathway, suggesting that exosomal miR-510 plays a crucial role in the tumor microenvironment of PC by modulating macrophage M2 polarization.

RBIO-02. DEVELOPMENT OF FLASH-CAR RADIOIMMUNOTHERAPY FOR PEDIATRIC BRAIN TUMORS

Abstract Pediatric brain tumors including medulloblastoma and high-grade glioma are generally refractory to T cell-based immunotherapy, largely due to an immune-hostile microenvironment infiltrated extensively with immunosuppressive macrophages. Ultra-high dose rate (FLASH) radiotherapy holds promise for treating solid tumors, given the potential lower toxicity in normal tissues and possible favorable impact on tumor immunity. Using a genetically engineered mouse model of medulloblastoma and glioma, we show that FLASH radiation stimulates pro-inflammatory polarization in tumor macrophages. Single-cell transcriptome analysis shows that FLASH proton beam radiation skews macrophages towards proinflammatory phenotypes and increases T cell infiltration in the medulloblastoma model. Further bulk transcriptome analyses reveal that FLASH radiation reduces peroxisome proliferator-activated receptor (PPAR)-γ and arginase-1 expression and inhibits immunosuppressive macrophage polarization under stimulus-inducible conditions. Mechanistically, FLASH radiation abrogates lipid oxidase expression and oxidized low-density lipid (oxLDL) generation to reduce PPARγ activity, while standard radiation induces reactive oxygen species (ROS)-dependent PPARγ activation in macrophages. Notably, FLASH radiotherapy improves infiltration and activation of chimeric antigen receptor (CAR) T cells and sensitizes tumors to GD2 CAR T immunotherapy in the autochthonous medulloblastoma and syngeneic glioma models. These findings suggest that FLASH radiotherapy may reprogram macrophage lipid metabolism to reverse tumor immunosuppression and that combination FLASH-CAR radioimmunotherapy may offer exciting opportunities for treatment of pediatric brain tumor.

EPCO-54. MULTIOMIC ANALYSIS OF SPINAL EPENDYMOMAS REVEALS MECHANISMS OF TUMOR AGGRESSIVENESS WITH MYCN AMPLIFICATION

Abstract Grade-3 spinal ependymomas, particularly those harboring MYCN amplification, have dismal prognoses despite optimal treatment regimens. MYCN functions as a transcription factor (TF) to drive the expression of key tumor driver genes. Our aim is to identify mechanisms underlying tumor aggressiveness and treatment resistance in MYCN-amplified ependymomas. MYCN amplification in spinal ependymomas was identified with custom next generation sequencing panels and bulk methylation analysis. We then conducted paired single-nucleus RNA (snRNA-seq) and chromatin accessibility (snATAC-seq) sequencing of grade-2 (n=2, 1 patient) and grade-3 spinal ependymomas (n=6, 6 patients, 4 MYCN-amplified). Comparator snRNA-seq datasets (grade-2/grade-3; 8/2) were included from a publicly available dataset (GSE163686; all non-MYCN-amplified). Canonical cell classes were identified with a combination of cluster- and cell-based strategies. Downstream analysis was conducted using R 4.3 (Seurat, Signac, clusterProfiler, CellChat), and Python 3.11 (scanpy) packages. We identified ependymoma tumor cells distinct from canonical immune cell classes with gene expression analysis and confirmed a distinct copy-number-variation pattern in these cells with chromosome 1 and 2p gain, and chromosome 6p and 22q loss. MYCN-amplified tumor cells (compared to non-MYCN-amplified grade2/3 tumor cells) showed increased differential accessibility at genes including VEGFA. Increased MYCN TF activity was confirmed with gene overexpression of downstream targets including NOTCH3, CREB5, PLK1, and VEGFA. Other genes that were highly accessible and overexpressed in MYCN-amplified tumor cells were related to oxidative stress, differentiation, and anti-apoptotic pathways. MYCN-amplified tumor cells modified the tumor microenvironment with increased VISTA signaling between MYCN-amplified tumor cells and tumor-associated macrophages, creating an immunosuppressive environment. We confirmed these effects by observing a higher proportion of M2-like macrophages in MYCN-amplified tumors (29.6% vs 18.2%). In conclusion, MYCN amplification in spinal ependymomas upregulates tumor cell proliferation, angiogenesis, and M2-like macrophage recruitment/programming.

EPCO-46. SPATIAL TRANSCRIPTOMICS ANALYSIS OF GLIOBLASTOMA REVEALS THREE DISTINCT REGIONAL PROGRAMS OF T-CELL INFILTRATION

Abstract Glioblastoma (GBM) resists T-cell killing by developing an immunosuppressive tumor microenvironment. Transcriptomic technologies, such as single-cell RNA sequencing (scRNA-seq), have characterized immune cell phenotypes and putative cell-cell interactions in GBM. However, the tumor microenvironment surrounding T-cells is still not well characterized, motivating the need for spatial analyses. Two publicly-available GBM spatial transcriptomic datasets were analyzed, with one discovery cohort (N=18) and one validation cohort (N=12). Using cell deconvolution with a scRNA-seq reference dataset, areas of high T-cell infiltration were identified and clustered into regional programs based on transcriptomic signatures. To highlight which ligand-receptor interactions are most prominent in these regional programs, a novel spatial enrichment algorithm was applied. The impact of spatially enriched ligand-receptor interactions on overall survival was evaluated using bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA). Three distinct T-cell regional programs (transitional, immunosuppressive, and infiltrative) were found across both the discovery and validation cohort. The infiltrative T-cell regional program resided on the periphery of the tumor with a neural progenitor phenotype (NRGN, VSNL1, SLC17A7). In contrast, the immunosuppressive T-cell region had a higher proportion of malignant cells and a greater mesenchymal tumor signature (ANXA1, CD44, complement pathway). Furthermore, ligand-receptor enrichment analysis showed that the immunosuppressive region had higher tumor and tumor-associated macrophage (TAM) signaling towards CD4 T-cells along the collagen, midkine, and osteopontin pathways. TCGA survival analysis showed that higher collagen and midkine signals were associated with worse overall and disease free survival. Our results identified three distinct regional programs in GBM and highlight a set of signaling pathways associated with T-cell suppression. Our findings suggest that the disruption of these pathways could reduce immunosuppressive signals from tumor and TAMs to T-cells. Subsequent validation with spatial proteomics data in glioblastoma is ongoing.

Compromised macrophages contribute to progression of MASH to hepatocellular carcinoma in FGF21KO mice.

Metabolic dysfunction-associated steatohepatitis is well accepted as a potential precursor of hepatocellular carcinoma. Previously, we reported that fibroblast growth factor 21 (FGF21) revealed a novel anti-inflammatory activity via inhibiting the TLR4-IL-17A signaling, which could be a potential anticarcinogenetic mechanism to prevent to MASH-HCC transition. Here, we set out to determine whether FGF21 has a major impact on Kupffer cells' (KCs) ability during MASH-HCC transition. We found aberrant hepatic FGF21 and KC pool in human MASH-HCC. Lack of FGF21 up-regulated ALOX15, which converted the oxidized fatty acids to induce excessive KC death and mobilization of monocyte-derived macrophages (MoMFs) for KC replacement. Lack of FGF21 oversupplied free fatty acids for sphingosine-1-phosphate (S1P) cascade synthesis to mediate MASH-HCC transition via S1P-YAP signaling and cross-talk between tumor cells and macrophages. In conclusion, lack of FGF21 accelerated MASH-HCC transition via the S1P-AP signaling. Compromised MoMFs could present as tumor-associated macrophage phenotype rendering tumor immune microenvironment for MASH-HCC transition.

Colon Cancer-Derived Exosomal LncRNA-XIST Promotes M2-like Macrophage Polarization by Regulating PDGFRA.

Colon cancer ranks second in overall cancer-related deaths and poses a serious risk to human life and health. In recent years, exosomes are believed to play an important and significant role in cancer, especially tumor-derived exosomes (TDEs). Previous studies have highlighted the pivotal role of exosomes in tumor development, owing to their ability to mediate communication between tumor cells and macrophages, induce macrophage M2 polarization, and facilitate the progression of tumorigenesis. In this study, we revealed that colon cancer-derived exosomes promoted M2-like macrophage polarization. Moreover, exosome-induced M2-like macrophages, in turn, promoted the proliferation, migration, and invasion abilities of colon cancer cells. Specifically, CT26- and HCT116-derived exosomes led to the activation of AKT, ERK, and STAT3/6 signaling pathways in THP-1(Mφ) cells. Furthermore, our findings showed that colon cancer-derived exosomes secreted lncXIST to sponge miR-17-5p, which, in turn, promoted the expression of PDGFRA, a common gene found in all three signaling pathways, to facilitate M2-like macrophage polarization. Dual-luciferase reporter assays confirmed the binding relationship between lncXIST and miR-17-5p, as well as miR-17-5p and PDGFRA. Collectively, our results highlight the novel role of lncXIST in facilitating macrophage polarization by sponging miR-17-5p and regulating PDGFRA expression.

The acid-sensing receptor GPR65 on tumor macrophages drives tumor growth in obesity.

Multiple cancers, including colorectal cancer (CRC), are more frequent and often more aggressive in individuals with obesity. Here, we showed that macrophages accumulated within tumors of patients with obesity and CRC and in obese CRC mice and that they promoted accelerated tumor growth. These changes were initiated by oleic acid accumulation and subsequent tumor cell-derived acid production and were driven by macrophage signaling through the acid-sensing receptor GPR65. We found a similar role for GPR65 in hepatocellular carcinoma (HCC) in obese mice. Tumors in patients with obesity and CRC or HCC also exhibited increased GPR65 expression, suggesting that the mechanism revealed here may contribute to tumor growth in a range of obesity-associated cancers and represent a potential therapeutic target.

SCG2 Mediates HNSCC Progression With CCL2/TGFβ1high M2 Macrophage Infiltration.

This study aims to unravel the mechanisms underlying M2 macrophage polarization in head and neck squamous cell carcinoma (HNSCC), and identify potential therapeutic targets. We conducted an integrated bioinformatic analysis using HNSCC bulk transcriptomes from TCGA and GEO databases to pinpoint critical factors influencing M2 macrophage polarization and tumor prognosis. The significance of these genes was validated in function analysis, single-cell transcriptome datasets, and invitro experiments. Their mechanisms in modulating M2 macrophage polarization were further explored by gene knockdown, cell coculture, and other assays for quantification. We identified a novel prognostic signature of five genes associated with M2 macrophage infiltration, in which SCG2 emerged as a pivotal factor in M2 macrophage polarization in HNSCC. High expression of SCG2 in tumor patients correlated with poorer prognoses, and knocking down SCG2 reduced the proliferation and migration of HNSCC cells, disrupting M2 macrophage polarization. Furthermore, interference of SCG2 resulted in a significant decrease in the secretion of pro-tumor cytokines such as CCL2 and TGFβ1. Our findings provide deeper insights into the pathogenesis of HNSCC and offer promising therapeutic targets for HNSCC, especially SCG2, to inhibit M2 macrophage polarization and modulate cytokine secretion.

Selective activation of PPARα by pemafibrate mitigates peritoneal inflammation and fibrosis through suppression of NLRP3 inflammasome and modulation of inflammation

Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-β1 (TGF-β1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-β1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated THP-1 cells. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.