Tumor-associated Myeloid Cells Research Articles

Abstract B046: Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma

Abstract Glioblastoma (GBM) is often fatal within a year despite treatment, with immune checkpoint blockade therapies benefiting only about 10% of patients. Non-responsive GBMs exhibit an immunosuppressive profile and higher levels of tumor-associated myeloid cells (TAMs). Addressing this immunosuppressive microenvironment is a major challenge in GBM immunotherapy. Our recent studies have identified a positive correlation between Proline-Rich Tyrosine Kinase 2 (Pyk2) activation in GBM tumor cells and the cytokine expression profile of TAMs. This identified Pyk2 as a potential prognostic marker for the immune state in GBM tumor microenvironment. We hypothesize that Pyk2 and MEK/Erk signaling in GBM cells regulate the release of chemokines and cytokines through extracellular vesicles (EVs) by modulating the actin cytoskeleton, thereby impacting TAM activation. The study aimed to investigate the role of Pyk2/MEK/Erk signaling in EV release in GBM cells, potentially revealing new therapeutic targets. Two humans primary GBM cell lines, with and without Pyk2 CRISPR/Cas9 knockout (Pyk2KO), were used. Flow cytometric analysis of EVs, enriched from cell-conditioned medium, identified a shift towards larger in diameter EVs populations in Pyk2 KO cells, compared to wild-type (WT) cells. Analysis using Integrin as a plasma membrane marker showed that 84.70% of Integrin+ and 15.30% of Integrin- EVs were derived from WT cells, whereas Pyk2KO cells produced 89.07% Integrin+ and 10.93% Integrin- EVs. Treatment of WT cells with the Erk/MEK inhibitor Avutometinib (1µM) altered the EV population’s ratio to 79.11% Integrin+ and 20.89% Integrin-. Similarly, Avutometinib treatment of Pyk2KO cells resulted in 78.83% Integrin+ and 21.17% Integrin- EVs, similar to the ratio observed in Avutometinib-treated WT cells. Western blot and PCR analysis of EVs content revealed a significant reduction in CCL2, CCL5, tumor necrosis factor (TNF), and vascular endothelial growth factors (VEGF) in EVs from Pyk2KO GBM cells, compared to WT. In vivo studies using GL261/C57Bl/6 mouse glioma implantation model and flow cytometric analysis of TAMs from tumors generated with WT and Pyk2KO GL261 cells demonstrated increased infiltration of TNF+/IFNg+ CD8+ lymphocytes and Ly6C+/CD206- myeloid cells, alongside a reduction in CD45+/CD11b+/CD33+/MHCII- myeloid-derived suppressor cells. PCR analysis of TAM isolated from Pyk2KO tumors revealed lower gene expression of TNF, CCL5, CCL2, VEGFa, and epidermal growth factor (EGF) compared to WT tumors. The study highlights that Pyk2/MEK/Erk signaling regulates the immune microenvironment in GBM by influencing EV release, which impacts TAM cell activation. Pyk2 primarily regulates the release of Integrin- EVs, while MEK/Erk predominantly regulates EVs shed from the plasma membrane. The findings underscore the interplay between Pyk2 and MEK/Erk signaling in regulating EV biogenesis and composition. Citation Format: Neisha M. Ramirez. Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma [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 B046.

Abstract C021: Tumor-associated microbiota suppresses anti-tumor immunity by driving cDC1 dysfunction in lung cancer

Abstract Type 1 conventional dendritic cells (cDC1s) are critical for initiating and sustaining tumor-reactive CD8 T cells; lack of functional cDC1s is a key driver for failed tumor immunosurveillance and immunotherapy. However, what factors cause the cDC1 insufficiency in the tumor microenvironment (TME) remain poorly understood. Commensal microbiota has emerged as a key regulator of TME and the efficacy of cancer immunotherapies. Nonetheless, most studies have focused on the intestinal microbiome; it remains underexplored whether and how the local microbiota within tumors influences the anti-cancer immunity. Using the autochthonous Kras LSL-G12D/+ ; p53 flox/flox (KP) mouse model of lung cancer, we found an increased local bacterial burden and altered bacterial composition associated with lung tumors. Importantly, this local microbiota suppresses the anti-tumor CD8 T cell response both at baseline and in response to ICB therapies. Specifically, single-cell RNA-seq analysis revealed that the microbiota shapes the functional subsets of tumor-reactive CD8 T cells. Bacterial depletion promotes the accumulation of stem-like Tpex cells (precursor of exhausted T cells) in the TME, which serve as a reservoir of effector CD8 T cells and mediate an effective response upon ICB treatment. To distinguish the function of the local microbiota within TME from the distal gut microbiota, we developed a new method of aerosolized antibiotic treatment, which selectively ablates the intra-tumoral lung microbiota while preserving the intact gut microbiota. Using this approach, we demonstrated that removal of the intra-tumoral lung microbiota markedly improves the tumor-reactive T cell response and sensitizing “cold” lung tumors to ICB therapies. Mechanistically, we identified cDC1s as a central player in microbiota-mediated suppression of anti-tumor immunity. Local microbiota diminishes cDC1 quality and quantity in the lung TME, resulting in impaired T cell priming and Tpex maintenance. At the molecular level, our data revealed that bacterial products derived from the local microbiota induce type I interferon expression in tumor-associated myeloid cells and enhance GM-CSF production from cancer cells: chronic type I interferon and GM-CSF signaling acts cooperatively to reduce the functional cDC1 population. Combining in vitro cDC1 assays, competitive bone marrow chimera experiments and cDC1-specific knockout models, we establish the causal relationships between the local microbiota, IFN-I and GM-CSF signaling in cDC1s, and the tumor response to ICB. Altogether, our study uncovered a dominant role of tumor-associated microbiota in inhibiting cDC1s and suppressing anti-tumor immunity through type I IFN and GM-CSF dependent pathways in lung cancer. Our findings not only identify the intra-tumoral microbiota as a critical therapeutic target for lung cancer treatment, but also provide new insights into the tissue-specific, context-dependent regulatory mechanisms of cDC1s in the TME. Citation Format: Qiang Dong, Chengcheng Jin. Tumor-associated microbiota suppresses anti-tumor immunity by driving cDC1 dysfunction in lung 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 C021.

EXTH-58. BLOCKING IMMUNE CHECKPOINT— LAIR1: DISRUPTING THE LAIR1—FXIII-A—COLLAGEN IMMUNOSUPPRESSIVE CIRCUIT FOR ENHANCED ANTITUMOR THERAPEUTICS

Abstract BACKGROUND Recent evidence indicates that tumor-associated myeloid cells (TAMCs), including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), play a crucial role in glioblastoma (GBM) immunosuppression and progression. These cells can constitute up to 50% of the total mass in GBM tumors and are pivotal in dampening the immune response, which negatively impacts patient survival. Targeting TAMCs represents a promising strategy to overcome the limitations of current cancer treatments. However, drug development in this area remains limited. Our study focuses on Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR1), a novel immune checkpoint overexpressed on TAMCs within GBM. We comprehensively analyze LAIR1’s inhibitory role in cancer progression, highlighting its potential as a therapeutic target. METHODS Both in vitro and in vivo experiments were conducted. LAIR1 wild-type (LAIR1+/+) and knock-out (LAIR1-/-) tumor models were tested for antitumor response and intratumoral immune landscape. We also utilized 2D and 3D cultures to evaluate the effects of antibody blockade (aLAIR1) alone and in combination with CAR-T therapy (8R-70CAR) in both human and mouse GBM models. RESULTS We have uncovered a previously unrecognized immunosuppressive LAIR1→ Factor XIII A (FXIII-A)→ Collagen IV circuit across cancer types. Inhibiting LAIR1, either through genetic LAIR1-/- or aLAIR1, effectively disrupts this immunosuppressive pathway and results in enhanced peripheral and tumor-infiltrating memory CD8 T cell populations, a phenotypic shift of TAMs towards an M1 profile, and a decrease in tumor collagen deposition. These collective effects contribute to the normalization of the TME and facilitate improved interactions between T cells and tumor cells, leading to a more effective antitumor response. Notably, using aLAIR1 as a standalone intervention or combined with CAR T cell therapy demonstrates enhanced antitumor efficacy in preclinical models resistant to chemo-radiation and anti-PD-1 treatments. CONCLUSIONS We discovered a novel mechanism by which LAIR1 contributes to GBM progression. LAIR1 blockade holds promise in reversing suppressive TME and serving as a therapeutic strategy.

IMMU-45. MYELOID-ASSOCIATED WILD-TYPE IDH1 PROMOTES IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Tumor-associated myeloid cells (TAMCs) promote immunosuppression in the GBM tumor microenvironment (TME) and resistance to immune checkpoint therapy (ICT). However, the metabolic and epigenetic mechanisms underlying TAMC activation and the therapeutic potential of fine-tuning TAMC activation remain largely unexplored. Our analyses of GBM patient single-cell RNA sequencing (scRNA-Seq) datasets demonstrated that in addition to tumor cells, wild-type isocitrate dehydrogenase (wt-IDH1) is also highly expressed in TAMCs. Using a murine model with myeloid cell-specific deletion of wt-IDH1 (LysM-cre-ERT2; wt-IDH1L/L), we demonstrated that the loss of wt-IDH1 promoted inflammatory myeloid phenotypes, increased CD8+ T cells in the TME, improved survival of GBM tumor-bearing mice, and enhanced anti-tumor effects of PD1 checkpoint blockade. Further, the pharmacological inhibition of wt-IDH1 using systemic administration of a first-in-class wt-IDH1-specific, brain-penetrant small molecule inhibitor or intrathecal delivery of siRNA targeted to wt-IDH1 mirrored the functional phenotype of wt-IDH1-deleted myeloid cells, overcame ICT resistance, and promoted long-term survival and anti-tumor immune memory. Mechanistically, in myeloid cells, the genetic or pharmacological inactivation of wt-IDH1 inhibited oxidative phosphorylation, reduced myeloid cell proliferation, increased their tumoricidal activity, and altered chromatin accessibility of key transcription factors implicated in the regulation of myeloid cell states, including AP-1, IRFs, STAT1/2, NFATs, and CEPBPs. These studies define wt-IDH1 as a metabolic checkpoint of myeloid cell activation and credential the inhibition of wt-IDH1 as a novel immunotherapeutic strategy to increase ICT efficacy for patients with GBM.

IMMU-44. EXTRINSIC ELECTRICAL MODULATION OF THE GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Approximately 30 to 50% of patients with glioblastoma (GBM) present with glioma-related epilepsy (GRE). Despite morbidity associated with seizures, GRE constitutes an independent positive predictive predictor for survival in GBM. Our prior results showed that repeated seizure induction through extrinsic electrical modulation (EEM, a technique in which an electrical stimulus sufficient to produce a therapeutic seizure is applied) slows intracranial tumor growth and prolongs overall survival in immunocompetent CT-2A glioma-bearing mice. However, the overall survival advantage following EEM treatment was diminished in immunocompromised mice bearing human patient derived xenografts. We hypothesized that EEM induces immune-related tumor microenvironment (TME) changes that prolong survival in mice. METHODS C57BL/6J mice were orthotopically implanted with CT-2A xenografts and subjected to EEM of 2.0 millicoulombs or sham treatment. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed at 3 and 24 hours to determine mRNA level of inflammatory cytokines. Immunohistochemistry (IHC) and bulk RNA sequencing analysis were utilized to assess CD3+ T-cell infiltration and identify immune-related secreted genes, respectively. Flow cytometry at 3 and 96 hours was utilized to characterize tumor-infiltrating immune cell populations. Samples were subjected to Percoll gradient enrichment of immune cell populations and stained for extracellular (CD45, CD4, CD8, CD11b, F4/80, CD80, Ly6G, Ly6C, and CX3CR1) and intracellular (TNF-a, Il-1b, CCL2, and Arg1) antigens. RESULTS RT-qPCR showed an increase in pro-inflammatory cytokines TNF-α (P = 0.0087) and IL-1β (P = 0.000349). IHC analysis demonstrated an increase in intratumoral CD3+ T-cells in EEM-treated samples (P = 0.0171). From 1855 upregulated genes, 3 immune-related secreted genes (C1QTNF4, FAM19A4, and GREM2) were identified. Flow cytometric analysis showed a significant increase in CD11b+, CX3RC1+ microglia (P = 0.0306) and CD45+, CD11b+ tumor-associated myeloid cells (TAMCs) (P = 0.0192) at 3 hours post-EEM. CONCLUSIONS EEM treatment promotes an inflammatory TME with increased intratumoral microglia, TAMCs and T-cell infiltration. These inflammatory changes may prolong survival of CT-2A glioma-bearing mice.

TMIC-21. PYK2/MAPK/ERK SIGNALING PATHWAY REGULATES EXTRACELLULAR VESICLE RELEASE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203

TMIC-83. ABERRANT EGFR ACTIVATION SUPPORTS ENHANCED TUMOR-MYELOID CROSSTALK AND IMPAIRS EFFECTOR T CELL INFILTRATION AND FUNCTION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is known to display a highly suppressive tumor immune microenvironment (TIME) dominated by myeloid cells with low T cell infiltration. Aberrant epidermal growth factor receptor (EGFR) activation is observed in ~50% of GBM patients via amplification and the constitutively active EGFRvIII variant, however the impact of EGFR activation on the brain TIME and T cell-mediated anti-tumor immunity remains poorly understood. Single cell transcriptomic and receptor-ligand interaction analyses of paired newly diagnosed GBM tumor and intratumoral CD45+ immune cells revealed increased crosstalk between EGFR activated tumors and EGFR ligand-expressing tumor-associated myeloid cells. Therefore, a novel orthotopic murine EGFRvIII-expressing glioma model wherein the mEGFRvIII gene is under a tetracycline-off system (MADR-mEGFRvIII) was leveraged to identify how EGFR signaling impacts overall TIME composition and the phenotype of intratumoral and systemic T cells in vivo. Temporal flow cytometric immunophenotyping following tumor or mock (saline) implantation identified no significant shift in T cell infiltration within the brain or cervical lymph nodes (cLNs). In contrast, one week of doxycycline-mediated genetic EGFR ablation conferred a significant survival benefit and was associated with enhanced intratumoral T cell infiltration as well as diminished tumor-associated F4/80+ myeloid cells compared to vehicle treated animals. Further characterization revealed genetic EGFR ablation enhanced recruitment of CXCR3+, TNFα+, and TIGIT+ CD4 T cells to the tumor, while the prevalence of these populations contracted in CD4 and CD8 T cells within the draining cLNs. In vitro, pharmacologic EGFR inhibition enhanced antigen-specific Pmel-1 T cell killing of gp100-expressing MADR-mEGFRvIII tumor cells and was associated with increased IFNγ secretion by ELISA. Collectively, our work demonstrates that aberrant EGFR signaling promotes an immunosuppressive brain TIME with reduced T cell infiltration potentially via tumor-myeloid signaling. Targeting this oncogenic axis may therefore reprogram the TIME and directly enable enhanced trafficking of effector T cells to the tumor.

Pan-Cancer Single-Cell Transcriptomic Analysis Reveals Divergent Expression of Embryonic Proangiogenesis Gene Modules in Tumorigenesis.

Angiogenesis is indispensable for the sustained survival and progression of both embryonic development and tumorigenesis. This intricate process is tightly regulated by a multitude of pro-angiogenic genes. The presence of gene modules facilitating angiogenesis has been substantiated in both embryonic development and the context of tumor proliferation. However, it remains unresolved whether the pro-angiogenic gene modules expressed during embryonic development also exist in tumors. This study performed a pan-cancer single-cell RNA sequencing (scRNA-seq) analysis on samples from 332 patients across seven cancer types: thyroid carcinoma, lung cancer, breast cancer, hepatocellular carcinoma, colorectal cancer, ovarian carcinoma, and prostate adenocarcinoma. Data processing was carried out using the Seurat R package, with rigorous quality control to filter high-quality cells and mitigate batch effects across datasets. We used principal component analysis (PCA), shared nearest neighbor graph-based clustering, and Uniform Manifold Approximation and Projection (UMAP) to visualize cell types and identify distinct cell clusters. Myeloid cell subpopulations were further analyzed for the expression of embryonic pro-angiogenic gene modules (EPGM) and tumor pro-angiogenic gene modules (TPGM). The analysis identified nine major cell types within the tumor microenvironment, with myeloid cells consistently exhibiting elevated expression of both tumor pro-angiogenic gene modules (TPGM) and EPGM across all tumor types. In particular, myeloid cells, including macrophages and monocytes, showed high EPGM expression, indicating an active role of embryonic pro-angiogenesis pathways in tumors. A subset analysis revealed 20 distinct myeloid subtypes with varying EPGM and TPGM expression across different cancers. Treatment and disease stage influenced these gene expressions, with certain subtypes, such as HSPAhi/STAT1+ macrophages in breast cancer, displaying reduced pro-angiogenic gene activity post-treatment. This study provides evidence that tumors may exploit EPGM to enhance vascularization and support sustained growth, as evidenced by the elevated EPGM expression in tumor-associated myeloid cells. The consistent presence of EPGM in TAMs across multiple cancer types suggests a conserved mechanism wherein tumors harness embryonic angiogenic pathways to facilitate their progression. Distinct EPGM expression patterns in specific myeloid cell subsets indicate potential therapeutic targets, particularly in cases where EPGM activation contributes to resistance against anti-angiogenic therapies. These findings shed new light on the molecular mechanisms underlying tumor angiogenesis and highlight the prognostic relevance of EPGM expression in cancer, underscoring its potential as a biomarker for clinical applications.

Amplifying glioblastoma immunotherapy: T cell shielding through Nitric oxide/reactive oxygen species scavenging nanoparticles Potentiates anti-PD-1

Despite the success of immune checkpoint blockade (ICB) therapy in various cancers, its efficacy faces challenges in glioblastoma (GBM) due to the immunosuppressive cold-tumor microenvironment. The scarcity of tumor-infiltrating T cells and the suppression of T cell activity significantly limit therapeutic outcomes in GBM. Nitric oxide (NO) and reactive oxygen species (ROS) from tumor-associated myeloid cells (TAMCs) are key contributors to T cell suppression, reducing ICB therapy effectiveness. In this study, we developed NO-ROS scavenging micelles that effectively scavenge both NO and ROS, protecting T cells from their exhausting effects. This leads to a significant increase in T cell infiltration and activation. Moreover, when combined with αPD-1, the survival rate increases to 40 % up to 120 days, enhancing therapeutic efficacy compared to αPD-1 alone. This approach not only protects T cells from the inhibitory effects of NO and ROS but also has the potential to reshape the tumor microenvironment, overcoming T cell suppression in cold tumors.

Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells.

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) play a critical role in resistance to immunotherapy. In this study, we identified epidermal growth factor-like 6 (Egfl6) as a new regulator of myeloid cell functions. Our analyses indicated that Egfl6, via binding with β3 integrins and activation of p38 and SYK signaling, acts as a chemotactic factor for myeloid cells migration and promotes their differentiation towards an immunosuppressive state. In syngeneic mouse models of ovarian cancer (OvCa), tumor expression of Egfl6 increased the intra-tumoral accumulation of polymorphonuclear (PMN) MDSCs and TAMs and their expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Consistent with this, in an immune 'hot' tumor model, Egfl6 expression eliminated response to a-PD-L1 therapy, while Egfl6 neutralizing antibody decreased the accumulation of tumor-infiltrating CD206+ TAMs and PMN-MDSCs and restored the efficacy of a-PD-L1 therapy. Supporting a role in human tumors, in human OvCa tissue samples, areas of high EGFL6 expression co-localized with myeloid cell infiltration. scRNAseq analyses revealed a correlation between EGFL6 and immune cell expression of immunosuppressive factors. Our data provide mechanistic insights into the onco-immunologic functions of EGFL6 in mediating tumor immune suppression and identified EGFL6 as a potential novel therapeutic target to enhance immunotherapy in OvCa patients.

STING-Activating Polymer-Drug Conjugates for Cancer Immunotherapy.

The stimulator of interferon genes (STING) pathway links innate and adaptive antitumor immunity and therefore plays an important role in cancer immune surveillance. This has prompted widespread development of STING agonists for cancer immunotherapy, but pharmacological barriers continue to limit the clinical impact of STING agonists and motivate the development of drug delivery systems to improve their efficacy and/or safety. We developed SAPCon, a STING-activating polymer-drug conjugate platform based on strain-promoted azide-alkyne cycloaddition of a novel dimeric amidobenzimidazole (diABZI) STING prodrug to hydrophilic poly(dimethylacrylamide-co-azido-ethylmethacrylate) polymer chains through a cathepsin B-responsive linker to increase circulation time and enable passive tumor accumulation. We found that intravenously administered SAPCon accumulated at tumor sites, where it was endocytosed by tumor-associated myeloid cells, resulting in increased STING activation in the tumor tissue. Consequently, SAPCon promoted an immunogenic tumor microenvironment characterized by increased frequency of activated macrophages and dendritic cells and improved infiltration of CD8+ T cells, resulting in inhibition of tumor growth, prolonged survival, and enhanced response to anti-PD-1 immune checkpoint blockade in orthotopic breast cancer models. Collectively, these studies position SAPCon as a modular and programmable platform for improving the efficacy of systemically administered STING agonists for cancer immunotherapy.

Tumor biomechanics as a novel imaging biomarker to assess response to immunotherapy in a murine glioma model

Glioblastoma is the most common and aggressive primary malignant brain tumor with poor prognosis. Novel immunotherapeutic approaches are currently under investigation. Even though magnetic resonance imaging (MRI) is the most important imaging tool for treatment monitoring, response assessment is often hampered by therapy-related tissue changes. As tumor and therapy-associated tissue reactions differ structurally, we hypothesize that biomechanics could be a pertinent imaging proxy for differentiation. Longitudinal MRI and magnetic resonance elastography (MRE) were performed to monitor response to immunotherapy with a toll-like receptor 7/8 agonist in orthotopic syngeneic experimental glioma. Imaging results were correlated to histology and light sheet microscopy data. Here, we identify MRE as a promising non-invasive imaging method for immunotherapy-monitoring by quantifying changes in response-related tumor mechanics. Specifically, we show that a relative softening of treated compared to untreated tumors is linked to the inflammatory processes following therapy-induced re-education of tumor-associated myeloid cells. Mechanistically, combined effects of myeloid influx and inflammation including extracellular matrix degradation following immunotherapy form the basis of treated tumors being softer than untreated glioma. This is a very early indicator of therapy response outperforming established imaging metrics such as tumor volume. The overall anti-tumor inflammatory processes likely have similar effects on human brain tissue biomechanics, making MRE a promising tool for gauging response to immunotherapy in glioma patients early, thereby strongly impacting patient pathway.

Non-Coding RNA in Tumor Cells and Tumor-Associated Myeloid Cells-Function and Therapeutic Potential.

The RNA world is wide, and besides mRNA, there is a variety of other RNA types, such as non-coding (nc)RNAs, which harbor various intracellular regulatory functions. This review focuses on small interfering (si)RNA and micro (mi)RNA, which form a complex network regulating mRNA translation and, consequently, gene expression. In fact, these RNAs are critically involved in the function and phenotype of all cells in the human body, including malignant cells. In cancer, the two main targets for therapy are dysregulated cancer cells and dysfunctional immune cells. To exploit the potential of mi- or siRNA therapeutics in cancer therapy, a profound understanding of the regulatory mechanisms of RNAs and following targeted intervention is needed to re-program cancer cells and immune cell functions in vivo. The first part focuses on the function of less well-known RNAs, including siRNA and miRNA, and presents RNA-based technologies. In the second part, the therapeutic potential of these technologies in treating cancer is discussed, with particular attention on manipulating tumor-associated immune cells, especially tumor-associated myeloid cells.

Development of NR0B2 as a therapeutic target for the re-education of tumor associated myeloid cells

Immune checkpoint blockade (ICB) has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. A paper co-published in this issue describes how NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Here, we develop NR0B2 as a potential therapeutic target. NR0B2 in tumors is associated with improved survival for several cancer types including breast. Importantly, NR0B2 expression is also prognostic of ICB success. Within breast tumors, NR0B2 expression is inversely associated with FOXP3, a marker of Tregs. While a described agonist (DSHN) had some efficacy, it required high doses and long treatment times. Therefore, we designed and screened several derivatives. A methyl ester derivative (DSHN-OMe) emerged as superior in terms of (1) cellular uptake, (2) ability to regulate expected expression of genes, (3) suppression of Treg expansion using in vitro co-culture systems, and (4) efficacy against the growth of primary and metastatic tumors. This work identifies NR0B2 as a target to re-educate myeloid immune cells and a novel ligand with significant anti-tumor efficacy in preclinical models.

DIPG-51. DIFFUSE MIDLINE GLIOMAS HIJACK MYELOID CELL POPULATIONS TO INITIATE AN ANTI-INFLAMMATORY TUMOR IMMUNE MICROENVIRONMENT

Abstract BACKGROUND Diffuse midline gliomas (DMG) are universally fatal pediatric brain tumors, for which no effective treatment exists. These tumors are known for their “cold” non-inflammatory tumor-immune microenvironment (TIME), which is thought to be a major hurdle for immunotherapy efficacy. METHODS/RESULTS To further investigate the mechanisms that drive this cold environment, we allografted immunocompetent mice with Histone 3.3 Lysine 27 (H3.3K27M) and 3.1 Lysine 27 (H3.1K27M) mutant murine DMG tumors, created through intra uterine electroporation, and performed single-cell analyses on the tumors that developed. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) we mapped the single-cell RNA and protein expression profiles of the TIME of these tumors and healthy pontine regions, with a dominant focus on the myeloid cell populations. Through differential expression and pathway enrichment analyses, we found that DMG-microglia populations upregulate anti-inflammatory pathways and, over time, are pushed out by infiltrating anti-inflammatory macrophages. We confirmed these findings in single-cell sequencing data from DMG patients, taken at diagnosis and at late stage disease. Next, we established microglia cultures in vitro, by differentiating monocytes from healthy donors into microglia. We discovered that treatment of these microglia with tumor-conditioned medium, taken from primary DMG cultures, diminished their pro-inflammatory potential after stimulation with lipopolysaccharides (LPS), as observed by cytokine release arrays that showed a strong downregulation of IL-6 and TNF-alpha release, amongst others. Subsequently, using a Seahorse metabolic analyzer, we found that glycoATP potential was diminished in DMG-primed microglia after LPS stimulation, further suggesting that DMG-excreted factors are responsible for blocking the pro-inflammatory potential of microglia. CONCLUSIONS Taken together, these findings increase our understanding on how DMG cells reprogram tumor-associated myeloid cells to generate an immune-cold TIME, which will aid the scientific community to find therapeutic solutions to reverse this phenomenon and improve the efficacy of immunotherapy in these devastating malignancies.

Tumor-Associated Myeloid Cells Selective Delivery of a Therapeutic Tumor Nano-Vaccine for Overcoming Immune Barriers for Effective and Long-Term Cancer Immunotherapy.

Therapeutic cancer vaccines have the potential to induce regression of established tumors, eradicate microscopic residual lesions, and prevent metastasis and recurrence, but their efficacy is limited by the low antigenicity of soluble antigens and the immunosuppressive tumor-associated macrophages (TAMs) that promote tumor growth. In this study, a novel strategy is reported for overcoming these defenses: a dual-targeting nano-vaccine (NV) based on hepatitis B core antigen (HBcAg) derived virus-like particles (VLPs), N-M2T-gp100 HBc NV, equipped with both SIGNR+ dendritic cells (DCs)/TAMs-targeting ability and high-density display of tumor-associated antigen (TAA). N-M2T-gp100 HBc NVs-based immunotherapy has demonstrated an optimal interaction between tumor-associated antigens (TAAs) and the immune composition of the tumor microenvironment. In a melanoma model, N-M2T-gp100 HBc VLPs significantly reducing in situ and abscopal tumor growth, and provide long-term immune protection. This remarkable anti-tumor effect is achieved by efficiently boosting of T cells and repolarizing of M2-like TAMs. This work opens exciting avenues for the development of personalized tumor vaccines targeting not just melanoma but potentially a broad range of cancer types based on functionalized VLPs.

Baseline heterogeneity of myeloid and stroma cells to predict pCR, baseline lymph node (LN) metastasis, and 1-year EFS in locally advanced ESCC with neoadjuvant immunotherapy: Final results from a single-arm, phase II clinical trial (SEEK-01).

e14661 Background: Response of neoadjuvant immunotherapy can vary among patients in locally advanced ESCC, with the lack of a promising biomarker which need fully capture the heterogeneity of TME. Now, based on final results of the SEEK-01 study (2023 ASCO, P#4047), we have, for the first time, established a precise correlation from baseline TME to pathological response, baseline LN status, and 1-year EFS. Methods: In SEEK-01 study, 22 locally advanced ESCC patients (cT2-4NxM0) were enrolled. Following neoadjuvant chemotherapy (paclitaxel-albumin+carboplatin) combined with tislelizumab, patients underwent minimal invasive esophagectomy (MIE). Fresh tumor tissues were obtained via endoscopy prior to neoadjuvant therapy for scRNAseq analysis. Pathological response was evaluated using the irRVT score, leading to classification into pCR (irRVT=0%), MPR (0<irRVT<10%), and non-responder (irRVT>60%). Baseline LN status was determined based on pathological diagnosis after surgery. We identified subclusters of tumor-associated myeloid cells and stroma cells that significantly predicted pathological response, baseline LN metastasis and recurrence within 1 year. Prediction model of efficacy for neoadjuvant immunotherapy was developed using baseline expression ratios of differential subclusters. Results: Among 22 enrolled patients, 18 completed neoadjuvant therapy and MIE. 4 achieved pCR, 2 got MPR and 6 were non-responder. 12 of 18 proved baseline LN metastasis. 5 of 18 suffered recurrence within 1 year. Myeloid cells, cancer-associated fibroblast (Caf) and endothelium (Endo) shew heterogeneity that can predict pathological response, baseline LN metastasis or recurrence within 1 year. Upgrade of CXCL9+ tumor-associated-macrophage (TAM), LAMP3+mregDC3 was a strong predictor of pCR, while the upgrade of STAT1+TAM, C1q+TAM, IL1b+TAM, IL1b+ Neutrophil, C1q+ Neutrophil was a solid biomarker of non-responder. Besides, presence of PTGS1+Caf, RGS5+Caf, S100B+Caf, RND1+Endo and S100+Endo highly correlated with baseline LN metastasis and recurrence within 1 year, which was better than PET-CT. On this basis, we constructed predictive models for pathological response. With further validated by public scRNAseq and TCGA database, it showed excellent predictive ability and robustness. Conclusions: The final results of the SEEK-01 study demonstrate the differential baseline infiltration of myeloid cells that can predict pathological response of neoadjuvant immunotherapy. The characteristic changes in Caf and Endo in TME can predict occult LN metastasis and 1-year EFS. A predictive model for efficacy of neoadjuvant immunotherapy in locally advanced ESCC was developed to facilitate individualized and precise neoadjuvant therapy before treatment. Clinical trial information: NCT05807542 .

Abstract PR011: Imaging mass cytometry captures patient heterogeneity enabling BCG response stratification in non-muscle invasive bladder cancer

Abstract BCG immunotherapy is the standard of care for high-risk non-muscle invasive bladder cancer (NMIBC). The mechanisms of response to BCG are not fully understood. Patient response to BCG is highly variable with ~30-40% of individuals experiencing recurring or progressing disease after treatment within 24 months. Prognostic markers of response to treatment are not fully understood and remain insufficient for patient stratification. In order to develop an understanding of BCG response by comprehensive assessment of the tumor context. We performed single cell spatial proteomic profiling via imaging mass cytometry (IMC) on tissue microarrays of tumor biopsies from 53 bladder cancer patients (36 responding and 17 non-responding). We then validated our finding on an external cohort of whole pathology slides from 10 patients (4 responding and 6 non-responding). The IMC panel was developed based on scRNAseq profiles of bladder tumor samples to provide an unbiased cellular context of NMIBC. Additionally, IMC data is accompanied by hematoxylin and eosin (H&E) images of adjacent cuts and annotated by a clinical pathologist to distinguish tumor, immune, and stromal regions. We first developed a convolutional neural network (CNN) to reproduce pathologist annotations of tumor, stroma, and immune regions from H&E data with high accuracy (AUC>93%). We then registered IMC and annotated H&E images to train a random forest model to predict pathologist annotations using IMC data (AUC>95%). To determine the cellular composition of TMA spots, we used sparse non-negative matrix factorization (sNMF) to encode and cluster IMC data within tumor, stroma, and immune regions. NMF analysis revealed that a component defined primarily by KRT5, CD11b, and CD14, specifically within tumor-annotated regions adjoining COL1A1+ stromal regions, is a prognostic marker of poor response (p-value<0.05). This is consistent with previous findings that tumor-associated myeloid cells indicate poor prognosis. Additional NMF components of the immune annotated region showed weak associations with response. Immune regions defined by CD68, CD11b, CD31, and PDPN corresponded with poor BCG response, indicating myeloid-stromal-vascular interactions outside the tumor influence outcome, while vascular immune regions defined by high aSMA presence correspond with better response. Collectively, combining NMF components to generate a single response score for responding and non-responding patients (p-value<0.05) proved more effective at patient stratification than any single NMF component alone. The external cohort validated our random forest model for separating tumor, stroma, and immune regions, our sNMF model for clustering IMC images, and our response score (p-value<0.1). Citation Format: Ali Foroughi Pour, Dylan Baker, Santhosh Sivajothi, Bonnie Choy, Khyati Meghani, Yanni Yu, Leigh A. Fall, Benjamin Ristau, Joshua J. Meeks, Paul Robson, Jeffrey H. Chuang. Imaging mass cytometry captures patient heterogeneity enabling BCG response stratification in non-muscle invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR011.

Nanodrug modified with engineered cell membrane targets CDKs to activate aPD-L1 immunotherapy against liver metastasis of immune-desert colon cancer

Immunotherapy based on the PD-1/PD-L1 axis blockade has no benefit for patients diagnosed with colon cancer liver metastasis (CCLM) for the microsatellite stable/proficient mismatch repair (MSS/pMMR)) subtype, which is known as an immune-desert cancer featuring poor immunogenicity and insufficient CD8+ T cell infiltration in the tumor microenvironment. Here, a multifunctional nanodrug carrying a cyclin-dependent kinase (CDK)1/2/5/9 inhibitor and PD-L1 antibody is prepared to boost the immune checkpoint blockade (ICB)-based immunotherapy against MSS/pMMR CCLM via reversing the immunosuppressive tumor microenvironment. To enhance the MSS/pMMR CCLM-targeting efficacy, we modify the nanodrug with PD-L1 knockout cell membrane of this colon cancer subtype. First, CDKs inhibitor delivered by nanodrug down-regulates phosphorylated retinoblastoma and phosphorylated RNA polymerase II and meanwhile arrests the G2/M cell cycle in CCLM to promote immunogenic signal release, stimulate dendritic cell maturation, and enhance CD8+ T cell infiltration. Moreover, CDKi suppresses the secretion of immunosuppressive cytokines in tumor-associated myeloid cells sensitizing ICB therapy in CCLM. Notably, the great efficacy to activate immune responses is demonstrated in the patient-derived xenograft model and the patient-derived organoid model as well, revealing a clinical application potential. Overall, our study represents a promising therapeutic approach for targeting liver metastasis, remolding the tumor immune microenvironment (TIME), and enhancing the response of MSS/pMMR CCLM to boost ICB immunotherapy.

CAF-associated genes putatively representing distinct prognosis by in silico landscape of stromal components of colon cancer.

Comprehensive understanding prognostic relevance of distinct tumor microenvironment (TME) remained elusive in colon cancer. In this study, we performed in silico analysis of the stromal components of primary colon cancer, with a focus on the markers of cancer-associated fibroblasts (CAF) and tumor-associated endothelia (TAE), as well as immunological infiltrates like tumor-associated myeloid cells (TAMC) and cytotoxic T lymphocytes (CTL). The relevant CAF-associated genes (CAFG)(representing R index = 0.9 or beyond with SPARC) were selected based on stroma specificity (cancer stroma/epithelia, cS/E = 10 or beyond) and expression amounts, which were largely exhibited negative prognostic impacts. CAFG were partially shared with TAE-associated genes (TAEG)(PLAT, ANXA1, and PTRF) and TAMC-associated genes (TAMCG)(NNMT), but not with CTL-associated genes (CTLG). Intriguingly, CAFG were prognostically subclassified in order of fibrosis (representing COL5A2, COL5A1, and COL12A1) followed by exclusive TAEG and TAMCG. Prognosis was independently stratified by CD8A, a CTL marker, in the context of low expression of the strongest negative prognostic CAFG, COL8A1. CTLG were comprehensively identified as IFNG, B2M, and TLR4, in the group of low S/E, representing good prognosis. Our current in silico analysis of the micro-dissected stromal gene signatures with prognostic relevance clarified comprehensive understanding of clinical features of the TME and provides deep insights of the landscape.