Tumor-associated Myeloid Cells Research Articles

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.

Abstract 5304: ABL407, a LILRB4x4-1BB bispecific antibody with a wild type Fc, exhibits potent antitumor activity by modulating immune system in multiple ways involving T cells, myeloids, and regulatory T cells

Abstract Tumor-associated myeloid cells such as myeloid-derived suppressor cells (MDSC) accumulate in the tumor microenvironment (TME) and suppress anti-tumor immune responses in a broad range of cancers. LILRB4 (Leukocyte Immunoglobulin-Like Receptor Subfamily B Member 4), an emerging myeloid target, is specifically expressed in tumor-associated myeloid cells and suppresses the immune response, thus promoting cancer progression and metastasis. ABL407 is a First-in-Class bispecific antibody designed to simultaneously target LILRB4 and 4-1BB, enabling LILRB4 expression-dependent 4-1BB activation. By antagonizing LILRB4 signaling, ABL407 inhibited LILRB4 mediated immunosuppression, restoring T cell activity. In addition, ABL407 inhibited fibronectin, a potential LILRB4 ligand, -mediated suppression of myeloid cells. To evaluate the efficacy of ABL407, we established EL4 murine tumor model overexpressing LILRB4 (EL4/LILRB4) in h4-1BB transgenic mice and hLILRB1/4-h4-1BB triple transgenic mice. We observed LILRB4 was overexpressed in M-MDSC in the blood and liver-metastasized tumors. A significant decrease of M-MDSC population and tumor growth inhibition was observed in mice treated with anti-LILRB4 antibody and ABL407. ABL407 treatment resulted in a proportion increment of T cells. However, the percentage of Treg cells and M-MDSC in the liver and blood was reduced by ABL407 treatment in hLILRB1/4-h4-1BB triple transgenic mice. Mice with complete remission (CR) were further protected from the rechallenge of previously exposed tumor after 3 months of cessation of ABL407 treatment, implicating that immunological memory might be generated. In conclusion, our findings suggest that ABL407, LILRB4x4-1BB bispecific antibody, may be a promising strategy for the treatment of cancer. It can be applied as a "LILRB4 antagonist" as well as a "LILRB4-dependent 4-1BB agonist" across a broad spectrum of cancers. Citation Format: Minkyu Seon, Yangsoon Lee, Hyunseong Youn, Yelim Park, Sora Kim, Kyungjin Park, Jonghwa Won. ABL407, a LILRB4x4-1BB bispecific antibody with a wild type Fc, exhibits potent antitumor activity by modulating immune system in multiple ways involving T cells, myeloids, and regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5304.

Abstract 2902: CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Background: Although recent advances in immunotherapy for metastatic melanoma have transformed the treatment paradigm for a disease that once carried a grim prognosis, nearly 50% of patients fail to reach a complete response to immune checkpoint blockade (ICB) due to multiple mechanisms of tumor-induced immune resistance. These challenges are amplified in the CNS tumor microenvironment (TME) where metastatic brain melanoma (MBM) demonstrates diminished clinical benefit to ICB treatment. A deeper understanding of these mechanisms are required to improve therapeutic response to ICB for these patients. Our group has previously identified that aberrant activation of the myddosomal cascade through downstream interleukin (IL)-1 receptor-associated kinase (IRAK-4) signaling results in chronic stimulation of MAPK and NF-κB in MBM. Further investigation reveals myddosome signaling emanates in large part from the TME, resulting in reflexive immune suppression. CA-4948 (emavusertib), a small molecule inhibitor of IRAK-4, is capable of reaching therapeutic dose levels in the brain where it demonstrates on-target inhibition of this pathway. These data highlight a unique therapeutic opportunity where selective inhibition of immune-suppressive signaling via targeted IRAK-4 blockade may sensitize MBM to anti-PD-1 ICB. Methods Multispectral imaging of patient MBM samples were analyzed for myddosome expression in the TME. Melanoma preclinical models, including both primary site (cutaneous) and metastatic brain disease, were evaluated for tumor burden and survival in response to CA-4948 treatment alone and in combination with anti-PD-1 ICB. Immunological responses of tumor to treatment were evaluated in two transgenic reporter models, CCR2RFPCX3CR1GFP and IFNγIRES-EYFP, to determine both the distribution and activation of infiltrating immune cells. Tumor-associated myeloid cells were assessed for phenotypic markers by multiparameter flow cytometry. Results Myddosome signaling was found to be elevated in tumor-associated immune cells in patient MBM specimens. Treatment of preclinical melanoma with CA-4948 combined with anti-PD-1 ICB resulted in significantly reduced tumor growth and improved survival in three independent models. CA-4948 treatment was associated with reduced tumor recruitment of peripherally derived tumor-associated macrophages, as well as increased lymphocyte infiltration and improved pro-inflammatory signaling measured by increased IFNγ expression in the combinatorial setting. Conclusion Targeted inhibition of IRAK-4 with CA-4948 improves immune surveillance and activation in preclinical models when combined with anti-PD-1 ICB, resulting in reduced tumor growth and increased survival. Thus, CA-4948 may be an effective adjuvant treatment strategy alongside anti-PD-1 ICB for the treatment of metastatic melanoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Amanda M. Acevedo, Jeet A. Patel, Savannah L. Carpenter, Alisha Bhatia, Reinhard W. von Roemeling, Jamison Hoffman, Maureen Lane, Duane A. Mitchell. CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2902.

Abstract 5287: Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids

Abstract Gliomas, a type of brain tumor originating from glial cells, comprise a heterogenous group of neoplasms. High-grade gliomas, in particular, present significant therapeutic challenges in the clinic. Unlike other solid tumors, the heterogenous glioma microenvironment is heavily enriched with tumor associated suppressor cells including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which can account for 30-40% of the cellular composition1. These pro-tumorigenic myeloid cells are thought to contribute to the resistance of glioma to immunotherapy. Efforts to target the suppressive nature of these dominating cell types are in development to yield more therapeutic options for patients with these devastating diseases. Constitutive activation of STAT3 in gliomas has been associated with increased tumor progression and reduced immune response. We have observed in other cancer models that a phenotypic switch occurs from STAT3 pathway activation in 3D spheroid culture to STAT3 inactivation when cultured in 2D. Therefore, we sought to determine the role of STAT3 activation and its role in the interplay between glioma and their immune suppressive environment using a 3D spheroid model. We modified our commercially available test platform, 3D-Predict™ Glioma, such that it is conducive to monitoring immune cell function. Newly diagnosed treatment naive primary samples from HGG patients were dissociated and cryogenically preserved and banked. Samples were profiled for the abundance of tumor associated suppressive myeloid cells using flow cytometry. We determined that relative proportions of tumor suppressor cells were detectable and varied across HGG primary patient samples with TAMs being the predominate immune cell type identified. Pro-tumorigenic TAM markers including CD163 and CD206 were correlated with temozolomide response and were associated with poor patient prognosis. When we evaluated the role of p-STAT in HGG primary samples, p-STAT was detected at baseline and abundance was related back to the presence of tumor associated immune suppressor cells. The impact of STAT3 signaling blockade by the inhibitor WP1066 was determined ex vivo, and efficacy was related back to MGMT promotor methylation status and patient clinical outcome. We detected shifts in cytokine signatures following treatment of HGG 3D spheroid models with WP1066 which are known to target tumor associated suppressor cell functions. This work demonstrates the critical role suppressive immune cells play in HGG clinical response. Given p-STAT can be altered in 2D culture compared to 3D culture, ex vivo therapeutic response profiling of HGG samples is necessary using the more physiologically relevant spheroid culture model. 1.Hambardzumyan et al. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 19, 20-27 (2016). Citation Format: Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers, Kathryn M. Appleton. Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5287.

Abstract 6535: Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity

Abstract Immune checkpoint inhibitors (ICB) have significantly improved outcomes across the spectrum of cancers, especially in patients with metastatic melanoma or non-small cell lung cancer (NSCLC). Despite encouraging efficacy in some patients, the development of primary and acquired resistance and severe toxicity associated with these treatment regimens, together with the overall low response rates, have limited their widespread use. Mining publicly available transcriptomic datasets, we have demonstrated that the response to ICB in patients with melanoma is influenced by the functionality of tumor associated macrophages (TAMs). Thus, it is of significance that we have demonstrated that 17-β estradiol (E2), working through myeloid cell intrinsic estrogen receptor alpha (ERα), promotes tumor growth in preclinical mouse models by polarizing tumor associated macrophages towards an immune-suppressive state in a manner that suppresses CD8+T cell functionality and limits ICB efficacy independent of gender. Transcriptomic analysis of tumor infiltrating myeloid cells isolated from E2-treated syngeneic tumors revealed that the type I interferon (IFN) signaling pathway was suppressed and that this activity could be reversed by the addition of fulvestrant a Selective Estrogen Receptor Downregulator. The importance of type I IFN signaling in E2-regulated tumor growth was confirmed in syngeneic mouse tumor models (B16F10-melanoma and LLC1-NSCLC cells) by showing that inhibiting type I interferon receptor (IFNAR) activity using blocking antibodies reversed the protective effects of E2 deprivation (ovariectomy) on tumor growth. Further, using in vitro cultures of macrophages from mouse bone marrow or peripheral blood mononuclear cells from deidentified human donors, we demonstrated that E2-dependent inhibition of type I IFN signaling could be attributed to increased efferocytosis by myeloid cells. These findings were recapitulated in vivo using an IFN reporter mouse (Mx1-egfp) implanted with labelled tumor cells (mCherry-Spectrin labelled LLC1), where we demonstrated that E2 mediated-suppression of myeloid cell intrinsic interferon signaling within the tumor microenvironment is contingent upon their engulfment of apoptotic cancer cells. Mechanistically, this enhanced efferocytosis was attributed to increased expression of the fractalkine receptor (CX3CR1) in E2-treated myeloid cells- as either genetic or pharmacological depletion of CX3CR1 in mouse reversed E2-mediated tumor growth (B16F10 and LLC1), myeloid cell polarization (from immunosuppressive to proinflammatory), myeloid cell intrinsic type I IFN signaling and reinvigorated CD8+T cell function. Taken together, our results highlight the potential clinical utility of targeting the E2/ER-CX3CR1 pathway as a means to enhance response to ICBs. Citation Format: Binita Chakraborty, Michael Brown, Prabuddha Chakraborty, Aditi Goyal, Ching Yi Chang, Donald McDonnell. Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6535.

Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging.

Deciphering cellular components and the spatial interaction network of the tumor immune microenvironment (TIME) of solid tumors is pivotal for understanding biologically relevant cross-talks and, ultimately, advancing therapies. Multiplexed tissue imaging provides a powerful tool to elucidate spatial complexity in a holistic manner. We established and cross-validated a comprehensive immunophenotyping panel comprising over 121 markers for multiplexed tissue imaging using MACSima™ imaging cyclic staining (MICS) alongside an end-to-end analysis workflow. Applying this panel and workflow to primary cancer tissues, we characterized tumor heterogeneity, investigated potential therapeutical targets, conducted in-depth profiling of cell types and states, sub-phenotyped T cells within the TIME, and scrutinized cellular neighborhoods of diverse Tcell subsets. Our findings highlight the advantage of spatial profiling, revealing immunosuppressive molecular signatures of tumor-associated myeloid cells interacting with neighboring exhausted, PD1high T cells in the TIME of hepatocellular carcinoma (HCC). This study establishes a robust framework for spatial exploration of TIMEs in solid tumors and underscores the potency of multiplexed tissue imaging and ultra-deep cell phenotyping in unraveling clinically relevant tumor components.

Shaping the immune-suppressive microenvironment on tumor-associated myeloid cells through tumor-derived exosomes.

Tumor-associated myeloid cells (TAMCs) play a crucial role in orchestrating the dynamics of the tumor immune microenvironment. This heterogeneous population encompasses myeloid-derived suppressor cells, tumor-associated macrophages and dendritic cells, all of which contribute to the establishment of an immunosuppressive milieu that fosters tumor progression. Tumor-derived exosomes (TEXs), small extracellular vesicles secreted by tumor cells, have emerged as central mediators in intercellular communication within the tumor microenvironment. In this comprehensive review, we explore the intricate mechanisms through which TEXs modulate immune-suppressive effects on TAMCs and their profound implications in cancer progression. We delve into the multifaceted ways in which TEXs influence TAMC functions, subsequently affecting tumor immune evasion. Furthermore, we elucidate various therapeutic strategies aimed at targeting TEX-mediated immune suppression, with the ultimate goal of bolstering antitumor immunity.

78 Targeting tumor-promoting myeloid cell functions in head & neck cancer

Myeloid cells are a major component of immune cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. Tumor-associated myeloid cells, such as neutrophils (or so-called MDSCs: suppressory neutrophils) contribute to cancer progression via multiple mechanisms. Neutrophils are the most abundant myeloid blood cells, which have been shown to exert heterogeneous phenotypic and functional states: reaching from anti- to protumoral. On the one hand these cells can suppress anti-tumor immune responses, support tumor angiogenesis or metastatic dissemination. On the other hand, neutrophils actively kill tumor cells, or activate T-cell dependent immune responses.There is still little information on tumor-infiltrating neutrophils and their clinical relevance. Nevertheless, neutrophils were demonstrated to be associated with poor clinical outcome in patients with different kinds of cancer, including renal cancer, non-small-cell lung carcinoma, melanoma and head and neck cancer. Enrichment of neutrophils was also associated with metastases in various tumor entities. The recruitment of neutrophils within the tumor microenvironment is strictly regulated by chemokines, such as CXCL2 or CXCL8/IL-8.Neutrophil activity strongly depends on the stimulus present in the microenvironment. Since these cells are highly plastic and can easily alter their functionality, they are an ideal target for designing anti-tumor therapies. However, most of therapeutic strategies to target neutrophils in tumor-bearing hosts are dealing with the blocking of their migration into the tumor site. The efficiency of such approaches is, however, limited, as it does not address heterogeneity of these cells, and thus depletes as well antitumoral cells, leading to cancer progression and infectious complications.Given a critical role of neutrophils in tumor progression, we tested several alternative strategies to neutralize pro-tumoral activity of these cells, including reprogramming and inhibition of their immunosuppressive functions. Our recent data using own designed CCL2 peptide MyeloMIB show promising therapeutic results in head and neck cancer preclinical models. Application of this peptide leads not only to the specific inhibition of neutrophil migration into tumor tissue, but also reprograms these cells into anti-tumoral state. These results suggest a dual-edged role of neutrophils as essential regulators of anti-cancer immune responses and argue for approaches fostering anti-cancer activity of these cells, not only their migration, during cancer immunotherapy.

Immuno-PET for Glioma Imaging: An Update

Despite significant advances in glioma diagnosis and treatment, overall outcomes remain suboptimal. Exploring novel therapeutic avenues show promise in advancing the field. Theranostics, an evolving discipline integrating diagnosis and therapy, emerges as a particularly auspicious approach. However, an unmet need exists for glioma-associated biomarkers as theranostic targets. Immuno-positron emission tomography (Immuno-PET), a pioneering method uniting PET diagnostic precision with antibody specificity, holds potential for identifying cancer-associated biomarkers. This review aims to provide an updated overview of immuno-PET applications in gliomas. Notably, [44Sc]-CHX-A″-DTPA-Cetuximab-Fab targeting Epidermal Growth Factor Receptor (EGFR) has displayed promise in glioma xenografts, enabling potential imaging at 4 h post-injection. Similarly, [89Zr]-bevacizumab targeting vascular endothelial growth factor (VEGF) yielded encouraging results in preclinical models and a pioneering clinical trial for pediatric patients with diffuse intrinsic pontine glioma (DIPG). Several cell differentiation markers, including CD146, indicative of tumor aggressiveness, and CD11b, reflecting tumor-associated myeloid cells (TAMCs), proved effective targets for immuno-PET. Additionally, immuno-PET directed at prostate-specific antigen (PSMA) demonstrated efficacy in imaging glioma-associated neovasculature. While holding promise for precise diagnosis and treatment guidance, challenges persist in achieving target specificity and selecting suitable radionuclides. Further studies are imperative to advance the field and bridge a translational gap from bench to bedside.