Tumor-associated Myeloid Cells Research Articles

A Population of TIM4+FOLR2+ Macrophages Localized in Tertiary Lymphoid Structures Correlates to an Active Immune Infiltrate Across Several Cancer Types.

TIM4 has previously been associated with antitumor immunity, yet the pattern of expression and the function of this receptor across human cancer tissues remain poorly explored. Here we combined extensive immunolabeling of human tissues with in silico analysis of pan-cancer transcriptomic data sets to explore the clinical significance of TIM4 expression. Our results unveil that TIM4 is expressed on a fraction of cavity macrophages (CATIM4+MΦ) of carcinoma patients. Moreover, we uncover a high expression of TIM4 on macrophages of the T-cell zone of the carcinoma-associated tertiary lymphoid structures (TLSTIM4+MΦ). In silico analysis of a pan-cancer data set revealed a positive correlation between TIM4 expression and markers of B cells, effector CD8+ T cells, and a 12-chemokine signature defining tertiary lymphoid structure. In addition, TLSTIM4+MΦ were enriched in cancers displaying microsatellite instability and high CD8+ T-cell infiltration, confirming their association with immune-reactive tumors. Both CATIM4+MΦ and TLSTIM4+MΦ express FOLR2, a marker of tissue-resident MΦ. However, CATIM4+MΦ had a higher expression of the immunosuppressive molecules TREM2, IL10, and TGFβ as compared with TLSTIM4+MΦ. By analyzing a scRNA sequence data set of tumor-associated myeloid cells, we identified two TIM4+FOLR2+ clusters coherent with CATIM4+MΦand TLSTIM4+MΦ. We defined specific gene signatures for each subset and found that the CATIM4+ MΦ signature was associated with worse patient survival. In contrast, TLSTIM4+MΦ gene signature positively correlates with a better prognosis. Together, these data illustrate that TIM4 marks two distinct macrophage populations with distinct phenotypes and tissue localization and that may have opposing roles in tumor immunity.

C3d(g), iron nanoparticles, hemin and cytochrome c may induce oxidative cytotoxicity in tumors and reduce tumor-associated myeloid cells-mediated immunosuppression

A variety of tumors are sensitive to cell death by ferroptosis, a type of regulated necrosis characterized by iron accumulation, failure of cellular antioxidant defence systems, unrestrained accumulation of lipid peroxides and membrane rupture. Efficient removal of apoptotic and necrotic cells via tumor-associated phagocytes results in local immune suppression. Phagocytosis inhibition reduces tumor growth and metastasis. Here I hypothesize that accumulating iron nanoparticles, hemin, cytochrome c and preformed lipid peroxides in tumors combined with phagocytosis inhibition may induce the generation of oxidizing species to high levels within tumor tissue, thus causing increased oxidative damage and tumor cell death. I propose that C3d(g), the final degradation product of the third component of complement (C3) can be utilized to limit phagocytosis and stimulate anti-tumor responses. Furthermore, the plasticity and the multiple subtypes of tumor-associated myeloid cells mainly represented by tumor-associated macrophages and myeloid-derived suppressor cells hinder inhibition by targeted approaches or monotherapies. Here I provide insights into a novel, integrative therapeutic model that aims at both killing tumor cells and counteracting tumor-associated myeloid cells via the induction of excessive oxidative conditions in tumors. In conclusion, accumulating iron and lipid peroxides in both tumor- and myeloid cells and extracellularly in the tumor microenvironment combined with limiting phagocytosis due to C3d(g) may synergistically increase the oxidized burden and induce tumor cytotoxicity. Future directions include detailed suggestions on criteria for intermittent administration of the proposed therapeutics so that newly recruited myeloid cells could differentiate/be activated to functional anti-tumor phenotypes in a more antigenic tumor microenvironment. Furthermore, combination with other therapeutic forms might be promising. Optimizing the delivery of ION-containing liposomes at tumor sites is also important, thus enhancing their efficiency and safety profiles.

BSCI-20 STING EPIGENETIC SILENCING IN GLIOMAS CAN BE RESCUED BY METHYLTRANSFERASE INHIBITION: IMPLICATIONS FOR NOVEL THERAPEUTIC APPROACHES

The stimulator of interferon genes (STING) is a key component of the innate immune response to pathogenic cytosolic DNA, resulting in IRF3- and NFκB-dependent transcription of type I interferons (IFN) and pro-inflammatory cytokines. STING activation primes endogenous antitumor immunity and is disrupted in a variety of cancers. Here we investigate STING signalling in glioblastoma (GBM) patient samples. STING agonist treatment of ex vivo gliomas leads to inconsistent induction of type I IFN responses that are restricted to tumor associated myeloid cells. Indeed, single-cell transcriptome and multiplex immunofluorescence analyses demonstrate that STING expression is suppressed in neoplastic cells but not tumor-associated immune cells or stroma. Methylation analyses reveal a STING promoter region that is highly methylated in bulk tumor samples from glioma and other neuroectoderm-derived cancers, but not in most extracranial cancers. Methylation in this region strongly correlates inversely with STING RNA expression. STING epigenetic silencing is also present in normal fetal and adult brains. We demonstrate that STING expression in glioma cell lines may be rescued by decitabine, a DNA methyltransferase inhibitor (DNMTi) that is commonly used to treat hematologic malignancies. However, transduction of a STING-expressing vector into these glioma cell lines is insufficient to reconstitute STING signalling, suggesting that additional decitabine-stimulated mechanisms are necessary for STING pathway rescue. Collectively, our results suggest that epigenetic silencing of STING occurs early in brain development and may provide an immunosuppressive context for the genesis of brain tumors. Furthermore, our work raises the potential of epigenetic modulation to reconstitute STING signalling as a therapeutic strategy for glioblastoma and potentially other STING-silenced, immunologically-cold cancers.

Abstract 5542: Characterization of a long-acting IL-13 super-antagonist engineered to target tumor associated macrophages and myeloid cells

Abstract Introduction: Regulation of Th1/Th2 balance in the tumor microenvironment (TME) plays an important role in both tumor progression and response to immunotherapy. The IL-4/IL-13 pathway induces an anti-inflammatory (Th2) response by stimulating M2 skewing of tumor associated macrophage (TAM) and myeloid derived suppressor cells (MDSCs), thereby supporting tumor growth by suppressing Th1 response. Therefore, inhibition of the IL-4/IL-13 pathway has the potential to inhibit tumor growth by shifting the TME towards a pro-inflammatory response. To achieve this, we engineered a long-acting IL-13 super-antagonist (i.e., Fc-MDNA413) to target the IL-13Rα1 component of type II IL-4 receptor (IL-4Rα/IL-13α1) expressed on TAMs and MDSCs to inhibit their differentiation and expansion for immuno-oncological applications. Experimental Procedure: Studies conducted include binding analyses by Biacore/SPR, signaling analyses in IL-4/IL-13 cell reporters, in vitro cell proliferation, in vitro M1/M2 macrophage polarization and in vivo efficacy in syngeneic tumor models. Summary of Data: IL-13Rα1 and IL-4Rα constitute the type II IL-4R that drives a Th2 response upon activation by IL-4 and IL-13. Binding studies showed that in comparison to wild-type Fc-IL13, Fc-MDNA413 has higher affinity for IL-13Rα1 but lower affinity for the decoy IL-13Rα2 receptor, indicating a more favorable receptor selectivity. In a cell-based reporter assay, Fc-MDNA413 inhibits both IL-4 and IL-13 induced p-STAT6 signaling, a pathway that drives towards a Th2 response. The antagonism of Fc-MDNA413 was also observed in an IL-13 dependent TF-1 proliferation assay where dose-dependent inhibition of cell proliferation was demonstrated. In a macrophage polarization assay with human primary myeloid cells, Fc-MDNA413 inhibited both IL-4 and IL-13 induced M2 polarization in a dose-dependent manner, consistent with an inhibitory effect on IL-4/IL-13 signaling. Treatment of tumor bearing mice with Fc-MDNA413 inhibited tumor growth in multiple syngeneic tumor models (e.g., EMT6 breast cancer and KLN205 lung cancer) known to have higher percentage of TAMs and MDSCs in TME, hence demonstrating the therapeutic potential of this long-acting IL-4/IL-13 super-antagonist. Conclusion: Fc-MDNA413 is a long-acting IL-4/IL-13 super-antagonist that inhibits IL-4 and IL-13 induced signaling and M2 skewing of macrophages, resulting in tumor growth inhibition in vivo. Ongoing studies include testing in additional syngeneic tumor models, deciphering in vivo mechanism of action and investigating potential synergy with other therapies. Next generation multifunctional superkines that incorporate IL-4/IL-13 antagonism are under development to target immunologically challenging tumors. Citation Format: Aanchal Sharma, Manjunatha Ankathatti Munegowda, Fahar Merchant, Minh D. To. Characterization of a long-acting IL-13 super-antagonist engineered to target tumor associated macrophages and myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5542.

Abstract LB216: Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors

Abstract Suppressive myeloid cells inhibit antitumor immunity by preventing T cell responses. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. Our team recently identified fibronectin as a functional ligand for ILT3. The fibronectin-ILT3 interaction serves as a “stromal checkpoint” through which the extracellular matrix actively promotes myeloid cell suppression in the tumor microenvironment. We now report the pre-clinical characterization of NGM831, a humanized IgG1 (N297G) antibody that binds to ILT3 of human and cynomolgus monkey origin with high specificity and sub-nM affinity and blocks the interaction of ILT3 with both its reported ligands, fibronectin and ApoE. In a gene expression profiling experiment, treatment of tolerogenic dendritic cells with NGM831 in the presence of fibronectin decreased their expression of inhibitory and scavenger receptors (CD163, MRC1, MARCO, STAB1, LILRB2) and markers of dendritic cell immaturity (CD14, CD209, FOLR2) and increased their expression of genes involved in antigen presentation (TNFSF14, DCSTAMP, CD82, OLR1) and TH1 polarization (SPP1, TBX21), suggesting that these cells had been remodeled to a more stimulatory phenotype. Consistent with these observations, treatment of tolerogenic dendritic cells with NGM831 increased their production of pro-inflammatory cytokines in response to FcɣR ligation, restored their responsiveness to maturation stimuli such as lipopolysaccharide (LPS) and CD40 ligand (CD40L), and increased their production of chemokines that could induce the migration of other immune cells to sites of inflammation. In addition, NGM831 acted synergistically with pembrolizumab to increase the ability of dendritic cells to stimulate allogeneic T cells in mixed lymphocyte reactions. Taken together, these results demonstrate that NGM831 can block fibronectin-mediated immunosuppression and promote myeloid cell reprogramming. These data support the clinical development of the ILT3 antagonist antibody NGM831 alone and in combination with immune checkpoint blockade. NGM831 is expected to enter clinical trials for the treatment of advanced solid tumors in early 2022. Citation Format: Peirong Chen, Suzanne C. Crawley, Vicky Y. Lin, Varun Kapoor, Kevin J. Paavola, Hung-I Chen, Joshua S. Lichtman, Betty Li, Kalyani Mondal, Alan K. Kutach, Yan Wang, Daniel D. Kaplan, Julie M. Roda. Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB216.

Abstract 2053: Phenotypic and functional analyses of response of lung tumor-associated myeloid cells to systemic oncolytic virotherapy

Abstract Introduction: Oncolytic virotherapy represents a promising cancer treatment modality. One of the appeals of oncolytic virotherapy approaches is the capacity of viruses to change tumor microenvironment, frequently interpreted as de-polarization of tumor-associated macrophages (TAMs) from highly pro-tumorigenic to less pro-tumorigenic phenotypes. The goal of this study was to analyze how systemic therapy with oncolytic adenovirus AVID-317 modulates complexity, phenotypes, and pro-tumorigenic function of tumor-associated myeloid cells in a mouse model of orthotopic metastatic lung cancer. Methods: We utilized single cell RNAseq and a 37-marker mass-cytometry (Cy-TOF) analyses to explore phenotypic and functional states of distinct myeloid cell populations in the lungs of tumor-bearing mice before and after virotherapy. We further performed correlation analyses to reveal functional contribution of each identified myeloid cell population to tumor control or tumor progression after virotherapy on a large groups of mice treated with the virus (N=43) or buffer (N=23). Summary of the results: Our phenotypic immunoprofiling identified 13 discrete TAM and DCs cell populations and 5 discrete populations of granulocytic cells, which were present in the lungs of lung tumor-bearing mice prior to virotherapy treatment. We found that in all TAM populations, the expression of CD274, ARG1, IL-10, and surprisingly, CD127 correlated with a higher tumor burden. In 7 out of 13 TAM populations, virotherapy further increased the expression of these pro-tumorigenic markers. Moreover, virotherapy led to a significant increase in the abundance of TAMs that correlated with tumor burden increase. In addition, in response to virotherapy, TAMs increased the expression of pro-tumorigenic growth factors. These data indicate the virotherapy indeed activated TAMs but did not trigger their de-polarization to lesser pro-tumorigenic phenotypes. To find whether virus-induced tumor cell death may drive TAM activation, we next treated tumor-bearing mice with a UV-inactivated AVID-317 virus that lacks the capacity of killing tumor cells. Our analyses confirmed that virus-mediated tumor cells death but not the exposure of TAMs to the virus drives TAM activation. Conclusion: Our analyses showed that oncolytic virotherapy activates TAMs, which in response produce pro-tumorigenic growth factors that may promote tumor progression. Our findings do not support the universal assumption of de-polarization of TAMs to a lesser pro-tumorigenic phenotypes in response to oncolytic virotherapy. The clinical success of oncolytic virotherapy will require careful consideration of the TAM activation in response to virotherapy and finding effective drug combinations to suppress pro-tumorigenic function of myeloid cells prior to administration of viruses with potent anti-tumor activity. Citation Format: Svetlana Atasheva, Jia Yao, Dmitry Shayakhmetov. Phenotypic and functional analyses of response of lung tumor-associated myeloid cells to systemic oncolytic virotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2053.

Abstract 3366: Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer

Abstract The increased cancer incidence and associated mortality call for the development of novel treatment modalities to address needs of cancer patients, which do not respond to existing therapies. Oncolytic viruses emerge as a new modality in cancer therapy due to their natural ability to infect and kill cancer cells, and to stimulate patient's own immune system to eliminate cancer cells throughout the body. For this study, we used novel engineered oncolytic adenovirus, Ad5/35-3M (AVID-317), which demonstrated greatly-improved safety profile after systemic virus administration due to a set of targeted mutations in the adenovirus capsid (Atasheva et al, Science Translational Medicine, 2020, eabc6659). Systemic administration of AVID-317 to NCr/Nude mice with disseminated lung adenocarcinoma slowed tumor progression and significantly extended survival, compared to buffer-treated control tumor-bearing mice. In AVID-317 treated group, about 25% of mice demonstrated complete rejection of disseminated tumors and the rest of mice showed significantly extended survival. To understand the mechanism of AVID-317-mediated tumor suppression and to design more effective combination therapies, we analyzed disseminated tumors from the lungs in NCr/Nude mice using single cell RNA-seq and Mass Cytometry (Cy-TOF) after virotherapy. Our scRNA-seq analysis showed that in a subset of mice responding poorly to AVID-317 therapy, tumor-associated CSF1R-expressing myeloid cells expressed higher levels of MHC-II, Ctsb, and Arg1. In addition, CD3e+ T cells expressed functional exhaustion marker, PD-1, exclusively in mice that responded poorly to virotherapy. Mass Cytometry analysis with 37 metal isotope-labeled antibodies confirmed elevated expression of activation markers on myeloid cells after virotherapy. Next, we analyzed the efficacy of a combination therapy where in addition to AVID-317 administration, tumor-bearing mice were treated with anti PD-1 antibody to block immune-suppressive signaling in T cells, anti-CSF1R antibody to block CSF1R signaling, and anti-Asialo GM1 antibody to deplete anti-viral NK cells. Our analyses showed that combination therapy regimen significantly improved therapeutic outcomes in mice with disseminated lung cancer, by extending median survival to 95 days from 55-56 days observed in mice treated with either virus alone or combination drugs without the virus. Taken together, our study demonstrated feasibility of using AVID-317 oncolytic adenovirus for systemic therapy of disseminated lung cancer. Our study also showed that a combination of virotherapy with a defined select of immunotherapeutic drugs allows for the significant improvement of therapeutic efficacy in pre-clinical model of disseminated lung cancer. Citation Format: Jia Yao, Svetlana Atasheva, Dmitry Shayakhmetov. Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3366.

Abstract LB006: In vivo programming of myeloid cells by mRNA mediated delivery of novel Fc alpha fusion receptor activates anti-tumor immunity

Abstract Immunotherapy has revolutionized cancer treatment. However, for the majority of patients with advanced solid tumors, sustained clinical benefit has yet to be achieved. Myeloid cells such as monocytes and macrophages readily accumulate in tumors, in some cases contributing up to 75% of the tumor mass. Reprogramming circulating and tumor associated myeloid cells to activate their ability to elicit anti-tumor adaptive immunity by phagocytosis, cytokine secretion and antigen presentation is an attractive approach to harness and orchestrate systemic anti-tumor immunity. It remains challenging to specifically target and activate myeloid cells in vivo. To overcome this hurdle, we have developed a novel in vivo myeloid cell engineering platform: Fc-alpha Receptor Fusion Constructs. Unlike other chimeric antigen receptors (CARs) the construct was engineered by fusing a tumor recognition scFv with the alpha chain of human Fc receptors. The stable expression and function of these receptors requires endogenously expressed Fc receptor gamma chain, a protein with limited expression to immune cells, mostly myeloid cells. Here, we present that intravenous infusion of lipid-nanoparticle (LNP) encapsulating the Fc-alpha Receptor Fusion Construct mRNA results in the uptake and expression of the construct by myeloid cells. In immunodeficient xenograft models of hepatocellular carcinoma and triple negative breast cancer, delivery of LNP mRNA encoding for GPC3 or TROP2 targeted Fc-alpha Receptor Fusion Constructs resulted in tumor killing, confirming the ability of this approach to program myeloid cells. Furthermore, in the B16 syngeneic melanoma model, treatment with the melanoma antigen GP75 targeted Fc-alpha Receptor Fusion Constructs was also associated with the initiation of broad systemic immune responses, characterized by tumoral accumulation of activated CD8+ T cells, reduced tumor associated Tregs and activation of antigen presenting cells in spleen. Together these studies highlight the potential of Fc-alpha Receptor Fusion Construct delivered directly in vivo to program myeloid cells to recognize and kill cancer. Citation Format: Hongyun Zhao, Inna Shcherbakova, Thomas Prod’homme, Bruce McCreedy, Yuxiao Wang, Daniel Getts. In vivo programming of myeloid cells by mRNA mediated delivery of novel Fc alpha fusion receptor activates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB006.

Abstract 2911: The CEA-targeted ISAC, BDC-2034, shows preclinical efficacy associated with innate immune activation, phagocytosis, and myeloid reprogramming

Abstract Introduction: Immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors through engagement of cell surface antigens to activate tumor-associated myeloid cells and initiate a broad innate and adaptive anti-tumor immune response. We are developing the ISAC BDC-2034 to target the tumor antigen, CEA (CEACAM5), which is expressed in many solid tumors. BDC-2034 comprises our proprietary antibody, CEA1, covalently conjugated to a potent TLR7/8 agonist via a non-cleavable linker. The strong pro-phagocytic capacity and slow internalization rate of CEA1 makes it an ideal antibody for the ISAC approach. Methods: The ability of BDC-2034 to induce the secretion of pro-inflammatory cytokines was evaluated in vitro using co-cultures of tumor cells and primary immune cells (including monocytes and dendritic cells). Anti-tumor efficacy was demonstrated in vivo with xenograft and syngeneic mouse tumor models. In parallel studies, the mechanism of BDC-2034 action was assessed by quantifying intratumoral immune infiltration, cytokine levels, and transcript profile. Results: In co-cultures of CEA-expressing tumor cells and immune cells, BDC-2034 induced the secretion of cytokines and chemokines that are essential for a broad anti-tumor immune response, including IL-12p70, CXCL10, and TNFα. These effects were accompanied by myeloid cell activation as demonstrated by elevation in costimulatory surface markers such as CD40. In vivo, BDC-2034 inhibited tumor growth in multiple xenograft and syngeneic mouse tumor models having CEA expression comparable to human cancers. Consistent with the proposed mechanism of action, BDC-2034 induced intratumoral immune cell infiltration, inflammatory cytokine secretion, and myeloid re-programming in a dose-dependent and CEA-dependent fashion. The constellation of BDC-2034 effects translated to durable therapeutic activity. Conclusions: These preclinical findings demonstrate the potential of BDC-2034 to generate anti-tumor activity in CEA-expressing cancers through direct innate immune activation and the induction of adaptive anti-tumor immunity. Citation Format: Lisa K. Blum, Cecelia I. Pearson, Laughing Bear Torrez Dulgeroff, Rishali Gadkari, Angela Luo, Andrew Luo, Jennifer E. Melrose, Jess L. Nolin, Hai Li, Arthur Lee, Matthew N. Zhou, Puneet Anand, Ganapathy Sarma, Karla A. Henning, Steven J. Chapin, Shelley E. Ackerman, Romas Kudirka, Yuyi Shen, Bruce Hug, Edith A. Perez, Marcin Kowanetz, Michael N. Alonso, Brian S. Safina, David Dornan, William G. Mallet. The CEA-targeted ISAC, BDC-2034, shows preclinical efficacy associated with innate immune activation, phagocytosis, and myeloid reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2911.

Abstract 3146: EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy

Abstract Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is Epidermal growth factor-like 6 (EGFL6). EGFL6 is a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. Similarly, EGFL6 promotes endothelial cell migration and proliferation. Indicating a potential role for EGFL6 as a myeloid cell regulatory factor, we found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of Syk/ERK signaling, (i) acts as a chemotactic factor for human myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling and flow cytometry analysis of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Consistent with Egfl6 driving an immune suppressive TME, EGFL6 expression in an otherwise immune ‘hot’/anti-PD-L1 responsive tumor model completely inhibited response to anti-PD-L-1 therapy. We are currently evaluating the impact of Egfl6 neutralizing antibody on the efficacy of ICI therapy and anti-tumor immunity. Combined our data show that EGFL6 acts as a chemotactic factor to both recruit myeloid cells to the ovarian TME and subsequently promotes their differentiation to an immunosuppressive phenotype. This suggests EGFL6 is a potential novel therapeutic target to ovarian tumor mediated immunosuppression and enhance response to immune therapy in ovarian cancer patients. Citation Format: Sarah Sinno, Shoumei Bai, Claudia Coronnello, Anda Vlad, Ronald J. Buckanovich, Sandra Cascio. EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3146.

Zebrafish Xenograft Model for Studying Pancreatic Cancer-Instructed Innate Immune Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has up to half the tumor mass of tumor-associated myeloid cells. Myeloid innate immune cells play important roles in regulating cancer cell recognition and tumor growth. PDAC cells often mold myeloid cells into pro-tumoral state to fuel cancer growth and induce immune suppression. However, how tumor cells educate the innate immune responses remains largely unknown. In this study, we used four different human PDAC cell lines (PANC1, BxPC3, AsPC1, and CFPAC1) to establish the zebrafish xenograft model and investigated the interaction between pancreatic cancer and innate immune cells. The primary tumor-derived cancer cells PANC1 and BxPC3 activated innate immune anti-tumoral responses efficiently, while cancer cells from metastatic tissues AsPC1 and CFPAC1 induced an innate immune suppression and educated innate immune cells towards pro-tumoral state. Chemical conversion of innate immune cells to anti-tumoral state inhibited tumor growth for AsPC1 and CFPAC1. Moreover, genetic and pharmacological inhibition of macrophages also significantly reduced tumor growth, supporting the important roles of macrophages in innate immune suppression. REG4 expression is high in AsPC1 and CFPAC1. Knockdown of REG4 induced innate immune activation and reduced tumor growth in the xenografts, indicating that REG4 is a beneficial target for PDAC therapy. Our study provides a fast in-vivo model to study PDAC-innate immune interaction and their plasticity that could be used to study the related mechanism as well as identify new drugs to enhance immunotherapy.

Characterization of tumor antigen expression and myeloid immune profiles to inform the development of immune stimulating antibody conjugates (ISACs).

2557 Background: Immuno-oncology (IO) has historically focused on T cell-driven effects, but a growing class of myeloid therapies are under investigation. This class includes immune-stimulating antibody conjugates (ISACs), which comprise a tumor-targeting antibody conjugated to an immune-stimulating payload. ISACs may require both tumor-associated antigens and tumor-resident myeloid cells for activity. As new IO strategies such as TLR-activating ISACs are developed, understanding the myeloid landscape is relevant for cancer biology and drug development. Methods: Tumor microarrays with formalin-fixed paraffin embedded sections of breast (BC), colorectal (CRC), gastric (GC), head and neck squamous cell (HNSCC), and non-small cell lung (NSCLC) cancers were analyzed by immunohistochemistry for tumor antigens (HER2, CEA, PD-L1), myeloid cell markers (CD68/CD163/CD11c/BDCA2), and CD8+ T cells. Results: Tumor infiltrating myeloid cells and CD8+ T cells varied across indications (Table). Low densities of intra-tumoral CD8+ T cells (<100/mm2) were observed in CRC, with higher CD8+ T cell counts observed in other cohorts. In contrast to T cells, substantial myeloid infiltrates were observed across tumor types. While MSI status was associated with higher CD8+ T cell infiltration in CRC, myeloid cells were abundant in both MSI and MSS samples. Monocyte-derived (mDC) and conventional DCs (cDC), which respond strongly to TLR activation, were present in all indications, with highest numbers observed in NSCLC. HER2+ BC samples had high infiltration of plasmacytoid DCs, a key TLR7-responsive population. PD-L1 expression on immune cells was associated with higher CD8+ T cell and myeloid cell numbers. In contrast to HER2 and PD-L1, CEA expression was independent of immune infiltration. Conclusions: Abundant myeloid infiltrates were observed across solid tumors, independent of T-cell infiltration. The presence of myeloid cells in multiple tumor types offers broadscale therapeutic targets for ISACs and other myeloid-directed therapies that can activate the innate immune system as a bridge to adaptive immunity.[Table: see text]

Overcoming resistance to oncolytic virus M1 by targeting PI3K-γ in tumor-associated myeloid cells

Oncolytic viruses (OVs) have become a category of promising anticancer immunotherapeutic agents over the last decade. However, the fact that many individuals fail to respond to OVs highlights the importance of defining the barely known immunosuppressive mechanisms that lead to treatment resistance. Here we found that the immunosuppression mediated by tumor-associated myeloid cells (TAMCs) directly quenches the antitumor effect of oncolytic virus M1 (OVM). OVM induces myeloid cells to migrate into tumors and strengthens their immunosuppressive phenotypes. Mechanically, tumor cells treated with OVM secrete interleukin-6 (IL-6) to activate the phosphatidylinositol 3-kinase (PI3K)-γ/Akt axis in TAMCs, promoting infiltration of TAMCs and aggravating their inhibition on cytotoxic CD8+ T lymphocytes. Pharmacologically targeting PI3K-γ relieves TAMC-mediated immunosuppression and enhances the efficacy of OVM. Additional treatment with immune checkpoint antibodies eradicates multiple refractory solid tumors and induces potent long-term antitumor immune memory. Our findings indicate that OVM functions as a double-edged sword in antitumor immunity and provide insights into the rationale for liberating Tcell-mediated antitumor activity by abolishing TAMC-mediated immunosuppression.

Repolarization of Tumor-Infiltrating Myeloid Cells for Augmentation of CAR T Cell Therapies.

Although CAR T cell therapies have proven to be effective in treating hematopoietic cancers, their abilities to regress solid tumors have been less encouraging. Mechanisms to explain these disparities have focused primarily on differences in cancer cell heterogeneity, barriers to CAR T cell penetration of solid tumors, and immunosuppressive microenvironments. To evaluate the contributions of immunosuppressive tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) on CAR T cell efficacies, we have exploited the ability of a folate-targeted Toll-like receptor 7 agonist (FA-TLR7-1A) to specifically reactivate TAMs and MDSCs from an immunosuppressive to pro-inflammatory phenotype without altering the properties of other immune cells. We report here that FA-TLR7-1A significantly augments standard CAR T cell therapies of 4T1 solid tumors in immune competent mice. We further show that co-administration of the FA-TLR7-1A with the CAR T cell therapy not only repolarizes TAMs and MDSCs from an M2-like anti-inflammatory to M1-like pro-inflammatory phenotype, but also enhances both CAR T cell and endogenous T cell accumulation in solid tumors while concurrently increasing their states of activation. Because analogous myeloid cells in healthy tissues ar not altered by administration of FA-TLR7-1A, no systemic activation of the immune system nor accompanying weight loss is observed. These data argue that immunosuppressive myeloid cells contribute prominently to the failure of CAR T cells to eradicate solid tumors and suggest that methods to reprogram tumor associated myeloid cells to a more inflammatory phenotype could significantly augment the potencies of CAR T cell therapies.

Immune Checkpoint Inhibition in GBM Primed with Radiation by Engineered Extracellular Vesicles.

The lack of safe and effective delivery across the blood-brain barrier and the profound immune suppressive microenvironment are two main hurdles to glioblastoma (GBM) therapies. Extracellular vesicles (EVs) have been used as therapeutic delivery vehicles to GBM but with limited efficacy. We hypothesized that EV delivery to GBM can be enhanced by (i) modifying the EV surface with a brain-tumor-targeting cyclic RGDyK peptide (RGD-EV) and (ii) using bursts of radiation for enhanced accumulation. In addition, EVs were loaded with small interfering RNA (siRNA) against programmed cell death ligand-1 (PD-L1) for immune checkpoint blockade. We show that this EV-based strategy dramatically enhanced the targeting efficiency of RGD-EV to murine GBM, while the loaded siRNA reversed radiation-stimulated PD-L1 expression on tumor cells and recruited tumor-associated myeloid cells, offering a synergistic effect. The combined therapy significantly increased CD8+ cytotoxic T cells activity, halting tumor growth and prolonging animal survival. The selected cell source for EVs isolation and the presented functionalization strategy are suitable for large-scale production. These results provide an EV-based therapeutic strategy for GBM immune checkpoint therapy which can be translated to clinical applications.

The emerging role of triggering receptor expressed on myeloid cell-2 in malignant tumor

Triggering receptor expressed on myeloid cell-2 (TREM2) is a transmembrane receptor which is specifically expressed on myeloid cells. To date, TREM2 has been confirmed as a key factor in many pathologies, such as Alzheimer's disease, obesity-related metabolic syndrome, fatty liver and atherosclerosis. However, the role of TREM2 in tumors remains poorly understood. TREM2 is highly expressed in more than 200 primary and metastatic tumors, a feature that makes TREM2 a potential clinical target for tumor immunotherapy. The tumor microenvironment (TME) is the "soil" which tumors survive on and exhibits immunosuppressive characteristics. During the development of a tumor, TME will secrete various chemotactic factors to recruit myeloid cells. It is clear now that cancer progression and metastasis depend on the interactions between cancer cells and myeloid cell infiltration in TME. As an important receptor involved in inflammatory suppression signaling pathways, TREM2 may play an important role in immune escape by the tumor. Recently, several studies have illustrated that TREM2 expressed on tumor infiltrated myeloid cells acts as a crucial regulator of the antitumor immune response. In this review, we systematically summarize recent publications about the latest advances in knowledge of TREM2 in cancer, especially focusing on its role in tumor associated myeloid cells and tumor immunotherapy.

Tumor-associated macrophages, dendritic cells, and neutrophils: biological roles, crosstalk, and therapeutic relevance.

Tumor-associated myeloid cells constitute a series of plastic and heterogeneous cell populations within the tumor microenvironment (TME), and exhibit different phenotypes and functions in response to various microenvironmental signals. In light of promising preclinical data indicating that myeloid-based therapy can effectively suppress tumor growth, a series of novel immune-based therapies and approaches are currently undergoing clinical evaluation. A better understanding of the diversity and functional roles of different myeloid cell subtypes and of how they are associated with TME remodeling may help to improve cancer therapy. Herein, we focus on myeloid cells and discuss how tumor cells can simultaneously reprogram these cells through tumor-derived factors and metabolites. In addition, we discuss the interactions between myeloid cells and other cells in the TME that have the potential to directly or indirectly regulate tumor initiation, invasion, or angiogenesis. We further discuss the current and future potential applications of myeloid cells in the development of focused therapeutic strategies in cancer treatment.

VEGFR2 activity on myeloid cells mediates immune suppression in the tumor microenvironment.

Angiogenesis, a hallmark of cancer, is induced by vascular endothelial growth factor–A (hereafter VEGF). As a result, anti-VEGF therapy is commonly used for cancer treatment. Recent studies have found that VEGF expression is also associated with immune suppression in patients with cancer. This connection has been investigated in preclinical and clinical studies by evaluating the therapeutic effect of combining antiangiogenic reagents with immune therapy. However, the mechanisms of how anti-VEGF strategies enhance immune therapy are not fully understood. We and others have shown selective elevation of VEGFR2 expression on tumor-associated myeloid cells in tumor-bearing animals. Here, we investigated the function of VEGFR2+ myeloid cells in regulating tumor immunity and found VEGF induced an immunosuppressive phenotype in VEGFR2+ myeloid cells, including directly upregulating the expression of programmed cell death 1 ligand 1. Moreover, we found that VEGF blockade inhibited the immunosuppressive phenotype of VEGFR2+ myeloid cells, increased T cell activation, and enhanced the efficacy of immune checkpoint blockade. This study highlights the function of VEGFR2 on myeloid cells and provides mechanistic insight on how VEGF inhibition potentiates immune checkpoint blockade.

Abstract P104: Therapeutic targeting of TREM1 with PY159 promotes myeloid cell reprogramming and unleashes anti-tumor immunity

Abstract Myeloid cells present in the tumor microenvironment can exist in immunosuppressive states that impede productive anti-tumor immunity. One strategy for targeting these immunosuppressive mechanisms is reprogramming of myeloid cells from immunosuppressive to immunostimulatory, resulting in the removal of the immune inhibition and unleashing of anti-tumor immunity. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor expressed primarily on neutrophils and subsets of monocytes and tissue macrophages. TREM1 signals through the association with DAP12 adaptor protein and mediates proinflammatory signaling, amplifies the host immune response to microbial pathogens, and has been implicated in the development of acute and chronic inflammatory diseases. TREM1 is also enriched in tumors, specifically on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, we developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody. We found that PY159 does not deplete TREM1-expressing cells, but rather acts as a TREM1 agonist. In vitro human blood assays showed that PY159 treatment upregulated activation markers on monocytes and stimulated neutrophil chemotaxis, as assayed by flow cytometry, transcriptional analysis, and in vitro migration assays. Furthermore, PY159 induced a selective set of proinflammatory cytokines and chemokines, which was dependent on PY159 afucosylation. We validated TREM1 expression in human tumors by single-cell RNAseq, immunohistochemistry, and flow cytometry, and found that it is expressed on myeloid populations, including tumor-associated neutrophils (TAN), tumor-associated macrophages (TAM), and monocytic myeloid-derived suppressive cells (mMDSC). We showed that PY159 can also induce proinflammatory cytokines and chemokines in dissociated human tumors in vitro, demonstrating that PY159 can reprogram tumor-associated myeloid cells. Finally, in vivo treatment of mice with a surrogate anti-mouse TREM1 antibody, PY159m, promoted anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with checkpoint inhibitors, such as anti-PD-1 antibody. Together, these results demonstrate that therapeutic targeting of TREM1 with a TREM1 agonist antibody, PY159, promotes myeloid cell reprogramming and anti-tumor immunity. PY159 safety and tolerability have been demonstrated in non-human primates, and safety and efficacy of PY159 are currently being evaluated in first-in-human clinical trial (NCT04682431) including solid tumors that are resistant and refractory to standard of care therapies. Citation Format: Erin Mayes, Vladi Juric, Mikhail Binnewies, Pamela Canaday, Tian Lee, Subhadra Dash, Joshua L. Pollack, Joshua Rudolph, Vicky Huang, Xiaoyan Du, Nadine Jahchan, Asa J. Ramoth, Shilpa Mankikar, Manith Norng, Carlos Santamaria, Kevin P. Baker, Linda Liang. Therapeutic targeting of TREM1 with PY159 promotes myeloid cell reprogramming and unleashes anti-tumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P104.

Abstract P158: Phenotypic analysis of myeloid cells in a 3D image-based repolarization assay with tumor spheroids

Abstract Background The immunosuppressive tumor microenvironment (TME) involves multiple cell types and a better understanding of the interplay between these cells could potentially unleash the full potential of many different types of immunotherapies. Tumor infiltrating myeloid cells have both cancer-restraining and cancer-promoting functions. Therefore, to further increase the biological relevance of in vitro platform, we incorporated the myeloid cell compartment into 3D co-cultures of tumor cells and T cells to measure the effects of immune-modulators. Using our proprietary image analysis software and machine learning, a set of morphological features was identified that allowed discrimination between undifferentiated monocytes, M1 and M2 macrophages, and dendritic cells. In addition, the phenotypic profiles of the myeloid cells could be analyzed in the presence of the tumor supernatants or in co-cultures with tumor cells. This assay allows a better understanding of the suppressive tumor microenvironment including multiple cell types and is suitable to test different cancer immunotherapies. Materials and Methods Different myeloid cell populations were generated from healthy PBMCs. Polarized M1 and M2 macrophages, DCs, and undifferentiated monocytes were then co-cultured with tumor conditioned media, spheroids derived from different cancer cell lines, or colorectal cancer organoids (CRC), growing in protein hydrogel in 384 well-plates for 1-7 days. In addition, purified T cells were also incorporated in these cultures. The cellular interactions were visualized using high-content microscopy and OMiner® software, which was trained to identify phenotypic profiles of different myeloid cell populations in 3D, was used to quantify their similarity to the defined subsets upon treatment. Results Myeloid cell populations were classified according to their phenotypic features identified by 3D image analysis, which verified the repolarization of M2 macrophages by their shift into phenotypic space of the M1 type macrophages upon treatment with CSF1r inhibitor or STING agonist. Repolarization was confirmed with an increased similarity score towards M1 macrophages upon treatment. This approach was further used to reveal the different effects of tumor cells and their immunosuppressive TME on myeloid cell phenotypes, showing that each tumor differently influenced the repolarization of the myeloid cells. In addition, triple co-cultures with T cells revealed the suppressive effect of tumor associated myeloid cells on the proliferation and infiltration of T cells. Conclusions The phenotypic analysis of different myeloid cells in 3D co-cultures could be visualized and quantified elucidating the bi-directional interplay between tumor and immune cells, and the functional reprograming of the suppressive tumor associated population towards an M1 phenotype induced by drug candidates. This advanced platform for testing cancer immunotherapies combines the ability to examine the complexity of the TME with the robustness of a high-throughput screening platform. Citation Format: Gera Goverse, Nataliia Beztsinna, Benjamin Visser, Tomas Veenendaal, Marjan van de Merbel, Emma Spanjaard, Ashgard Weterings, Kuan Yan, Leo Price, Lidia Daszkiewicz. Phenotypic analysis of myeloid cells in a 3D image-based repolarization assay with tumor spheroids [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P158.