Immunosuppressive M2 Macrophages Research Articles

TMIC-50. IMPACT OF ZOTIRACICLIB ON GLIOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Zotiraciclib, an orally administered multi-kinase inhibitor, has shown anti-glioma effect via suppression of transcriptional process and mitochondrial function. Here, we investigate the impact of zotiraciclib on the glioma immune microenvironment. METHODS Mouse glioma was generated by intracranial injection of mouse syngeneic cells (IDH1-wildtype NPA cells or IDH1R132H-mutant NPAI cells) in C57BL/6 mice. Zotiraciclib was administered (30 mg/kg, intraperitoneal injection, twice per week) starting seven days after tumor implantation. NPA and NPAI tumors were dissected after 10 and 22 days’ treatment, respectively. Immune cells in the tumor tissue were isolated and examined using flow cytometry. Primary human CD8+ cells and CD14+ monocytes isolated from the blood of a healthy donor were used to further investigate the effect of zotiraciclib (15 nM and 50 nM) on human cytotoxic T cell activation and immunosuppressive M2 macrophage polarization, respectively, using flow cytometry. RESULTS No significant difference in the abundance of T cells, myeloid cells, NK cells, NKT cells, DCs, and B cells between zotiraciclib-treated and vehicle-treated groups was detected in neither NPA nor NPAI tumors. The treatment-induced change in the abundance of cytotoxic T cells, helper T cells, macrophages, monocytes, neutrophils, M-MDSCs, PMN-MDSCs, Tregs, and M2 macrophages wasn’t observed. Zotiraciclib reduced the expansion of human CD8+ cytotoxic T cells, but did not affect the expression of CD25, HLA-DR, TNFα and IFNγ after activation in vitro. The viability of human M2 macrophages during differentiation and polarization was not affected, while the expression of CD163 and CD206 was reduced by zotiraciclib. CONCLUSIONS The immunophenotyping performed for this study demonstrates that zotiraciclib does not alter the mouse glioma immune microenvironment. Complementary studies using human immune cells shows that zotiraciclib does not suppress the activation of cytotoxic T cells but reduces the polarization of immunosuppressive M2 macrophages, supporting combining zotiraciclib and immune stimulatory approach in glioma treatment.

Biomarker Analyses Investigating Disease Biology and Associations with Outcomes in the JAVELIN Merkel 200 Trial of Avelumab in Metastatic Merkel Cell Carcinoma.

Avelumab (anti-PD-L1) became the first approved treatment for metastatic Merkel cell carcinoma (mMCC) based on results from the phase II JAVELIN Merkel 200 trial. In this study, we report exploratory biomarker analyses from the trial. Patients with mMCC (n = 88) with or without prior first-line chemotherapy received avelumab 10 mg/kg every 2 weeks. We conducted analyses on somatic mutations, mutational signatures, and tumor mutational burden using paired whole-exome sequencing. Additionally, we examined gene and gene set expression, immune content from RNA sequencing profiles, as well as tumor PD-L1 and CD8 statuses from IHC and CD8 status from digital pathology. Tumors positive for Merkel cell polyomavirus (MCPyV) were characterized by an absence of driver mutations and a low tumor mutational burden, consistent with previous studies. A novel MCPyV-specific host gene expression signature was identified. MCPyV+ tumors had increased levels of immunosuppressive M2 macrophages in the tumor microenvironment, which seemed to correlate with PD-L1 expression; high CD8+ T-cell density in these tumors did not predict response to avelumab. Conversely, in patients with MCPyV- tumors, higher CD8+ T-cell density seemed to be associated with response to avelumab. Mutations in several genes were associated with treatment outcomes. Compared with tumors sampled before chemotherapy, tumors sampled after chemotherapy had downregulated gene signatures for immune responses, including reduced expression of IFNγ-related pathways. Levels of activated dendritic cells in responding patients were higher in patients assessed after versus before chemotherapy. Exploratory analyses provide insights into mMCC biology and potential associations with response to avelumab. Chemotherapy seems to negatively modulate the immune microenvironment.

Dietary restriction and fasting alleviate radiation-induced intestinal injury by inhibiting cGAS/STING activation

Radiation injury to the intestine is one of the most common complications in patients undergoing abdominal or pelvic cavity radiotherapy, limiting the clinical application of this treatment. Evidence shows the potential benefits of dietary restriction in improving metabolic profiles and age-related diseases. The present study investigated the effects and mechanisms of dietary restriction in radiation-induced intestinal injury. The mice were randomly divided into the control group, 10 Gy total abdominal irradiation (TAI) group, and groups pretreated with 30% caloric restriction (CR) for 7 days or 24 h fasting before TAI. After radiation, the mice were returned to ad libitum. The mice were sacrificed 3.5 days after radiation, and tissue samples were collected. CR and fasting reduced radiation-induced intestinal damage and promoted intestinal recovery by restoring the shortened colon length, improving the impaired intestinal structure and permeability, and remodeling gut microbial structure. CR and fasting also significantly reduced mitochondrial damage and DNA damage, which in turn reduced activation of the cyclic GMP-AMP synthase/stimulator of interferon gene (cGAS/STING) pathway and the production of type I interferon and other chemokines in the jejunum. Since the cGAS/STING pathway is linked with innate immunity, we further showed that CR and fasting induced polarization to immunosuppressive M2 macrophage, decreased CD8+ cytotoxic T lymphocytes, and downregulated proinflammatory factors in the jejunum. Our findings indicated that CR and fasting alleviate radiation-induced intestinal damage by reducing cGAS/STING-mediated harmful immune responses.

Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect

BackgroundThe dynamic interplay between tyrosine kinase inhibitors (TKIs) and the tumor immune microenvironment (TME) plays a crucial role in the therapeutic trajectory of non-small cell lung cancer (NSCLC). Understanding the...

Phosphoglycerate mutase 1 promotes breast cancer progression through inducing immunosuppressive M2 macrophages.

Immunosuppressive tumor microenvironment (TME) contributes to tumor progression and causes major obstacles for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a key enzyme involved in cancer metabolism while its role in remodeling TME remains unclear. In this study, we reported that PGAM1 suppression in breast cancer (BC) cells led to a decrease in M2 polarization, migration, and interleukin-10 (IL-10) production of macrophages. PGAM1 regulation on CCL2 expression was essential to macrophage recruitment, which further mediated by activating JAK-STAT pathway. Additionally, the CCL2/CCR2 axis was observed to participate in PGAM1-mediated immunosuppression via regulating PD-1 expression in macrophages. Combined targeting of PGAM1 and the CCL2/CCR2 axis led to a reduction in tumor growth in vivo. Furthermore, clinical validation in BC tissues indicated a positive correlation between PGAM1, CCL2 and macrophage infiltration. Our study provides novel insights into the induction of immunosuppressive TME by PGAM1 and propose a new strategy for combination therapies targeting PGAM1 and macrophages in BC.

GATA6 identifies an immune-enriched phenotype linked to favorable outcomes in patients with pancreatic cancer undergoing upfront surgery

This study underscores GATA6's role in distinguishing classical and basal-like pancreatic ductal adenocarcinoma (PDAC) phenotypes. Retrospective studies associate GATA6 immunohistochemistry (IHC) expression with survival outcomes, warranting prospective validation. In a prospective treatment-naive cohort of patients with resected PDAC, GATA6 IHC proves a prognostic discriminator, associating high GATA6 expression with extended survival and the classical PDAC phenotype. However, GATA6's prognostic significance is numerically lower after gemcitabine-based neoadjuvant chemoradiotherapy compared to its significance in patients treated with upfront surgery. Furthermore, GATA6 is implicated in immunomodulation, although a comprehensive investigation of its immunological role is lacking. Treatment-naive PDAC tumors with varying GATA6 expression yield distinct immunological landscapes. Tumors highly expressing GATA6 show reduced infiltration of immunosuppressive regulatory Tcells and M2 macrophages but increased infiltration of immune-stimulating, antigen-presenting, and activated Tcells. Our findings caution against solely relying on GATA6 for molecular subtyping in clinical trials and open avenues for exploring immune-based combination therapies.

Tea polyphenol-engineered hybrid cellular nanovesicles for cancer immunotherapy and androgen deprivation therapy.

Androgen deprivation therapy (ADT) is a crucial and effective strategy for prostate cancer, while systemic administration may cause profound side effects on normal tissues. More importantly, the ADT can easily lead to resistance by involving the activation of NF-κB signaling pathway and high infiltration of M2 macrophages in tumor microenvironment (TME). Herein, we developed a biomimetic nanotherapeutic platform by deriving cell membrane nanovesicles from cancer cells and probiotics to yield the hybrid cellular nanovesicles (hNVs), loading flutamide (Flu) into the resulting hNVs, and finally modifying the hNVs@Flu with Epigallocatechin-3-gallate (EGCG). In this nanotherapeutic platform, the hNVs significantly improved the accumulation of hNVs@Flu-EGCG in tumor sites and reprogramed immunosuppressive M2 macrophages into antitumorigenic M1 macrophages, the Flu acted on androgen receptors and inhibited tumor proliferation, and the EGCG promoted apoptosis of prostate cancer cells by inhibiting the NF-κB pathway, thus synergistically stimulating the antitumor immunity and reducing the side effects and resistance of ADT. In a prostate cancer mouse model, the hNVs@Flu-EGCG significantly extended the lifespan of mice with tumors and led to an 81.78% reduction in tumor growth compared with the untreated group. Overall, the hNVs@Flu-EGCG are safe, modifiable, and effective, thus offering a promising platform for effective therapeutics of prostate cancer.

Abstract 2909: ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms

Abstract CCAAT/Enhancer Binding Protein β (C/EBPβ) is a transcription factor that is an established driver of cellular transformation in several cancer types with poor prognosis, including glioblastoma (GBM), triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). These cancers’ growth is often supported by an immunosuppressive (IS) tumor microenvironment (TME), characterized by high expression of TAMs, MDSCs, Tregs, and in the case of GBM, IS microglia. In addition to its cell-autonomous role of promoting survival and proliferation in cancer cells, C/EBPβ is also critical for IS M2 macrophage polarization, while its role in other TME populations is less understood. ST101 is a peptide antagonist of C/EBPβ that is being evaluated in a Phase 2 clinical study in patients with recurrent and newly diagnosed glioblastoma (NCT04478279). Here we explored the impact of ST101 on IS TME cell populations and on activation of cytotoxic T-cells in macrophage co-culture systems. ST101 increased the M1:M2 ratio up to 40-fold in hPBMC-derived macrophages cultured under conditions that typically drive M2 polarization. Further, ST101 promoted M2 repolarization to the M1 cell type, demonstrating that C/EBPβ is required for maintenance of the M2 program. When added to co-cultures of T cells with M2 macrophages, ST101 induced a 3-4-fold increase in intracellular IFN-γ staining indicating enhanced T cell activation. Notably, in M1 cultures ST101 further enhanced expression of the immune activity marker CD80 and T cell activation. Similar to its effect on M2 repolarization, ST101 induced activation of iPs-derived IS human microglia cultures toward an M1-like program. ST101 also impacted IS cell populations in vivo, as demonstrated in an orthotopic TNBC model in which ST101 exposure was associated with a reduction of the TAM fraction, increased intratumoral M1/M2 ratios, reduction of Tregs, increase of CD8:Treg fraction, and enhanced activity in combination with anti-PD-1. Similar observations were observed in clinical samples, where following ST101 exposure a significant decrease in expression of factors involved in M2 polarization, including CD209, SIGLEC5, and IL-24, and T cell suppression, including FoxP3 and inhibitory KIR proteins, were observed. Gene expression analysis suggests that ST101 induced a decrease in the intratumoral Treg:TIL ratio, indicating a shift towards a more immune-active TME. Overall, these results provide novel evidence for the contribution of C/EBPβ to the expression of multiple IS cancer-associated cell types and support the use of ST101 to antagonize C/EBPβ and promote an immune-active TME. Further, these data provide rational for evaluating the clinical impact of ST101 in combinations with immune checkpoint inhibitors. Citation Format: Claudio Scuoppo, Julia Diehl, Ricardo Ramirez, Mark Koester, Erin Gallagher, Siok Leong, Jerel Gonzales, Karen Mendelson, Zach Mattes, Lila Ghamsari, Gene Merutka, Abi Vainstein-Haras, Barry J. Kappel, Jim A. Rotolo. ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms [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 2909.

Targeting PHGDH reverses the immunosuppressive phenotype of tumor-associated macrophages through α-ketoglutarate and mTORC1 signaling

Phosphoglycerate dehydrogenase (PHGDH) has emerged as a crucial factor in macromolecule synthesis, neutralizing oxidative stress, and regulating methylation reactions in cancer cells, lymphocytes, and endothelial cells. However, the role of PHGDH in tumor-associated macrophages (TAMs) is poorly understood. Here, we found that the T helper 2 (Th2) cytokine interleukin-4 and tumor-conditioned media upregulate the expression of PHGDH in macrophages and promote immunosuppressive M2 macrophage activation and proliferation. Loss of PHGDH disrupts cellular metabolism and mitochondrial respiration, which are essential for immunosuppressive macrophages. Mechanistically, PHGDH-mediated serine biosynthesis promotes α-ketoglutarate production, which activates mTORC1 signaling and contributes to the maintenance of an M2-like macrophage phenotype in the tumor microenvironment. Genetic ablation of PHGDH in macrophages from tumor-bearing mice results in attenuated tumor growth, reduced TAM infiltration, a phenotypic shift of M2-like TAMs toward an M1-like phenotype, downregulated PD-L1 expression and enhanced antitumor T-cell immunity. Our study provides a strong basis for further exploration of PHGDH as a potential target to counteract TAM-mediated immunosuppression and hinder tumor progression.

Abstract B149: Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity

Abstract Beta-catenin represents a significant challenge as a therapeutic target due to its multifaceted role in tumor development as well as normal cell homeostasis. Dysregulation of the Wnt/beta-catenin signaling pathway is implicated in various cancers, including colorectal, hepatocellular, breast, ovarian, and pancreatic cancers. We developed ST316 as a peptide antagonist of the interaction of beta-catenin with BCL9, a co-activator implicated in oncogenic beta-catenin signaling. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) that is currently enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/beta-catenin signaling pathway. In vitro studies indicate that ST316 selectively attenuates Wnt transcriptional activity in beta-catenin-dependent but not -independent cell lines. Gene expression profiling using RNA sequencing in beta-catenin mutant HCT116 cells and APC mutant Colo320DM cells demonstrates a significant downregulation of Wnt pathway target genes, oncogenic signature genes and pro-tumor immunologic gene sets within 24 hours of ST316 treatment. Consequently, ST316 induced a dose-dependent decrease in viability of beta-catenin-dependent cells while beta-catenin-independent cells were resistant, indicating that the anticancer effects of ST316 are mediated by inhibition of the Wnt/beta-catenin pathway. To evaluate the impact of ST316 in vivo, a 4T1 triple negative breast cancer orthotopic tumor model was employed. Once weekly injection of ST316 (5mg/kg SC) over eight weeks suppressed expression of Wnt target genes CDK4 and cMyc and resulted in a significant 84.3% inhibition of tumor growth compared to the control group. In addition to direct anti-tumor activity, ST316 triggers a pro-inflammatory immune microenvironment. ST316 induces polarization of human macrophages derived from peripheral blood mononuclear cells, resulting in >100-fold shift from immunosuppressive M2 macrophages towards the anti-tumor M1 phenotype. Co-cultures of M2 macrophages with T cells treated with ST316 demonstrate a significant three-fold increase in T-cell activation, as indicated by intracellular IFN-γ staining. Finally, ST316 enhances the anti-tumor activity of anti-PD-1 therapy in a syngeneic orthotopic 4T1 mouse model, where combination ST316 and anti-PD-1 antibody results in enhanced tumor growth inhibition compared to either single agent alone (p<0.01). These findings demonstrate the antitumor and immunostimulatory effects of ST316 and highlight its potential as a therapeutic agent for targeting cancers with aberrantly activated Wnt signaling pathways. Citation Format: Lila Ghamsari, Claudio Scuoppo, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Zachary Mattes, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein, Jim Rotolo. Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B149.

A Gene‐Editable Palladium‐Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy

AbstractMacrophage phagocytosis of tumor cells has emerged as an attractive strategy for tumor therapy. Nevertheless, immunosuppressive M2 macrophages in the tumor microenvironment and the high expression of anti‐phagocytic signals from tumor cells impede therapeutic efficacy. To address these issues and improve the management of malignant tumors, in this study we developed a gene‐editable palladium‐based bioorthogonal nanoplatform, consisting of CRISPR/Cas9 gene editing system‐linked Pd nanoclusters, and a hyaluronic acid surface layer (HBPdC). This HBPdC nanoplatform exhibited satisfactory tumor‐targeting efficiency and triggered Fenton‐like reactions in the tumor microenvironment to generate reactive oxygen species for chemodynamic therapy and macrophage M1 polarization, which directly eliminated tumor cells, and stimulated the antitumor response of macrophages. HBPdC could reprogram tumor cells through gene editing to reduce the expression of CD47 and adipocyte plasma membrane‐associated protein, thereby promoting their recognition and phagocytosis by macrophages. Moreover, HBPdC induced the activation of sequestered prodrugs via bioorthogonal catalysis, enabling chemotherapy and thereby enhancing tumor cell death. Importantly, the Pd nanoclusters of HBPdC were sufficiently cleared through basic metabolic pathways, confirming their biocompatibility and biosafety. Therefore, by promoting macrophage phagocytosis, the HBPdC system developed herein represents a highly promising antitumor toolset for cancer therapy applications.

A Gene-Editable Palladium-Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy.

Macrophage phagocytosis of tumor cells has emerged as an attractive strategy for tumor therapy. Nevertheless, immunosuppressive M2 macrophages in the tumor microenvironment and the high expression of anti-phagocytic signals from tumor cells impede therapeutic efficacy. To address these issues and improve the management of malignant tumors, in this study we developed a gene-editable palladium-based bioorthogonal nanoplatform, consisting of CRISPR/Cas9 gene editing system-linked Pd nanoclusters, and a hyaluronic acid surface layer (HBPdC). This HBPdC nanoplatform exhibited satisfactory tumor-targeting efficiency and triggered Fenton-like reactions in the tumor microenvironment to generate reactive oxygen species for chemodynamic therapy and macrophage M1 polarization, which directly eliminated tumor cells, and stimulated the antitumor response of macrophages. HBPdC could reprogram tumor cells through gene editing to reduce the expression of CD47 and adipocyte plasma membrane-associated protein, thereby promoting their recognition and phagocytosis by macrophages. Moreover, HBPdC induced the activation of sequestered prodrugs via bioorthogonal catalysis, enabling chemotherapy and thereby enhancing tumor cell death. Importantly, the Pd nanoclusters of HBPdC were sufficiently cleared through basic metabolic pathways, confirming their biocompatibility and biosafety. Therefore, by promoting macrophage phagocytosis, the HBPdC system developed herein represents a highly promising antitumor toolset for cancer therapy applications.

Fenbendazole Exhibits Differential Anticancer Effects In Vitro and In Vivo in Models of Mouse Lymphoma.

Fenbendazole (FBZ) has been safely used as an antiparasitic agent in animals for decades, and the anticancer effects of FBZ have been studied through various mechanisms. However, there is a lack of in vivo studies that include lymphoma. Therefore, this study examined the effects of FBZ on EL-4 cells and a mouse T lymphoma model. FBZ induced G2/M phase arrest in EL-4 cells, resulting in cell death and decreased metabolic activity. However, FBZ had no anticancer effects on an EL-4 mouse lymphoma model in vivo, as evident by rapid weight loss and tumor growth comparable to the control. The FBZ-treated EL-4 cells expressed higher levels of PD-L1 and CD86, which are associated with T cell immunity in the tumor microenvironment (TME), than the controls. Furthermore, the hematoxylin and eosin staining of the FBZ-treated tumor tissues showed a starry sky pattern, which is seen in actively proliferating cancer tissues, and an immunohistochemical analysis revealed a high percentage of immunosuppressive M2 macrophages. These changes in the immune activity in the TME contradict the results of the in vitro experiments, and further studies are needed to determine the detailed mechanisms by which FBZ induces these responses.

M1 macrophage-derived exosome for reprograming M2 macrophages and combining endogenous NO gas therapy with enhanced photodynamic synergistic therapy in colorectal cancer

Reprogramming immunosuppressive M2 macrophages into M1 macrophages in tumor site provides a new strategy for the immunotherapy of colorectal cancer. In this study, M1 macrophage-derived exosome nanoprobe (M1UC) with Ce6-loaded upconversion material is designed to enhance the photodynamic performance of Ce6 while reprogramming M2 macrophages at tumor site and producing NO gas for three-mode synergistic therapy. Under the excitation of near-infrared light at 808 nm, the probe can generate 660 nm up-conversion fluorescence, which enables the photosensitizer Ce6 to produce ROS efficiently. In addition, the probe leads the production of NO by nitric oxide synthase on exosomes. Confocal laser and flow cytometry results show that M1UC probe reprograms M2 macrophages into M1 macrophages with an efficiency of 95.12%. The cell experiments show that the apoptosis rate of the three-mode synergistic therapy group is 78.8%, and the therapeutic effect is significantly higher than those of the other single treatment groups. In vivo experiments results show that M1UC probes maximally gather at the tumor site after 12 h of intravenous injection in orthotopic colorectal cancer mice. After 808 nm laser irradiation, the survival rate of mice is 100% and the recurrence rate was 0 within 60 d, and the therapeutic effect is significantly higher than those of other single treatment groups, which is also confirmed by immunohistochemistry. This M1 macrophage-derived exosome nanoplatform which is based on the three modes of immunotherapy, gas therapy and photodynamic therapy, provides a new design idea for the diagnosis and treatment of deep tumors.

THU499 The Mesenchymal Stem Cells Induce Immunosuppressive Microenvironment In Pituitary Tumors

Abstract Disclosure: D. Marrero-Rodríguez: None. V. Cortes-Morales: None. K. Taniguchi-Ponciano: None. A. Valenzuela-Perez: None. S. Vela-Patiño: None. A. Cano-Zaragoza: None. F. Martinez-Mendoza: None. J. Kerbel: None. S. Andonegui-Elguera: None. J. Montesinos: None. M. Mercado: None. The tumor microenvironment (TME) includes diverse cellular components such as mesenchymal stem cells (MSC) and immune cells. MSC are a subset of heterogeneous cell populations characterized by the expression of CD105, CD73, and CD90 that can differentiate into chondrocytes, osteoblasts, and adipocytes in vitro. They are known to have immunomodulatory capabilities. Our aim was to isolate and characterize the immunomodulatory capabilities of MSC from pituitary tumors (PT) and non-tumoral gland as well as to describe the immune TME. We isolated and cultivated MSC from LH/FSH-PT and ACTH-PT, as well as non-tumoral pituitary glands. The MSC obtained from PT and non-tumoral gland were co-cultured in the presence of monocytes from healthy donors and their ability to transform into M0, M1 or M2 macrophages was assessed by flow cytometry. The results showed that pituitary tumors derived MSC induce an immunosuppressive M2 macrophage state, by increasing the expression of CD14 and CD206 markers, and decreasing HLA-DR. Upon co-culturing them with naïve T cells, PT MSC were capable inducing a Treg phenotype. To identify PT TME, we performed tumor whole transcriptome characterization and cellular deconvolution using CIBERSORT. Our analysis was indicative of a strong presence of M2 macrophages, TCD4+ and TCD8+ cells, which was confirmed by immunofluorescence, suggesting an immunosuppressive TME. Thus, the MSC immunosuppressive induction correlates directly with immune TME. ACTH-PT derived MSC showed a stronger anti-inflammatory activity than LH/FSH-PT. We also characterized the transcriptomic differences between non-tumoral and PT derived MSC by means of RNAseq. We found differences between the non-tumoral MSC and pituitary tumor derived MSC, but mainly between ACTH- and LH/FSH-PT derived MSC. In conclusion our data suggest that the presence of MSC influence an immunosuppressive tumor microenvironment alongside with tumor cells. Presentation: Thursday, June 15, 2023

2238P Tebentafusp reprograms immunosuppressive tumor-associated M2 macrophages towards anti-tumoral M1 macrophages

2238P Tebentafusp reprograms immunosuppressive tumor-associated M2 macrophages towards anti-tumoral M1 macrophages

P06.03.A IMMORTALIZED MACROPHAGES AS A TOOL TO STUDY PHAGOCYTOSIS AND POLARIZATION IN VITRO

Abstract BACKGROUND Macrophages, an important element of the tumour-microenvironment (TME), are classified based on polarization state: tumour inhibiting ‘classically-activated macrophages’ M1 and tumour promoting ‘alternatively-activated macrophages’ M2. It is wellknown that the glioblastoma (GBM) TME is dominated by immunosuppressive M2 macrophages contributing to tumor growth by promoting angiogenesis, invasion and immunosuppression. However, macrophage culture is difficult to maintain as the cells do not proliferate and expand, making co-culture studies challenging. In this work, we successfully immortalized and expanded bone marrow derived mouse macrophages (BMDMs) that can be used as a tool to study phagocytosis and polarization in co-culture with tumor cells. MATERIAL AND METHODS Primary macrophages, obtained from the bone marrow of C57/BL6 mice, were transduced with two lentiviral vectors: hTERT with antibiotic hygromycin (Hy) and Simian virus 40 (SV40) with mCherry selective markers. Viral-titers were prepared by co-transfecting HEK293T with hTERT and SV40 constructs and plasmids MDL-X (Gag/Pol):Vpx. Vpx is a virion-packaged accessory protein that induces degradation of SAMHD1, a GTP- activated dNTP hydrolase that inhibits lentiviral infection of macrophages. BMDMs transduced with hTERT were subjected to gradient Hy selection after 5 days infection and SV40.mCherry positive cells were sorted by flow cytometry. To assess phagocytotic capacity, BMDMs were incubated with apoptotic bodies of a murine GBM cell-line (GL261) which was treated with HSV-TK/ganciclovir (GCV) suicide gene therapy. Phagocytosis was measured by fluorescence microscopy and flow cytometry. Further, BMDMs were externally induced with either E. coli lipopolysaccharide (LPS) and interferon gamma (IFNγ) or interleukin 4 (IL4) to activate M1 and M2 polarization. Flow cytometry was used to detect iNOS, CD40 and CD86 markers for M1 phenotype and Arginase1 and CD206 for M2. RESULTS hTERT.MDL-X.Vpx.Hy BMDM showed no clear signs of immortalization with limited proliferation followed by cell death. In contrast, SV40.MDL-X.Vpx.mCherry BMDM exhibited proliferative expansion from single colony, efficient freeze-thaw cycles and were used for further studies. BMDMs showed successful phagocytosis of apoptotic bodies from HSV-tk/GCV treated GL261 cells with an increasing capacity upto 18 hours of application. The phagocytic capacity was higher compared to commercial cell lines such as BV2 and Raw 264.7. Immortalized BMDMs tend to have a M2 phenotype without external induction and it is possible to activate M1 phenotype by LPS and IFNγ induction. CONCLUSION Here, we successfully immortalized bone-marrow derived macrophages and showed that they can be used as a tool to study macrophage polarization and phagocytic capability in a tumor immunotherapy context.

Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance.

Despite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy. Our results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages. Therapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.

A Vanadium-Based Nanoplatform Synergizing Ferroptotic-like Therapy with Glucose Metabolism Intervention for Enhanced Cancer Cell Death and Antitumor Immunity.

Ferroptosis activation has been considered a mighty weapon for cancer treatment, and growing attention is being paid to reinforcing tumor cells' sensitivity to ferroptosis. However, the existence of certain ferroptosis resistance mechanisms, especially the abnormal metabolism of tumor cells, has long been underestimated. We propose an enhanced ferroptosis-activating pattern via regulating tumor cells' glycometabolism and construct a nanoplatform named PMVL, which is composed of lonidamine (LND)-loaded tannic acid coordinated vanadium oxides with the camouflage of PD-L1 inhibiting peptide-modified tumor cell membrane. This work reveals that the mixed valence of vanadium (VIV and VV) in PMVL triggers ferroptosis due to the self-cyclic valence alteration of V, the process of which generates •OH for lipid peroxide accumulation (VIV → VV) and depletes glutathione (GSH) for glutathione peroxidase (GPX4) deactivation (VV → VIV). Notably, LND strengthens ferroptosis by dual suppression of glycolysis (decreasing ATP supply) and the pentose phosphate pathway (decreasing NADPH production), causing anabatic GSH consumption. Besides, the inhibited glycolysis generates less intracellular lactic acid and alleviates the acidity of tumor microenvironment, preventing immunosuppressive M2 macrophage polarization. In vitro and in vivo data demonstrate the glycometabolism-intervention-enhanced ferroptosis and boosted immunity activation, potentially providing opportunities and possibilities for synergetic cancer therapy.

Therapeutic effect of a VSIG4-specific mAb EU103 in a preclinical model of patient-derived ascites of ovarian cancer.

e14516 Background: V-set and immunoglobulin domain-containing 4 (VSIG4) is one of the B7 family-related proteins that includes PD-L1, VISTA, and CTLA4 ligand. VSIG4 overexpression is correlated with poor prognosis in patients with ovarian cancer, lung cancer, gastric cancer, high-grade glioma, and multiple myeloma. VSIG4 is also known to function as a receptor for complements and the negative regulation of T cell proliferation. Although VSIG4 is highly expressed in tissue-resident macrophages and tumor-associated macrophages (TAM), its role in the tumor microenvironment (TME) is not fully elucidated. Methods: We previously developed a therapeutic antibody, EU103, in a humanized form of a VSIG4-specific antibody. Most importantly, we demonstrated that EU103 directly acts on the tumor-associated macrophages (TAM) and mechanistically induces repolarization of TAM to tumor killing M1 macrophages and blocks VSIG4-mediated T cell suppression, eventually leading to CD8+ T cell proliferation and tumor suppression in a PBMC-humanized human lung cancer mouse model. Results: Expanding our previous findings with the therapeutic potential of EU103 in a non-small cell lung carcinoma (NSCLC), here, we also proved its therapeutic effect in human ovarian cancer. Firstly, TAMs isolated from ovarian cancer patients express high levels of VSIG4 with immunosuppressive M2 macrophage phenotype. Secondly, EU103 treatment of the TAMs in in vitro studies induces M2-to-M1 conversion and activation of T cells in the ascites leading to ovarian tumor cell killing in a dose-dependent manner. Thirdly, in vivo therapeutic efficacy of EU103 in ovarian cancer, especially through M2-to-M1 conversion, is also verified in both CD34+ cell and peripheral blood mononuclear cell (PBMC) humanized mouse models. Finally, we validate the results above by treating ovarian cancer patient-derived ascites with EU103. Conclusions: Almost 300,000 women are diagnosed with ovarian cancer worldwide each year, and ovarian cancer ranks fifth in cancer deaths among women. In the United States alone, we expect approximately 20,000 new diagnoses and more than 12,000 deaths for this year. Given our findings in this study and clinical significance, we propose here the therapeutic potential of EU103, previously developed by our proprietary antibody discovery and engineering technology, in ovarian cancer.