Tumor-associated Dendritic Cells Research Articles

Abstract 4747: Novel targets for BRAFV600E and BRAFV600ERAC1P29S drug resistant melanoma

Abstract The most common mutational drivers in malignant melanoma are the oncogene BRAFV600E/K (60%), NRASQ61L/R (30%) and the RHO GTPase RAC1P29S/L (9%). BRAFV600E/K melanoma standard of care comprises the use of checkpoint inhibitors (CPIs) such as anti-PD1 or anti-CTLA-4 in combination with targeted therapies against BRAF and MEK kinases. BRAF mutant melanomas develop intrinsic, acquired, or after treatment resistance to BRAF and MEK inhibitors : around half of targeted therapy combination treatments with anti-PD1 and 70% with anti-CTLA-4 will be unsuccessful due to melanoma intrinsic resistance. Among other causes, intrinsic drug resistance can be associated with additional genome mutations, such as the activating Rac1P29S mutation present in 6% of patients with BRAF positive melanoma. A deeper understanding on the biology, progression, and tumor microenvironment of drug resistant melanomas is needed to aid in the search of novel targets and to increase the effectivity of proposed treatments. We generated 3 sets of isogenic BRAFV600E and BRAFV600E/RAC1P29S cell lines by transfecting 501mel, 451Lu and YUMM1.7 cell lines with Rac1P29S. We then exposed them for 3 months to increasing concentrations of PLX4720 to generate drug resistant cell lines (DR). Rac1 P29S -transfected and PLX4720 resistant cell lines had an increase in the Mek/Erk signaling pathway. A differential analysis using a kinome and an epigenome drug screening library in the 501mel cell lines (501mel, 501mel Rac1P29S, 501mel-DR, 501mel Rac1P29S -DR) showed that BRAFV600E/RAC1P29S and the DR melanoma cell lines growth can be inhibited with mTor, PI3K, cdk9 and BRD4 inhibitors. BRD4 is an epigenetic regulator that recruits P-TEFb to stimulate RNA polymerase II elongation. BRD4 also interacts with SMAD3 and CDK9 to regulate transcription through eIF4G increasing oncogenic c-Myc and increasing Mek/Erk signaling pathways. We tested 501mel, 451Lu and YUMM1.7 isogenic cell line series against Rapamycin to confirm mTORC1 as an effective target. Drugs that target different subunits of the eIF4F complex (CR-1-31-B, Ribavirin and Briciclib) also decrease BRAFV600E/RAC1P29S and the DR melanoma cell lines growth specifically. Furthermore, silencing of eIF4A1 and Raptor genes reduces cell growth in the BRAFV600E/RAC1P29S, and the DR cells when compared to the parental cell lines. Silencing Rictor had no effect on cell growth on any cells. When tested in a syngraft model, CR-1-31-B and rapamycin reduced tumor size when using YUMM1.7, YUMM1.7- RAC1P29S and YUMM1.7 RAC1P29S-DR cell lines. Next, we sought to investigate the effect of inhibiting mTOR and eiF4F in the PD-1/PD-L1 axis in tumor cells and tumor associated dendritic cells and macrophages, and how targeting these modifies the tumor microenvironment. Citation Format: Cristina Uribe-Alvarez, Daniela Araiza-Olivera, Alexa Cannon, Jonathan Chernoff. Novel targets for BRAFV600E and BRAFV600ERAC1P29S drug resistant melanoma [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 4747.

Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

The crosstalk of CD8+ T cells and ferroptosis in cancer.

Ferroptosis is an iron-dependent, novel form of programmed cell death characterized by lipid peroxidation and glutathione depletion and is widespread in a variety of diseases. CD8+ T cells are the most important effector cells of cytotoxic T cells, capable of specifically recognizing and killing cancer cells. Traditionally, CD8+ T cells are thought to induce cancer cell death mainly through perforin and granzyme, and Fas-L/Fas binding. In recent years, CD8+ T cell-derived IFN-γ was found to promote cancer cell ferroptosis by multiple mechanisms, including upregulation of IRF1 and IRF8, and downregulation of the system XC-, while cancer cells ferroptosis was shown to enhance the anti-tumor effects of CD8+ T cell by heating the tumor immune microenvironment through the exposure and release of tumor-associated specific antigens, which results in a positive feedback pathway. Unfortunately, the intra-tumoral CD8+ T cells are more sensitive to ferroptosis than cancer cells, which limits the application of ferroptosis inducers in cancer. In addition, CD8+ T cells are susceptible to being regulated by other immune cell ferroptosis in the TME, such as tumor-associated macrophages, dendritic cells, Treg, and bone marrow-derived immunosuppressive cells. Together, these factors build a complex network of CD8+ T cells and ferroptosis in cancer. Therefore, we aim to integrate relevant studies to reveal the potential mechanisms of crosstalk between CD8+ T cells and ferroptosis, and to summarize preclinical models in cancer therapy to find new therapeutic strategies in this review.

Hyperglycosylation of prosaposin in tumor dendritic cells drives immune escape.

Tumors develop strategies to evade immunity by suppressing antigen presentation. In this work, we show that prosaposin (pSAP) drives CD8 T cell-mediated tumor immunity and that its hyperglycosylation in tumor dendritic cells (DCs) leads to cancer immune escape. We found that lysosomal pSAP and its single-saposin cognates mediated disintegration of tumor cell-derived apoptotic bodies to facilitate presentation of membrane-associated antigen and T cell activation. In the tumor microenvironment, transforming growth factor-β (TGF-β) induced hyperglycosylation of pSAP and its subsequent secretion, which ultimately caused depletion of lysosomal saposins. pSAP hyperglycosylation was also observed in tumor-associated DCs from melanoma patients, and reconstitution with pSAP rescued activation of tumor-infiltrating T cells. Targeting DCs with recombinant pSAP triggered tumor protection and enhanced immune checkpoint therapy. Our studies demonstrate a critical function of pSAP in tumor immunity and may support its role in immunotherapy.

Lycium barbarum L. polysaccharide LBP3 exerts the anti-tumor effect through enhancing the function of tumor-associated dendritic cells via inhibiting IRE1α -XBP1 pathway of ER stress

Liver cancer is a huge global challenge. It has been exacerbated by tumor-associated dendritic cells (TDCs) due to Endoplasmic Reticulum (ER) stress. We planned to investigate the effects of LBP3 on TDCs immune function, and to explore whether the underlying mechanism was associated with IRE1α-XBP1 pathway of ER stress regulating lipid metabolism. In our early studies, LBP3 (40–350 kDa) with significant anti-tumor and immunomodulatory activities was isolated from the polysaccharide of Lycium barbarum L. (LBP). Here, we determined that LBP3 had a great anti-tumor efficacy in H22-tumor bearing mice by functional activation of TDCs. In vivo and in vitro results showed that LBP3 could up-regulate the expression of MHC Ⅱ, CD80 and CD86 on TDCs, increase the level of TNF-α secreted by TDCs, enhance TDCs’ ability to improve the proliferation and immunosuppression of lymphocytes. Furthermore, LBP3 could decrease the high expression of ER stress-related proteins IRE1α, GRP78, XBP1 and CHOP, reduce the lipid accumulation, raise the low expression of costimulatory molecules on TDCs induced by thapsigargin (TG). Taken together, LBP3 could reduce intracellular lipid accumulation by inhibiting IRE1α-XBP1 pathway of ER stress, improve the function of TDCs to stimulate T cells, thence play an anti-tumor role.

Regulatory T Cell Inhibition by P60 Combined with Adenoviral AFP Transduced Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma

ABSTRACT Background & Aims Vaccination with tumor-associated antigen-pulsed dendritic cells leads to specific T-cell response against hepatocellular carcinoma. However, clinical response has been shown to be limited. High regulatory T-cell count is associated with poor prognosis and seems to mediate immune tolerance in hepatocellular carcinoma. Forkhead box P3-peptide inhibitor P60 has been shown to specifically inhibit regulatory T-cell function in murine models. Aim of this study was to investigate whether P60 can improve the immune response induced by vaccination with adenovirus-transduced dendritic cells expressing alpha-fetoprotein in subcutaneous and orthotopic murine models for hepatocellular carcinoma. Methods Mice developing subcutaneous or orthotopic HCC received daily treatment with P60 starting at different tumor stages. Additionally, mice were vaccinated twice with dendritic cells expressing alpha-fetoprotein. Results In a preventive setting prior to tumor engraftment, vaccination with alpha-fetoprotein-expressing dendritic cells significantly decreased tumor growth in a subcutaneous model (p = .0256), but no further effects were achieved by addition of P60. However, P60 enhanced the antitumoral effect of a vaccination with alpha-fetoprotein-expressing dendritic cells in established subcutaneous and orthotopic hepatocellular carcinoma characterized by high Treg levels (p = .011). Conclusion In this study, we showed that vaccination with alpha-fetoprotein-expressing dendritic cells in combination with a specific inhibition of regulatory T-cells by using P60 leads to synergistic tumor inhibition and prolonged survival. This emphasizes the importance of regulatory T-cells inhibition for obtaining an effective antitumoral immune response in hepatocellular carcinoma.

Dual effects of radiotherapy on tumor microenvironment and its contribution towards the development of resistance to immunotherapy in gastrointestinal and thoracic cancers.

Successful clinical methods for tumor elimination include a combination of surgical resection, radiotherapy, and chemotherapy. Radiotherapy is one of the crucial components of the cancer treatment regimens which allow to extend patient life expectancy. Current cutting-edge radiotherapy research is focused on the identification of methods that should increase cancer cell sensitivity to radiation and activate anti-cancer immunity mechanisms. Radiation treatment activates various cells of the tumor microenvironment (TME) and impacts tumor growth, angiogenesis, and anti-cancer immunity. Radiotherapy was shown to regulate signaling and anti-cancer functions of various TME immune and vasculature cell components, including tumor-associated macrophages, dendritic cells, endothelial cells, cancer-associated fibroblasts (CAFs), natural killers, and other T cell subsets. Dual effects of radiation, including metastasis-promoting effects and activation of oxidative stress, have been detected, suggesting that radiotherapy triggers heterogeneous targets. In this review, we critically discuss the activation of TME and angiogenesis during radiotherapy which is used to strengthen the effects of novel immunotherapy. Intracellular, genetic, and epigenetic mechanisms of signaling and clinical manipulations of immune responses and oxidative stress by radiotherapy are accented. Current findings indicate that radiotherapy should be considered as a supporting instrument for immunotherapy to limit the cancer-promoting effects of TME. To increase cancer-free survival rates, it is recommended to combine personalized radiation therapy methods with TME-targeting drugs, including immune checkpoint inhibitors.

Regulation of antigen-specific T cell infiltration and spatial architecture in multiple myeloma and premalignancy.

Entry of antigen-specific T cells into human tumors is critical for immunotherapy, but the underlying mechanisms are poorly understood. Here, we combined high-dimensional spatial analyses with in vitro and in vivo modeling to study the mechanisms underlying immune infiltration in human multiple myeloma (MM) and its precursor monoclonal gammopathy of undetermined significance (MGUS). Clustered tumor growth was a feature of MM but not MGUS biopsies, and this growth pattern was reproduced in humanized mouse models. MM biopsies exhibited intralesional as well as spatial heterogeneity, with coexistence of T cell-rich and T cell-sparse regions and the presence of areas of T cell exclusion. In vitro studies demonstrated that T cell entry into MM clusters was regulated by agonistic signals and CD2-CD58 interactions. Upon adoptive transfer, antigen-specific T cells localized to the tumor site but required in situ DC-mediated antigen presentation for tumor entry. C-type lectin domain family 9 member A-positive (CLEC9A+) DCs appeared to mark portals of entry for gradients of T cell infiltration in MM biopsies, and their proximity to T cell factor 1-positive (TCF1+) T cells correlated with disease state and risk status. These data illustrate a role for tumor-associated DCs and in situ activation in promoting the infiltration of antigen-specific T cells in MM and provide insights into spatial alterations in tumor/immune cells with malignant evolution.

Metabolism and senescence in the immune microenvironment of osteosarcoma: focus on new therapeutic strategies.

Osteosarcoma is a highly aggressive and metastatic malignant tumor. It has the highest incidence of all malignant bone tumors and is one of the most common solid tumors in children and adolescents. Osteosarcoma tissues are often richly infiltrated with inflammatory cells, including tumor-associated macrophages, lymphocytes, and dendritic cells, forming a complex immune microenvironment. The expression of immune checkpoint molecules is also high in osteosarcoma tissues, which may be involved in the mechanism of anti-tumor immune escape. Metabolism and senescence are closely related to the immune microenvironment, and disturbances in metabolism and senescence may have important effects on the immune microenvironment, thereby affecting immune cell function and immune responses. Metabolic modulation and anti-senescence therapy are gaining the attention of researchers as emerging immunotherapeutic strategies for tumors. Through an in-depth study of the interconnection of metabolism and anti- senescence in the tumor immune microenvironment and its regulatory mechanism on immune cell function and immune response, more precise therapeutic strategies can be developed. Combined with the screening and application of biomarkers, personalized treatment can be achieved to improve therapeutic efficacy and provide a scientific basis for clinical decision-making. Metabolic modulation and anti- senescence therapy can also be combined with other immunotherapy approaches, such as immune checkpoint inhibitors and tumor vaccines, to form a multi-level and multi-dimensional immunotherapy strategy, thus further enhancing the effect of immunotherapy. Multidisciplinary cooperation and integrated treatment can optimize the treatment plan and maximize the survival rate and quality of life of patients. Future research and clinical practice will further advance this field, promising more effective treatment options for patients with osteosarcoma. In this review, we reviewed metabolic and senescence characteristics in the immune microenvironment of osteosarcoma and related immunotherapies, and provide a reference for development of more personalized and effective therapeutic strategies.

Rapid recruitment and IFN-I-mediated activation of monocytes dictate focal radiotherapy efficacy.

The recruitment of monocytes and their differentiation into immunosuppressive cells is associated with the low efficacy of preclinical nonconformal radiotherapy (RT) for tumors. However, nonconformal RT (non-CRT) does not mimic clinical practice, and little is known about the role of monocytes after RT modes used in patients, such as conformal RT (CRT). Here, we investigated the acute immune response induced by after CRT. Contrary to non-CRT approaches, we found that CRT induces a rapid and robust recruitment of monocytes to the tumor that minimally differentiate into tumor-associated macrophages or dendritic cells but instead up-regulate major histocompatibility complex II and costimulatory molecules. We found that these large numbers of infiltrating monocytes are responsible for activating effector polyfunctional CD8+ tumor-infiltrating lymphocytes that reduce tumor burden. Mechanistically, we show that monocyte-derived type I interferon is pivotal in promoting monocyte accumulation and immunostimulatory function in a positive feedback loop. We also demonstrate that monocyte accumulation in the tumor microenvironment is hindered when RT inadvertently affects healthy tissues, as occurs in non-CRT. Our results unravel the immunostimulatory function of monocytes during clinically relevant modes of RT and demonstrate that limiting the exposure of healthy tissues to radiation has a positive therapeutic effect on the overall antitumor immune response.

Optimizing tumor-associated antigen-stimulated autologous dendritic cell and cytokine-induced killer cell coculture to enhance cytotoxicity for cancer immunotherapy in manufacturing

BackgroundDendritic Cell Cytokine-induced killer cell (DC-CIK) coculture treatment in cancer immunotherapy has been shown to be effective. However, the cost of DC- CIK therapy is prohibitive for many patients, and the lack of standard manufacturing processes and treatment strategies are major limitations. Our study used tumor lysate as a tumor-associated antigen source and DCs and CIK cells in coculture. We developed an efficient method to obtain autologous DCs- and CIK cells from peripheral blood. We used flow cytometry to assess DC activation and the cytometric bead array assay to quantify cytokines secreted by CIK cells.ResultsWe evaluated the antitumor activity of DC- CIK coculture in vitro with the K562 cell line. We demonstrated that a manufacturing process employing frozen immature DCs can yield the lowest loss with the highest economic benefits. DC-CIK coculture can effectively upgrade CIK cells’ immunological specificity to tumors in the presence of tumor-associated antigens.ConclusionIn vitro experiments revealed that when the DC- CIK cell ratio was 1: 20 in the coculture, CIK cells secreted the highest number of cytokines on the 14th day and the antitumor immune effect showed the highest potency. CIK cells’ cytotoxicity to K562 cells was highest when the CIK: K562 cell ratio was 25: 1. We developed an efficient manufacturing process for DC- CIK coculture, while also establishing the optimal DC- CIK cell ratio for immunological activity and the best cytotoxic CIK: K562 cell ratio.

Slimming and Reinvigorating Tumor-Associated Dendritic Cells with Hierarchical Lipid Rewiring Nanoparticles.

Dendritic cells (DCs) are crucial mediators of innate and adaptive antitumor immunity, whereas exogenously and endogenously driven lipid accumulation causes immune tolerance of tumor-associated DCs (TADCs) and thereby diminishes tumor responsiveness to various therapies. Herein, a type of multilevel lipid rewiring nanoparticles (NPs) for TADC revitalization is designed. These self-assembled NPs specifically bind to the lipid transport receptor Msr1 on the TADC surface and orchestrate the restriction of extracellular lipid uptake, cytoplasmic de novo lipid biosynthesis and nuclear lipogenic gene transcription. It is found that the slimming of TADCs via the three-in-one lipid metabolic reprogramming substantially promotes their maturation and rehabilitate their functions in inflammatory cytokine production, cytotoxic T cell recruitment, and tumor inhibition. Significantly, tumor resistance to immune checkpoint blockade therapy is further overcome. The study presents a non-canonical strategy to remodel tumor-infiltrating immune cells and paves a new path for improving the efficacy of cancer immunotherapy.

Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity.

In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.

An Intelligent Vascular Disrupting Dendritic Nanodevice Incorporating Copper Sulfide Nanoparticles for Immune Modulation-Mediated Combination Tumor Therapy.

Development of intelligent nanoplatforms that can simultaneously target multiple factors associated with tumor growth and metastasis remains an extreme challenge. Here, an intelligent dendritic nanodevice incorporating both copper sulfide nanoparticles (CuS NPs) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA, a vascular disrupting agent) within the dendrimer internal cavities and surface modified with a targeting agent LyP-1 peptide is reported. The resulting generation 5 (G5) dendrimer-based nanodevice, known as G5-PEG-LyP-1-CuS-DMXAA NPs (GLCD NPs), possess good colloidal stability, pH-sensitive drug release kinetics, and high photothermal conversion efficiency (59.3%). These functional GLCD NPs exert a LyP-1-targeted killing effect on breast tumors by combining CuS-mediated photothermal therapy (PTT) and DMXAA-induced vascular disruption, while also triggering antitumor immune responses through PTT-induced immunogenic cell death and DMXAA-mediated immune regulation via M1 polarization of tumor-associated macrophages and dendritic cell maturation. In addition, with the LyP-1-mediated proapoptotic activity, the GLCD NPs can specifically kill tumor lymphatic endothelial cells. The simultaneous disruption of tumor blood vessels and lymphatic vessels cuts off the two main pathways of tumor metastasis, which plays a two-pronged role in inhibiting lung metastasis of the breast cancer model. Thus, the developed GLCD NPs represent an advanced intelligent nanoformulation for immune modulation-mediated combination tumor therapy with potential for clinical translations.

TAM receptor tyrosine kinase MERTK as a therapeutic target in the tumor immune microenvironment in a murine Krasmutant lung cancer model

Abstract TAM receptor tyrosine kinase MERTK is involved in apoptotic cell clearance to maintain tissue homeostasis. During efferocytosis, MERTK activation also promotes an immunosuppressive phenotype, which can be hijacked by tumor cells to escape immune attacking. Here, we determined the impact of MERTK inhibition on immune cell subsets in the tumor microenvironment in mice with subcutaneous murine CMT167 (Kras-G12V) lung cancer grafts. Although MERTK expression was not detected on tumor associated dendritic cells (DCs), there was a significant increase in the incidence of DCs in tumors from Mertk knockout (KO) mice compared to wild type mice by day 5 after inoculation of tumor cells. Characterization of tumor-associated macrophages by flow cytometry revealed three distinct populations expressing different levels of MHCII (CD11b +MHCII high, CD11b +MHCII +and CD11b highMHCII neg). Tumors from Mertk KO mice had significantly greater numbers of MHCII highmacrophages by day 10 after tumor cell inoculation and reduced numbers of MHCII negmacrophages by day 19. Together, the increased incidence of dendritic cells and the shift toward a more antigen-presenting macrophage phenotype are expected to promote anti-tumor immunity in Mertk KO mice. Indeed, CMT167 tumor growth was significantly reduced in Mertk KO mice and this effect was abrogated in Mertk KO scid mice, which lack functional B and T cells. Treatment with MRX-2843, a MERTK-selective inhibitor currently in Phase 1 clinical trials, also reduced tumor growth in mice. Together, these data demonstrate a critical role for MERTK as a mediator of tumor progression in the tumor immune microenvironment and implicate MRX-2843 as an effective strategy to exploit this finding in the clinic. The research was supported by the National Cancer Institute of the National Institutes of Health (Emory Lung Cancer Spore, Award Number P50CA217691). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Dual TLR9 and PD-L1 targeting unleashes dendritic cells to induce durable antitumor immunity

BackgroundAlthough immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may...

Abstract 4600: Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor models

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4600.

Abstract 2812: NL-201, a de novo CD25-independent IL-2Rβγ agonist, synergizes with radiation to generate potent antitumor immunity

Abstract There is a growing interest in combining radiotherapy (RT) with immunotherapeutic agents to improve efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of high-dose recombinant IL-2 is limited by toxicity and the expansion of regulatory T cells (Tregs) through signaling events mediated by IL2Rα (CD25). Here we examined the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway dependency of type I interferon (IFN) response and anti-tumor effect of RT in combination with NL-201, a highly potent and stable CD25-independent IL-2 and IL-15 receptor agonist with enhanced affinity for IL2Rβ and IL2Rγc. In mice implanted with E0771 syngeneic tumors, NL-201 in combination with RT enhanced activation of the STING pathway. This was associated with increased type I IFN production as well as increased activation and number of tumor associated dendritic cells (DCs). In STING (-/-) mice implanted with E0771 tumors, Type I IFN production, tumor-growth inhibition (TGI), and survival advantage was significantly decreased and there were significantly fewer activated CD4+ and CD8+ T-cells. Similarly, CD11c+ knock-out mice implanted with E0771 tumors and treated with the combination exhibited similar TGI, survival, and IFN production compared to vehicle treated mice. Based on these results, we tested the ability of the combination of RT and NL-201 to enhance systemic immunotherapy with an anti-PD-1 checkpoint inhibitor in mice implanted with 4T1 breast tumor cells. Tumor-bearing mice treated with the triple combination exhibited superior TGI and survival, along with increased effector memory T-cell populations as compared to all other treatment groups. These results support our previous findings that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, synergizes with RT, likely via the direct activation of STING. Genetic knockout of STING in tumor-bearing mice abrogated the activity of the NL-201 and RT combination, thus confirming the crucial role of innate immune sensors in the combination treatment’s immune activation and resulting antitumor effect. These results also describe the important role of STING-mediated activation and tumor penetration of DCs, which were crucial for anti-tumor response. Additionally, when combined with anti-PD-1 antibodies, NL-201+RT combination treatment significantly improved TGI of metastatic breast cancers that were previously resistant to checkpoint blockade. This work provides rationale for the continued exploration of RT in combination with CD25-independent IL-2Rβγ agonism as a means to enhance the systemic immune response to treat immunologically challenging tumors. Citation Format: Kristin Huntoon, DaeYong Lee, Xuefeng Li, Yifan Wang, Wen Jiang, Betty Kim. NL-201, a de novo CD25-independent IL-2Rβγ agonist, synergizes with radiation to generate potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2812.

Prognostic and Immunological Significance of Rhomboid Domain Containing Protein 1 in Multiple Primary Cancers.

RHBDD1 is an intramembrane serine protease of the rhomboid superfamily that regulates diverse physiological and pathological processes. However, the relationship between RHBDD1 expression, tumor-infiltrating immune cells (TIICs), and cancer prognosis remains unclear. We comprehensively analyzed the prognostic and immunological significance of RHBDD1 in multiple primary cancers. RHBDD1 expression was investigated using Oncomine, TIMER, and UALCAN databases, after which the clinical prognostic value of RHBDD1 was assessed with online public databases. In addition, we explored the correlation between RHBDD1 and TIICs by TIMER and GEPIA and investigated the relationship between RHBDD1 expression and chemokines in cancers by TIMER. In general, compared to that in adjacent normal tissue, lower expression of RHBDD1 was found in various cancers and was correlated to pathological stages. Although RHBDD1 showed a protective effect on multiple solid tumors, a high expression level of RHBDD1 was detrimental to the survival of stomach adenocarcinoma patients. RHBDD1 was positively correlated to immune infiltration levels in various cancers, including lung, breast, ovarian, and gastric cancer. Furthermore, gene markers of TIICs, such as tumor-associated macrophages (TAMs), dendritic cells (DCs), and regulatory T cells, were also correlated to RHBDD1 expression. In addition, the RHBDD1 expression level was positively correlated to multiple chemokines in cancers, which could recruit diverse immune cells at the tumor site. RHBDD1, which correlates with immune infiltration, can be used as a potential prognostic biomarker in multiple primary cancers. Specifically, RHBDD1 expression potentially contributes to the recruitment of TAMs and DCs and the regulation of T cell functions in cancers.

Prognostic marker C3AR1 is associated with ovarian cancer cell proliferation and immunosuppression in the tumor microenvironment

BackgroundC3AR1 was reported in driving tumor immunity in multiple cancers. However, its roles in ovarian cancer remain unclear. This study aims to determine role of C3AR1 in prognosis and regulating tumor infiltrating immune cells of ovarian cancer (OC).Materials and methodsThe expression, prognosis and clinical data related to C3AR1 were collected from public databases such as The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA) and Clinical Proteomics Tumor Analysis Alliance (CPTAC), and further analyze their relationship with immune infiltration. Immunohistochemistry verified the expression of C3AR1 in ovarian cancer and control tissues. C3AR1 was forced expressed in SKOV3 cells by plasmid transfection, and verified by qRT-PCR and Western blot. Cell proliferation were evaluated by EdU assay.ResultsBioinformatics analysis (TCGA, CPTAC) and immunohistochemical staining of clinical samples confirmed higher C3AR1 expression in ovarian cancer than that in normal tissues. High C3AR1 expression predicted adverse clinical outcomes. KEGG and GO analysis showed that the biological processes of C3AR1 in ovarian cancer are mainly involved in T cell activation, cytokine and chemokine activation. C3AR1 expression was positively correlated with chemokines and their receptors in the tumor microenvironment, such as CCR1(R = 0.83), IL10RA (R = 0.92), and INFG (R = 0.74). In addition, increased C3AR1 expression predicted more infiltration of tumor-associated macrophages, dendritic cell and CD8 + T cell. Some important m6A regulators, such as IGF2BP2, ALKBH5, IGFBP3 and METL14, are significantly positively or negatively correlated with C3AR1. Finally, overexpression of C3AR1 significantly increased proliferation of SKOV3 cells.ConclusionIn summary, our study suggested that C3AR1 is associated with the prognosis and immune cell infiltration of ovarian cancer, and is a promising immunotherapeutic target.