Ovarian Cancer Microenvironment Research Articles

Extracellular vesicles targeting tumor microenvironment in ovarian cancer

Ovarian cancer (OC) is a prevalent neoplastic condition affecting women. Extracellular vesicles (EVs), nano-sized membrane vesicles, are secreted by various cells in both physiological and pathological states. The profound interplay between EVs and the tumor microenvironment (TME) in ovarian cancer is crucial. In this review, we explores the pivotal role of EVs in facilitating intercellular communication between cancer cells and the TME, emphasizing the potential of EVs as promising diagnostic markers and innovative therapeutic targets for ovarian cancer. The comprehensive analysis outlines the specific mechanisms by which EVs engage in communication with the constituents of the TME, including the modulation of tumor growth through EVs carrying matrix metalloproteinases (MMPs) and EV-mediated inhibition of angiogenesis, among other factors. Additionally, the we discuss the potential clinical applications of EVs that target the TME in ovarian cancer, encompassing the establishment of novel treatment strategies and the identification of novel biomarkers for early detection and prognosis. Finally, this review identifies novel strategies for therapeutic interventions, such as utilizing EVs as carriers for drug delivery and targeting specific EV-mediated signaling pathways. In summary, this manuscript offers valuable insights into the role of EVs in ovarian cancer and highlights the significance of comprehending intercellular communication in the realm of cancer biology.

The Emerging Role of the Single-Cell and Spatial Tumor Microenvironment in High-Grade Serous Ovarian Cancer.

The development of single-cell and spatial technologies has enabled a more detailed understanding of the tumor microenvironment and its role in therapy response and clinical outcome of high-grade serous ovarian cancer (HGSC). Interestingly, emerging evidence suggests that HGSCs with different genetic drivers harbor distinct tumor-immune microenvironments. Further, spatial cell-cell interactions have been shown to shape the CD8+ T-cell phenotypes and responses to immune checkpoint blockade therapies. The heterogeneous stroma consisting of cancer-associated fibroblast (CAF) subtypes, endothelia, and site-specific stromal types such as mesothelium modulates treatment responses via increasing stiffness and by producing ligands that promote drug resistance, angiogenesis, or immune escape. Chemotherapy itself shifts CAFs toward an inflammatory phenotype that associates with poor survival and immune-suppressive signaling. New emerging immunotherapies include combinational approaches and agents targeting, for example, the tumor-intrinsic endoplasmic reticulum pathway. A more detailed understanding of the spatial interplay of tumor, immune, and stromal cells in the tumor microenvironment is needed to develop more efficient immunotherapeutic strategies for HGSC.

Cancer-mesothelial and cancer-macrophage interactions in the ovarian cancer microenvironment.

The metastatic ovarian cancer microenvironment is characterized by an intricate interaction network between cancer cells and host cells. This complex heterotypic cancer-host cell crosstalk results in an environment that promotes cancer cell metastasis and treatment resistance, leading to poor patient prognosis and survival. In this review, we focus on two host cell types found in the ovarian cancer microenvironment: mesothelial cells and tumor-associated macrophages. Mesothelial cells make up the protective lining of organs in the abdominal cavity. Cancer cells attach and invade through the mesothelial monolayer to form metastatic lesions. Crosstalk between mesothelial and cancer cells can contribute to metastatic progression and chemotherapy resistance. Tumor-associated macrophages are the most abundant immune cell type in the ovarian cancer microenvironment with heterogeneous subpopulations exhibiting protumor or antitumor functions. Macrophage reprogramming toward a protumor or antitumor state can be influenced by chemotherapy and communication with cancer cells, resulting in cancer cell invasion and treatment resistance. A better understanding of cancer-mesothelial and cancer-macrophage crosstalk will uncover biomarkers of metastatic progression and therapeutic targets to restore chemotherapy sensitivity.

Molecular Characterization of Cuproptosis-related lncRNAs: Defining Molecular Subtypes and a Prognostic Signature of Ovarian Cancer.

Cuproptosis, a newly discovered form of programmed cell death, relies on mitochondrial respiration, the chain of which has been found to be altered in ovarian cancer (OC). The current work probed into the effects of Cuproptosis on the prognosis, immune microenvironment and therapeutic response of OC based on Cuproptosis-related lncRNAs. Data on OC gene expression and clinical characteristics were collected from TCGA, ICGC and GEO databases, and mRNA and lncRNA were distinguished. Cuproptosis-related lncRNAs were screened for consensus clustering analysis. Differentially expressed lncRNAs (DElncRNAs) were identified between clusters, and least absolute shrinkage and selection operator (LASSO) and Cox regression analysis were performed to establish a prognostic signature. Its potential value in OC was evaluated by Gene Set Enrichment Analysis (GSEA), tumor cell mutation and immune microenvironment analysis, and response to immunotherapy and antineoplastic drugs. According to the classification scheme of Cuproptosis-related lncRNAs, OC was divided into four molecular subtypes, which were different in survival time, immune characteristics and somatic mutation. The prognostic signature between subtypes included 10 lncRNAs, which were significantly correlated with the prognosis, immune microenvironment related indexes, the expression of immune checkpoint molecules and the sensitivity of antineoplastic drug Paclitaxel and Gefitinib of OC. We examined the expression of ten LncRNAs in OC cell lines and found that LINC00189, ZFHX4-AS1, RPS6KA2-IT1 and C9orf106 were expressed elevated in OC cell lines, and LINC00861, LINC00582, DEPDC1-AS1, LINC01556, LEMD1-AS1, TYMSOS expression was decreased in OC cell lines. The results of CCK8 showed that the cell viability of OC cells decreased after inhibition of C9orf106, whereas the cell viability of OC cells increased after inhibition of LEMD1-AS1. This work revealed new Cuproptosis-related lncRNA molecular subtypes exhibiting tumor microenvironment (TME) heterogeneity for OC and proposed a prognostic signature that may have benefits in understanding the prognosis, pathological features and immune microenvironment of OC patients.

Integrative analysis of circadian clock with prognostic and immunological biomarker identification in ovarian cancer.

Objective: To identify circadian clock (CC)-related key genes with clinical significance, providing potential biomarkers and novel insights into the CC of ovarian cancer (OC). Methods: Based on the RNA-seq profiles of OC patients in The Cancer Genome Atlas (TCGA), we explored the dysregulation and prognostic power of 12 reported CC-related genes (CCGs), which were used to generate a circadian clock index (CCI). Weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network were used to identify potential hub genes. Downstream analyses including differential and survival validations were comprehensively investigated. Results: Most CCGs are abnormally expressed and significantly associated with the overall survival (OS) of OC. OC patients with a high CCI had lower OS rates. While CCI was positively related to core CCGs such as ARNTL, it also showed significant associations with immune biomarkers including CD8+ T cell infiltration, the expression of PDL1 and CTLA4, and the expression of interleukins (IL-16, NLRP3, IL-1β, and IL-33) and steroid hormones-related genes. WGCNA screened the green gene module to be mostly correlated with CCI and CCI group, which was utilized to construct a PPI network to pick out 15 hub genes (RNF169, EDC4, CHCHD1, MRPL51, UQCC2, USP34, POM121, RPL37, SNRPC, LAMTOR5, MRPL52, LAMTOR4, NDUFB1, NDUFC1, POLR3K) related to CC. Most of them can exert prognostic values for OS of OC, and all of them were significantly associated with immune cell infiltration. Additionally, upstream regulators including transcription factors and miRNAs of key genes were predicted. Conclusion: Collectively, 15 crucial CC genes showing indicative values for prognosis and immune microenvironment of OC were comprehensively identified. These findings provided insight into the further exploration of the molecular mechanisms of OC.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

Development of a machine learning-based signature utilizing inflammatory response genes for predicting prognosis and immune microenvironment in ovarian cancer.

Ovarian cancer (OC) represents a significant health challenge, characterized by a particularly unfavorable prognosis for affected women. Accumulating evidence supports the notion that inflammation-related factors impacting the normal ovarian epithelium may contribute to the development of OC. However, the precise role of inflammatory response-related genes (IRRGs) in OC remains largely unknown. To address this gap, we performed an integration of mRNA expression profiles from 7 cohorts and conducted univariate Cox regression analysis to screen 26 IRRGs. By utilizing these IRRGs, we categorized patients into subtypes exhibiting diverse inflammatory responses, with subtype B displaying the most prominent immune infiltration. Notably, the elevated abundance of Treg cells within subtype B contributed to immune suppression, resulting in an unfavorable prognosis for these patients. Furthermore, we validated the distribution ratios of stromal cells, inflammatory cells, and tumor cells using whole-slide digitized histological slides. We also elucidated differences in the activation of biological pathways among subtypes. In addition, machine learning algorithms were employed to predict the likelihood of survival in OC patients based on the expression of prognostic IRRGs. Through rigorous testing of over 100 combinations, we identified CXCL10 as a crucial IRRG. Single-cell analysis and vitro experiments further confirmed the potential secretion of CXCL10 by macrophages and its involvement in lymphangiogenesis within the tumor microenvironment. Overall, the study provides new insights into the role of IRRGs in OC and may have important implications for the development of novel therapeutic approaches.

OncoTherad immunotherapy plus platelet rich plasma as potential therapeutic strategy for ovarian cancer: The role of toll-like receptors 2 and 4 signaling pathways.

e17602 Background: Ovarian cancer (OC) has the most devastating death rate of gynecological cancers with only 44% of women surviving 5 years after diagnosis. Toll-like receptors (TLRs) signaling can play an important role in the OC treatment. TLR activation in immune cells can help activate an effective antitumor response. In this scenario, a new perspective is represented by combined OncoTherad (MRB-CFI-1) immunotherapy and Platelet Rich Plasma (PRP). OncoTherad (MRB-CFI-1) leads to the distinct stimulation of the innate immune system mediated by TLR2 and TLR4, resulting in an increased activation of the IFN signaling pathway. Also, PRP acts on immune activation and exerts antitumor effects. This study characterized the histopathological and molecular effects of the OncoTherad associated with PRP in the treatment of OC chemically induced in rats and described the possible mechanisms of action of this association involving the TLRs 2 and 4 signaling pathways. Methods: Fischer 344 female rats were divided into 5 groups (n = 7 animals per group): Control (Sham surgery) group; Cancer (7,12 dimethylbenzoanthracene – DMBA injection in the ovarian bursa, 1.25 mg/kg) group; OncoTherad (20mg/kg) group; PRP (328.103 – 549.103 platelets/mm3) group and; OncoTherad+PRP (same doses of the isolated treatments) group. Intraperitoneal doses were administered twice a week for 4 weeks. Immunohistochemistry was analyzed as total immunoreactivity and intensity of immunoreaction. Results: Our results showed serous type ovarian carcinoma, follicular atresia, absence of corpora lutea and hyperplasia of the germinal epithelium and tunica albuginea in the Cancer group. The histopathological changes in the PRP group were similar as Cancer group. In contrast, the animals treated with OncoTherad singly or in combination with PRP showed decrease of ovarian neoplastic lesions and histopathological recovery. TLR4 and IL-6 immunoreactivities were significantly lower (p < 0.05) in the Cancer group. PRP treatment reduced (p < 0.05) TLR2. In the Oncotherad+PRP group, the immunoreactivities of TLR4, MyD88, IRF-3 and TNF-α were lower. OncoTherad treatment alone led to stimulation of the innate immune system mediated by TLR2 and TLR4, resulting in an increased activation of the MyD88 signaling pathway (intensified IL-6 and TNF-α immunoreactivities). Conclusions: OncoTherad immunotherapy was able to reduce ovarian lesions and stimulated the TLR2 and 4 signaling pathways in the OC microenvironment, leading to antitumor effects by activating the production of inflammatory cytokines. Thus, OncoTherad immunotherapy could be considered an important therapeutic strategy for OC.

Leucyl and Cystinyl Aminopeptidase as a Prognostic-Related Biomarker in OV Correlating with Immune Infiltrates.

It was indicated that tumor intrinsic heterogeneity and the tumor microenvironment (TME) of ovarian cancer (OV) influence immunotherapy efficacy and patient outcomes. Leucyl and cystinyl aminopeptidase (LNPEP) encodes a zinc-dependent aminopeptidase, which has been proved to participant in the vesicle-mediated transport and class I MHC mediated antigen processing and presentation. However, the function of LNPEP in TME of OV and its potential molecular mechanisms have not been determined. Therefore, we aimed to investigate a prognostic biomarker which may be helpful in identifying TME heterogeneity of ovarian cancer. In this study, bioinformatics databases were used to explore the expression profile and immune infiltration of LNPEP. Bioinformatics analyses of survival data and interactors of LNPEP were conducted to predict the prognostic value of LNPEP in OV. The protein levels of LNPEP were validated by Western blot and immunohistochemistry. Based on the TCGA data, our data displayed that the mRNA expression of LNPEP was markedly down-regulated in ovarian cancer than that in para-cancer tissues, contrary to the protein level. Importantly, high LNPEP expression was associated with poor prognosis in patients with OV. Furthermore, Cox regression analysis showed that LNPEP was an independent prognostic factor in OV. GO and KEGG pathway analyses indicated the co-expressed genes of LNPEP were mainly related to a variety of immune-related pathways, including Th1 and Th2 cell differentiation, Th17 cell differentiation, and immunoregulatory interaction. Our data also demonstrated that the expression of LNPEP was strongly correlated with immune infiltration levels, immunomodulators, chemokines and chemokine receptors. In our study, we identified and established a prognostic signature of immune-related LNPEP in OV, which will be of great value in predicting the prognosis of clinical trials and may become a new therapeutic target for immunological research and potential prognostic biomarker in OV.

Crosstalk of Immune Cells and Platelets in an Ovarian Cancer Microenvironment and Their Prognostic Significance.

Ovarian cancer (OC) is one of the deadliest gynecological cancers, largely due to the fast development of metastasis and drug resistance. The immune system is a critical component of the OC tumor microenvironment (TME) and immune cells such as T cells, NK cells, and dendritic cells (DC) play a key role in anti-tumor immunity. However, OC tumor cells are well known for evading immune surveillance by modulating the immune response through various mechanisms. Recruiting immune-suppressive cells such as regulatory T cells (Treg cells), macrophages, or myeloid-derived suppressor cells (MDSC) inhibit the anti-tumor immune response and promote the development and progression of OC. Platelets are also involved in immune evasion by interaction with tumor cells or through the secretion of a variety of growth factors and cytokines to promote tumor growth and angiogenesis. In this review, we discuss the role and contribution of immune cells and platelets in TME. Furthermore, we discuss their potential prognostic significance to help in the early detection of OC and to predict disease outcome.

A novel T-cell exhaustion-related feature can accurately predict the prognosis of OC patients.

The phenomenon of T Cell exhaustion (TEX) entails a progressive deterioration in the functionality of T cells within the immune system during prolonged conflicts with chronic infections or tumors. In the context of ovarian cancer immunotherapy, the development, and outcome of treatment are closely linked to T-cell exhaustion. Hence, gaining an in-depth understanding of the features of TEX within the immune microenvironment of ovarian cancer is of paramount importance for the management of OC patients. To this end, we leveraged single-cell RNA data from OC to perform clustering and identify T-cell marker genes utilizing the Unified Modal Approximation and Projection (UMAP) approach. Through GSVA and WGCNA in bulk RNA-seq data, we identified 185 TEX-related genes (TEXRGs). Subsequently, we transformed ten machine learning algorithms into 80 combinations and selected the most optimal one to construct TEX-related prognostic features (TEXRPS) based on the mean C-index of the three OC cohorts. In addition, we explored the disparities in clinicopathological features, mutational status, immune cell infiltration, and immunotherapy efficacy between the high-risk (HR) and low-risk (LR) groups. Upon the integration of clinicopathological features, TEXRPS displayed robust predictive power. Notably, patients in the LR group exhibited a superior prognosis, higher tumor mutational load (TMB), greater immune cell infiltration abundance, and enhanced sensitivity to immunotherapy. Lastly, we verified the differential expression of the model gene CD44 using qRT-PCR. In conclusion, our study offers a valuable tool to guide clinical management and targeted therapy of OC.

Characteristics of the receptor phenotype of tumor-associated immune cells of the epithelial-mesenchymal microenvironment of ovarian cancer

Among all histological types, serous carcinomas account for up to 85%. Due to pronounced heterogeneity (at the molecular and genetic level) and chemoresistance, difficulties arise in finding active targets for tumor elimination.
 Aim. To establish a link between the population composition of tumor-associated immune cells of the microenvironment and the stage of serous ovarian cancer.
 Materials and methods. The analysis of the pathologic and anatomical material in 74 patients with serous ovarian cancer was carried out. Monoclonal antibodies were used to determine antigens in the samples: CD3, CD4, CD8, CD11b, CD14 and CD16.
 Results. The obtained results of the immunohistochemical study showed that in the composition of the immune cells of the microenvironment, the largest number of cells, at all stages (IIV) of the oncological process, are represented by macrophages (CD11b+, CD14+), CD3+ lymphocytes are in second place in terms of the number of cells, followed by CD8+ and CD4+ and the smallest number of CD16+ cells.
 Conclusion. As a result of the immunohistochemical study, a multidirectional trend was found between the population composition of tumor-associated immune cells of the microenvironment and the stage of serous ovarian cancer. With an increase in the stage of the disease, the number of macrophages (CD11b+, CD14+) and lymphocytes (CD3+, CD16+) decreased regardless of the degree of differentiation of the tumor. With an increase in the tumor stage, the number of CD4+ and CD8+ populations decreased, but in this case, the degree of differentiation played a significant role, i.e. the higher the tumor stage and the lower the degree of differentiation, the fewer cells were detected.

Generation of repetitive element-specific T cells to target ovarian cancer

Abstract The immunosuppressive tumor microenvironment in ovarian cancer (OC) enables these tumors to evade the immune system, resulting in late diagnosis and poor therapeutic outcomes for patients. This immunosuppression can be partially reversed with epigenetic inhibitors, which stimulate an immunogenic interferon response in OC cells by inducing the transcription of repetitive elements (REs). However, epigenetic therapy alone is not curative in clinical trials and new treatments that further augment the immune response to OC are needed. While REs are epigenetically silent in terminally differentiated cells, their increased expression in OC cells implicate these genomic elements as an unexplored pool of tumor antigens. As epigenetic therapy also upregulates antigen processing and presentation in OC cells, the generation of T cells specific for over-expressed RE-derived antigens may boost the immune recognition of OC cells. Transducing healthy donor T cells with a T cell receptor construct that recognizes the RE ERV-K-Env resulted in antigen specificity only when co-cultured with B lymphoblastoid cells derived from the same donor that the T cell receptor was isolated from. Transduced T cells were not specific for ERV-K-Env in autologous systems. As previous reports have shown that ERV-K-Env-specific T cells can only be expanded from patients with OC, current studies are underway to expand T cells from OC patients. Upon successful expansion of ERV-K-Env-specific T cells from multiple OC patients, we will assess if specific recognition is enhanced by treating autologous tumor cells with epigenetic therapy. These efforts serve as a novel approach to enhance the immune response to OC and improve the overall OC patient survival rate. NIH F31 CA271788-01 DOD W81XWH2010273

A novel autophagy-related gene signature associated with prognosis and immune microenvironment in ovarian cancer

Ovarian cancer (OV), the most fatal gynecological malignance worldwide, has high recurrence rates and poor prognosis. Recently, emerging evidence supports that autophagy, a highly regulated multi-step self-digestive process, plays an essential role in OV progression. Accordingly, we filtered 52 potential autophagy-related genes (ATGs) among the 6197 differentially expressed genes (DEGs) identified in TCGA-OV samples (n = 372) and normal controls (n = 180). Based on the LASSO-Cox analysis, we distinguished a 2-gene prognostic signature, namely FOXO1 and CASP8, with promising prognostic value (p-value < 0.001). Together with corresponding clinical features, we constructed a nomogram model for 1-year, 2-year, and 3-year survival, which was validated in both in training (TCGA-OV, p-value < 0.001) and validation (ICGC-OV, p-value = 0.030) cohorts. Interestingly, we evaluated the immune infiltration landscape through the CIBERSORT algorithm, which indicated the upregulation of 5 immune cells, including CD8 + T cells, Tregs, and Macrophages M2, and high expression of critical immune checkpoints (CTLA4, HAVCR2, PDCD1LG2, and TIGIT) in high-risk group. Stepwise, high-risk group exhibited better sensitivity towards chemotherapies of Bleomycin, Sorafenib, Veliparib, and Vinblastine, though less sensitive to immunotherapy. Especially, based on the IHC of tissue microarrays among 125 patients in our institution, we demonstrated that aberrant upregulation of FOXO1 in OV was related to metastasis and poor prognosis. Moreover, FOXO1 could significantly promote tumor invasiveness, migration, and proliferation in OV cell lines, which was assessed through the Transwell, wound-healing, and CCK-8 assay, respectively. Briefly, the autophagy-related signature was a reliable tool to evaluate immune responses and predict prognosis in the realm of OV precision medicine.

Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression

ABSTRACT The tumor microenvironment (TME) in ovarian cancer (OC) is characterized by immune suppression, due to an abundance of suppressive immune cells populations. To effectively enhance the activity of immune checkpoint inhibition (ICI), there is a need to identify agents that target these immunosuppressive networks while promoting the recruitment of effector T cells into the TME. To this end, we sought to investigate the effect of the immunomodulatory cytokine IL12 alone or in combination with dual-ICI (anti-PD1 + anti-CTLA4) on anti-tumor activity and survival, using the immunocompetent ID8-VEGF murine OC model. Detailed immunophenotyping of peripheral blood, ascites, and tumors revealed that durable treatment responses were associated with reversal of myeloid cell-induced immune suppression, which resulted in enhanced anti-tumor activity by T cells. Single cell transcriptomic analysis further demonstrated striking differences in the phenotype of myeloid cells from mice treated with IL12 in combination with dual-ICI. We also identified marked differences in treated mice that were in remission compared to those whose tumors progressed, further confirming a pivotal role for the modulation of myeloid cell function to allow for response to immunotherapy. These findings provide the scientific basis for the combination of IL12 and ICI to improve clinical response in OC.

Abstract 5153: The role of CCL22-specific CD4 T cells in modulating the immunosuppressive tumor microenvironment in ovarian cancer

Abstract Background: CCL22, a macrophage-derived chemokine, is overexpressed in ovarian cancer and exerts immunosuppressive functions by the recruitment of regulatory T cells (Tregs) to the tumor microenvironment (TME). Our research group described naturally occurring T cells specific for an MHC class I epitope derived from CCL22. CD8+ CCL22-specific T cells isolated from cancer patients could recognize CCL22-expressing cells and decrease the levels of CCL22 in ovarian ascites in vitro. Expansion of CCL22-specific CD8+ T cells in animal models of cancer by peptide vaccination led to a therapeutic anti-tumor effect by reducing Treg recruitment into the TME. In this study we aimed at characterizing CD4+ CCL22-specific T cells and their potential for modulating the tumor microenvironment in ovarian cancer. Methods: CCL22-responses were screened with a library of overlapping 20mer peptides covering the entire sequence of CCL22 in PBMCs from healthy donors and ovarian cancer patients. CCL22-specific T cells were isolated from PBMCs and ovarian ascites for functional characterization through IFNγ ELISpot and ICS against peptides and CCL22-expressing target cells. Cytokine secretion was measured with protein multiplex immunoassays. Vaccination and therapeutic efficacy were evaluated in BALB/c CT26 and 4T1 syngeneic models. Results: We identified an immunogenic region in the CCL22 protein sequence and detected CD4 T-cell responses against CCL22-derived peptides in healthy donors and ovarian cancer patients through IFNγ ELISpot. CCL22-specific CD4+ cells have also been identified ex vivo in the ascites fluid from patients with ovarian cancer. We isolated and expanded CCL22-specific CD4+ T-cell clones from the ovarian ascites and demonstrated their reactivity against CCL22-producing cell lines. Furthermore, the stimulation of CCL22-specific CD4+ T cells in the PBMC culture and ascites samples was associated with a modulation of the cytokine environments. Additionally, vaccination with surrogate murine 20mer and MHC-II restricted CCL22-derived peptides lead to successful induction of specific T-cell responses in the spleens and draining lymph nodes of vaccinated animals. Activation of such CCL22-specific CD4 T cell responses was associated with a therapeutic effect seen as reduction of the tumor growth in different models of cancer. Conclusion: This study provides evidence for the ability of pro-inflammatory CD4+ CCL22-specific T cells to modulate the tumor microenvironment, particularly in ovarian cancer. Altogether we provide the rationale for the development of a CCL22-based immune modulatory vaccination for ovarian cancer. Citation Format: Ines Lecoq, Evelina Martinenaite, Marie C. Westergaard, Inge Marie Svane, Mads H. Andersen. The role of CCL22-specific CD4 T cells in modulating the immunosuppressive tumor microenvironment in ovarian cancer. [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 5153.

Abstract 3262: Single-cell spatial dynamics of the tumor-immune microenvironment of high-grade serous ovarian cancer during neoadjuvant chemotherapy

Abstract Neoadjuvant Platinum-based combination chemotherapy (NACT) is commonly offered to patients with high-grade serous ovarian cancer (HGSC) harboring unresectable disease in the primary setting. In order to develop effective immunotherapeutic and combinatorial treatment strategies for HGSC, a deeper knowledge on the effects of NACT on the spatial tumor-immune interactions is required. We performed genetic and single-cell spatial characterization of the tumor-immune microenvironment of 22 HGSC samples collected before and after NACT using Whole Genome Sequencing and tissue cyclic immunofluorescence (t-CycIF) highly-multiplexed imaging platform. We used image analysis and bioinformatics to analyze 30 antibodies in over 6 500 000 single-cells in the context of their spatial tissue architecture. The results were integrated with genomic features and clinical data. We identified four distinct tumor cell metaclusters, eight immune cell subtypes and six stromal cell subpopulations. Chemotherapy induced distinct phenotypic changes in the tumor and immune cells including glutamine metabolism, cell proliferation and antigen presentation. Using single-cell spatial analyses, we identified 18 unique tumor-immune-stromal communities, characterized by different tumor, stromal and immune cell proportions, as well as the tumor-stromal interface. Interestingly, we observed increased CD8+T-cell and decreased CD163+ macrophage infiltration specifically at the tumor-stroma interface induced by chemotherapy. Furthermore, NACT induced spatial CD8+T-cell and macrophage cell-cell interaction patterns specifically in the tumor-stromal interface. Further dissection of the functional properties of the spatial cell-cell interactions using spatial transcriptomics revealed distinct tumor-cell clonal dynamics and gene expression programs on the tumor-stroma interface during NACT. In conclusion, we identified distinct spatial features in the tumor-immune microenvironment indicating the tumor-stromal interface as the underpinning of spatial dynamics in HGSC during NACT. Using a spatial single-cell multi-omics approach has the potential to improve patient stratification and immunotherapeutic strategies in HGSC. Citation Format: Inga-Maria Pauliina Launonen, Angela Szabo, Fernando Perez, Julia Casado, Zoltan Maliga, Sampsa Hautaniemi, Jaana Oikkonen, Johanna Hynninen, Tuulia Vallius, Ajit Johnson Nirmal, Peter Sorger, Anna Vähärautio, Anniina Färkkilä. Single-cell spatial dynamics of the tumor-immune microenvironment of high-grade serous ovarian cancer during neoadjuvant chemotherapy [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 3262.

Abstract 617: Oncogenic CXCL10 triggers CD8+ T cell exhaustion in ovarian cancer

Abstract Ovarian cancer is one of the most severe gynecologic malignancies with high mortality. Although programmed cell death 1 (PD-1) or PD-ligand 1 (PD-L1) immune checkpoint blockade (ICBs) has been used therapeutically in gynecologic cancers and has a clinically significant response in endometrial cancer, the response rate remains poor and has no different from the conventional therapies in ovarian cancer. The highly immunosuppressive microenvironment in ovarian cancer, consisting of inhibitory ligands, suppressive mediators, and cell subsets, leads to CD8+ T cell dysfunction and exhaustion and contributes to the limited efficacy of immunotherapeutic approaches. Correlative human studies have highlighted the potential importance of chemokines on the status of CD8+ T cell infiltration into tumors and on patient survival. Here, we show that CXCL10, an IFN-γ induced chemokine, derived from ovarian cancer cells, contributes to impairing CD8+ T cell antitumor immunity by regulating CD8+ T cell exhaustion in the tumor microenvironment. Clinically, we found that CXCL10 and its receptor CXCR3 had a strong positive correlation with exhausted T cell signature genes in ovarian cancer patients in TCGA and in-house cohort. Functionally, we showed that CD8+ T cell exhaustion was induced by CXCL10 activation by CRISPRa from ovarian cancer cells, whereas inhibition by CRISPRi restored CD8+ T cell antitumor immunity ex vivo and in vivo. Mechanically, ovarian cancer cells-derived CXCL10 induced the expression of CD8+ T cells exhaustion transcription factors in a CXCR3-dependent manner, driving PD-1 and TIM-3 expression on CD8+ T cells. Therapeutically, suppressing CXCL10 secretion from ovarian cancer cells enhanced the response to anti-PD-1 immunotherapy in the orthotopic mouse model. The results suggest that selectively inhibiting the oncogenic CXCL10 production may work effectively with ICBs in ovarian cancer immunotherapy. Our findings advance the understanding of the low response rate of immunotherapy in ovarian cancer and explore the potential mechanism underlying the induction of CD8+ T cell exhaustion in the tumor microenvironment. Citation Format: Runying Long, Tao Ding, Michelle K.L Siu, David W Chan, Jiangnan He, Annie NY Cheung, Kwan Man, Hextan YS NGAN, Karen KL Chan. Oncogenic CXCL10 triggers CD8+ T cell exhaustion in ovarian cancer [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 617.

Abstract 4551: Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer: understanding the contribution of malignant cells

Abstract Following a ‘deconstruction’ of the metastatic tumor microenvironment (TME) of high grade serous ovarian cancer (HGSOC), we developed 3-dimensional (3D) human multicellular in vitro models using patient-derived primary cells and HGSOC malignant cell lines. In this study, we used these 3D in vitro human models to study how malignant cells influence the surrounding TME. Using patient-derived primary adipocytes, fibroblasts and mesothelial cells isolated from macroscopically normal omentum (the primary site of metastasis in HGSOC) from women undergoing gynaeco-oncology surgery, we built multicellular models combining those cells with different high grade serous malignant cell lines and monocytes isolated from healthy individuals. We found that peripheral blood monocytes were able to differentiate and polarize to macrophages in our pentacultures (i.e., five different cell types) with phenotypes also found in HGSOC omental metastases. In addition, the cells in our pentaculture models self-rearranged in ways that were dependent on the HGSOC malignant cell lines used in the culture. Macrophage phenotype and cytokine secretion from the multicellular cultures was also influenced by the different malignant HGSOC cell lines used in the model. RNAseq analysis of the pentacultures showed that the cultures clustered according to the malignant cell line used. In addition, we were able to identify some gene expression clusters in the macrophages and fibroblasts of the pentacultures that matched those found by single cell RNAseq analysis of human HGSOC biopsies. These human 3D in vitro multicellular models allow us to define the contribution of each cell type to tumor growth and spread, and also provide us a semi-high throughput platform to investigate antibody or drug targets. Citation Format: Beatrice Malacrida, Samar Elorbany, Eleni Maniati, Florian Laforets, Ranjit Manchanda, Frances R. Balkwill. Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer: understanding the contribution of malignant cells. [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 4551.

Abstract 527: Targeting citric acid cycle anaplerosis reverses the immunosuppressive phenotype of CD11b+Gr1+ myeloid cells in ovarian cancer

Abstract Immunosuppressive myeloid cells (CD11b+Gr1+) are a major constituent of the epithelial ovarian cancer (EOC) microenvironment that confer immunosuppression and promote tumor growth. We recently showed that the EOC microenvironment can induce metabolic reprogramming inCD11b+Gr1+ cells by increasing oxidative phosphorylation via TCA cycle, resulting in increased immunosuppressive ability. Increased TCA cycle activity was due to glutamine anaplerosis, which was facilitated by the upregulation of DLST (dihydrolipoamide succinyl transferase), the E2transferase subunit of α-KGDC (alpha-ketoglutarate dehydrogenase complex). We selected CPI-613 (Devimistat), an orphan metabolic drug, being actively tested in clinical trials to target the a-KGDC complex. We utilized the syngeneic ID8p53-/− model, a high grade serous ovarian cancer, to determine if CPI-613 may regulate CD11b+Gr1+ cell metabolism and immunosuppressive function and restore an antitumor immune response that inhibits EOC. One week after inducing tumors, mice were treated with CPI-613 (2.5mg/kg body weight) twice a week by IP injections or with vehicle (corn oil, control). CPI-613 treated mice showed significantly improved overall survival (median survival 75 days versus 48 days in control), decreased tumor progression and burden.CPI-613 decreased the number of CD11b+Gr1+ myeloid cells in ascites, and more importantly, reduced the intracellular immunosuppressive markers arginase 1 and IL-1β, indicating that the immunosuppressive potential was diminished, which was also validated by reversal of T cell suppression by CPI-613. The alleviation of immunosuppression was complemented by remarkable increase in CD8+ T effector cells. Further, combination of CPI-613 with anti-PD1(100ug/mice, every 5th day for 4 weeks) significantly improved survival and potentiated the CD8+mediated anti-tumor response. CPI-613 inhibited expression of DLST and immunosuppressive markers in the CD11b+Gr1+ myeloid cells, accompanied by decreased oxidative phosphorylation and decreased glutamine anaplerosis. Interestingly CPI-613 treatment also decreased the tumor associated macrophages, increased the M1 marker CD38 and decreased M2 marker EGR2, which reflected in increased M1/M2 macrophage ratio. Increased glutamine demand and addiction have been shown in variety of cancers, including EOC. We found that DLST expression corelates with worse progression free survival in the ovarian cancer TCGA dataset, implying that DLST is important in EOC cells. As a result, DLST targeting of aberrant glutamine metabolism will have the dual benefit of reducing CD11b+Gr1+ cell-mediated immunosuppression while also directly inhibiting tumor growth. Thus CPI-613 represents an attractive drug to target glutamine addiction and reduce immunosuppressive environment plaguing ovarian cancer. Citation Format: Mary Priyanka Udumula, Harshit Singh, Faraz Rashid, Miriana Hijaz, Radhika Gogoi, Philip Philip, Adnan Munkarah, Shailendra Giri, Ramandeep Rattan. Targeting citric acid cycle anaplerosis reverses the immunosuppressive phenotype of CD11b+Gr1+ myeloid cells in ovarian cancer [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 527.