Ovarian Cancer Tumor Microenvironment Research Articles

Novel protein-based prognostic signature linked to immunotherapeutic efficiency in ovarian cancer

BackgroundPersonalized medicine remains an unmet need in ovarian cancer due to its heterogeneous nature and complex immune microenvironments, which has gained increasing attention in the era of immunotherapy. A key obstacle is the lack of reliable biomarkers to identify patients who would benefit significantly from the therapy. While conventional clinicopathological factors have exhibited limited efficacy as prognostic indicators in ovarian cancer, multi-omics profiling presents a promising avenue for comprehending the interplay between the tumor and immune components. Here we aimed to leverage the individual proteomic and transcriptomic profiles of ovarian cancer patients to develop an effective protein-based signature capable of prognostication and distinguishing responses to immunotherapy.MethodsThe workflow was demonstrated based on the Reverse Phase Protein Array (RPPA) and RNA-sequencing profiles of ovarian cancer patients from The Cancer Genome Atlas (TCGA). The algorithm began by clustering patients using immune-related gene sets, which allowed us to identify immune-related proteins of interest. Next, a multi-stage process involving LASSO and Cox regression was employed to distill a prognostic signature encompassing five immune-related proteins. Based on the signature, we subsequently calculated the risk score for each patient and evaluated its prognostic performance by comparing this model with conventional clinicopathological characteristics.ResultsWe developed and validated a protein-based prognostic signature in a cohort of 377 ovarian cancer patients. The risk signature outperformed conventional clinicopathological factors, such as age, grade, stage, microsatellite instability (MSI), and homologous recombination deficiency (HRD) status, in terms of prognoses. Patients in the high-risk group had significantly unfavorable overall survival (p < 0.001). Moreover, our signature effectively stratified patients into subgroups with distinct immune landscapes. The high-risk group exhibited higher levels of CD8 T-cell infiltration and a potentially greater proportion of immunotherapy responders. The co-activation of the TGF-β pathway and cancer-associated fibroblasts could impair the ability of cytotoxic T cells to eliminate cancer cells, leading to poor outcomes in the high-risk group.ConclusionsThe protein-based signature not only aids in evaluating the prognosis but also provides valuable insights into the tumor immune microenvironments in ovarian cancer. Together our findings highlight the importance of a thorough understanding of the immunosuppressive tumor microenvironment in ovarian cancer to guide the development of more effective immunotherapies.

Construction of a PANoptosis-related Prognostic Signature for Predicting Prognosis, Tumor Microenvironment, and Immune Response in Ovarian Cancer.

The PANoptosis pathway is a recently identified mechanism of cellular death that involves the interaction and synchronization among cellular pyroptosis, apoptosis, and necrosis. More and more evidence suggests that PANoptosis is involved in the development and treatment of cancer. However, a comprehensive understanding of the influence of PANoptosis genes on prognostic value, tumor microenvironment characteristics, and therapeutic outcomes in patients with ovarian cancer (OC) remains incomplete. The present work was designed to devise a PANoptosis signature for OC prognosis and explore its potential molecular function. For this study, we obtained RNA sequencing and clinical data for ovarian cancer from the Cancer Genome Atlas (TCGA) and the GSE32062 cohort. Somatic variants of PANoptosis-related genes (PRGs) in OC were analyzed using GSCA. TCGA-OC and GSE32062 were used to construct training and validation cohorts for the model. Differential expression and correlation analyses were performed following the screening of genes with prognostic ability using univariate Cox analysis. Least Absolute Shrinkage and Selection Operator (LASSO) regression was performed to construct PRG signature based on genes that were differentially expressed and correlated with prognosis. CIBERSORT and ESTIMATE were used to analyze the relationship between the PRGs signature and immune infiltration. TIDE was used to analyze the relationship between the PRG signature and immune checkpoint genes. OncoPredict was used to analyze the relationship between the PRG signature and the drug sensitivity. Quantitative real-time PCR (qRT-PCR) was used to validate the expression of PRGs in OC. The PRG signature was constructed using three prognostic genes (AIM2, APAF1, and ZBP1) in both TCGA-OC. The results showed that the PRGs signature had an AUC of 0.521, 0.546, and 0.598 in TCGA-OC and 0.620, 0.586, and 0.579 in GSE32062 to predict to predict OS at 1-, 3-, and 5-year intervals. Furthermore, a higher PRG signature risk score was significantly associated with shorter OS (HR = 1.693, 95% CI: 1.303 - 2.202, p = 8.34 × 10^-5 in TCGA-OC and HR = 1.63, 95% CI: 1.13 - 2.35, p = 0.009 in GSE32062). The risk score was identified as the independent prognostic factor for OC. Patients categorized according to their risk score exhibited notable variations in immune status, response to immunotherapy, and sensitivity to drugs. AIM2, APAF1, and ZBP1 were significantly aberrantly expressed in OC cell lines. The PRG signature has the potential to serve as a prognostic predictor for OC and to provide new insights into OC treatment.

MiRNAs as Regulators of Immune Cells in the Tumor Microenvironment of Ovarian Cancer.

Ovarian cancer is one of the leading causes of cancer deaths among women. There is an ongoing need to develop new biomarkers and targeted therapies to improve patient outcomes. One of the most critical research areas in ovarian cancer is identifying tumor microenvironment (TME) functions. TME consists of tumor-infiltrating immune cells, matrix, endothelial cells, pericytes, fibroblasts, and other stromal cells. Tumor invasion and growth depend on the multifactorial crosstalk between tumor cells and immune cells belonging to the TME. MiRNAs, which belong to non-coding RNAs that post-transcriptionally control the expression of target genes, regulate immune responses within the TME, shaping the landscape of the intrinsic environment of tumor cells. Aberrant expression of miRNAs may lead to the pathological dysfunction of signaling pathways or cancer cell-regulatory factors. Cell-to-cell communication between infiltrating immune cells and the tumor may depend on exosomes containing multiple miRNAs. MiRNAs may exert both immunosuppressive and immunoreactive responses, which may cause cancer cell elimination or survival. In this review, we highlighted recent advances in the field of miRNAs shaping the landscape of immune cells in the TME.

Single-cell RNA sequencing reveals that MYBL2 in malignant epithelial cells is involved in the development and progression of ovarian cancer.

Ovarian carcinoma (OC) is a prevalent gynecological malignancy associated with high recurrence rates and mortality, often diagnosed at advanced stages. Despite advances in immunotherapy, immune exhaustion remains a significant challenge in achieving optimal tumor control. However, the exploration of intratumoral heterogeneity of malignant epithelial cells and the ovarian cancer tumor microenvironment is still limited, hindering our comprehensive understanding of the disease. Utilizing single-cell RNA sequencing (scRNA-seq), we comprehensively investigated the cellular composition across six ovarian cancer patients with omental metastasis. Our focus centered on analysis of the malignant epithelial cells. Employing CytoTRACE and slingshot pseudotime analyses, we identified critical subpopulations and explored associated transcription factors (TFs) influencing ovarian cancer progression. Furthermore, by integrating clinical factors from a large cohort of bulk RNA sequencing data, we have established a novel prognostic model to investigate the impact of the tumor immune microenvironment on ovarian cancer patients. Furthermore, we have investigated the condition of immunological exhaustion. Our study identified a distinct and highly proliferative subgroup of malignant epithelial cells, known as C2 TOP2A+ TCs. This subgroup primarily consisted of patients who hadn't received neoadjuvant chemotherapy. Ovarian cancer patients with elevated TOP2A expression exhibited heightened sensitivity to neoadjuvant chemotherapy (NACT). Moreover, the transcription factor MYBL2 in this subgroup played a critical role in ovarian cancer development. Additionally, we developed an independent prognostic indicator, the TOP2A TCs Risk Score (TTRS), which revealed a correlation between the High TTRS Group and unfavorable outcomes. Furthermore, immune infiltration and drug sensitivity analyses demonstrated increased responsiveness to Paclitaxel, Cisplatin, and Gemcitabine in the Low TTRS Group. This research deepens our understanding of malignant epithelial cells in ovarian cancer and enhances our knowledge of the ovarian cancer immune microenvironment and immune exhaustion. We have revealed the heightened susceptibility of the C2 TOP2A+ TCs subgroup to neoadjuvant chemotherapy and emphasized the role of MYBL2 within the C2 subgroup in promoting the occurrence and progression of ovarian cancer. These insights provide valuable guidance for the management of ovarian cancer treatment.

The Complex Tumor Microenvironment in Ovarian Cancer: Therapeutic Challenges and Opportunities.

The tumor microenvironment (TME) in ovarian cancer (OC) has much greater complexity than previously understood. In response to aggressive pro-angiogenic stimulus, blood vessels form rapidly and are dysfunctional, resulting in poor perfusion, tissue hypoxia, and leakiness, which leads to increased interstitial fluid pressure (IFP). Decreased perfusion and high IFP significantly inhibit the uptake of therapies into the tumor. Within the TME, there are numerous inhibitor cells, such as myeloid-derived suppressor cells (MDSCs), tumor association macrophages (TAMs), regulatory T cells (Tregs), and cancer-associated fibroblasts (CAFs) that secrete high numbers of immunosuppressive cytokines. This immunosuppressive environment is thought to contribute to the lack of success of immunotherapies such as immune checkpoint inhibitor (ICI) treatment. This review discusses the components of the TME in OC, how these characteristics impede therapeutic efficacy, and some strategies to alleviate this inhibition.

The Impact of Neoadjuvant Chemotherapy on Ovarian Cancer Tumor Microenvironment: A Systematic Review of the Literature.

Immunotherapy, particularly the use of immune checkpoint inhibitors (ICIs), has shown limited efficacy in treating ovarian cancer (OC), possibly due to diverse T cell infiltration patterns in the tumor microenvironment. This review explores how neoadjuvant chemotherapy (NACT) impacts the immune landscape of OC, focusing on tumor-infiltrating lymphocytes (TILs), PD-1/PD-L1 expression, and their clinical implications. A comprehensive literature search across four databases yielded nine relevant studies. These studies evaluated stromal (sTILs) and intra-epithelial (ieTILs) TILs before and after NACT. sTIL responses varied, impacting prognostic outcomes, and ieTILs increased in some patients without clear survival associations. PD-L1 expression after NACT correlated with improved overall survival (OS), and increases in granzyme B+ and PD-1 correlated with longer progression-free survival (PFS). Remarkably, reduced FoxP3+ TILs post-NACT correlated with better prognosis. NACT often increases sTIL/ieTIL and CD8+ subpopulations, but their correlation with improved PFS and OS varies. Upregulation of co-inhibitory molecules, notably PD-L1, suggests an immunosuppressive response to chemotherapy. Ongoing trials exploring neoadjuvant ICIs and chemotherapy offer promise for advancing OC treatment. Standardized measurements assessing TIL density, location, and heterogeneity are crucial for addressing genetic complexity and immunological heterogeneity in OC.

The endothelin-1-driven tumor-stroma feed-forward loops in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) tumor microenvironment (TME) is constellated by cellular elements and a network of soluble constituents that contribute to tumor progression. In the multitude of the secreted molecules, the endothelin-1 (ET-1) has emerged to be implicated in the tumor/TME interplay; however, the molecular mechanisms induced by the ET-1-driven feed-forward loops (FFL) and associated with the HG-SOC metastatic potential need to be further investigated. The tracking of the patient-derived (PD) HG-SOC cell transcriptome by RNA-seq identified the vascular endothelial growth factor (VEGF) gene and its associated signature among those mostly up-regulated by ET-1 and down-modulated by the dual ET-1R antagonist macitentan. Within the ligand-receptor pairs concurrently expressed in PD-HG-SOC cells, endothelial cells and activated fibroblasts, we discovered two intertwined FFL, the ET-1/ET-1R and VEGF/VEGF receptors, concurrently activated by ET-1 and shutting-down by macitentan, or by the anti-VEGF antibody bevacizumab. In parallel, we observed that ET-1 fine-tuned the tumoral and stromal secretome toward a pro-invasive pattern. Into the fray of the HG-SOC/TME double and triple co-cultures, the secretion of ET-1 and VEGF, that share a common co-regulation, was inhibited upon the administration of macitentan. Functionally, macitentan, mimicking the effect of bevacizumab, interfered with the HG-SOC/TME FFL-driven communication that fuels the HG-SOC invasive behavior. The identification of ET-1 and VEGF FFL as tumor and TME actionable vulnerabilities, reveals how ET-1R blockade, targeting the HG-SOC cells and the TME simultaneously, may represent an effective therapeutic option for HG-SOC patients.

Adipocyte pyroptosis occurs in omental tumor microenvironment and is associated with chemoresistance of ovarian cancer

BackgroundOvarian carcinoma (OC) is a fatal malignancy, with most patients experiencing recurrence and resistance to chemotherapy. In contrast to hematogenous metastasizing tumors, ovarian cancer cells disseminate within the peritoneal cavity, especially the omentum. Previously, we reported omental crown-like structure (CLS) number is associated with poor prognosis of advanced-stage OC. CLS that have pathologic features of a dead or dying adipocyte was surrounded by several macrophages is well known a histologic hallmark for inflammatory adipose tissue. In this study, we attempted to clarify the interaction between metastatic ovarian cancer cells and omental CLS, and to formulate a therapeutic strategy for advanced-stage ovarian cancer.MethodsA three-cell (including OC cells, adipocytes and macrophages) coculture model was established to mimic the omental tumor microenvironment (TME) of ovarian cancer. Caspase-1 activity, ATP and free fatty acids (FFA) levels were detected by commercial kits. An adipocyte organoid model was established to assess macrophages migration and infiltration. In vitro and in vivo experiments were performed for functional assays and therapeutic effect evaluations. Clinical OC tissue samples were collected for immunochemistry stain and statistics analysis.ResultsIn three-cell coculture model, OC cells-derived IL-6 and IL-8 could induce the occurrence of pyroptosis in omental adipocytes. The pyroptotic adipocytes release ATP to increase macrophage infiltration, release FFA into TME, uptake by OC cells to increase chemoresistance. From OC tumor samples study, we demonstrated patients with high gasdermin D (GSDMD) expression in omental adipocytes is highly correlated with chemoresistance and poor outcome in advanced-stage OC. In animal model, by pyroptosis inhibitor, DSF, effectively retarded tumor growth and prolonged mice survival.ConclusionsOmental adipocyte pyroptosis may contribute the chemoresistance in advanced stage OC. Omental adipocytes could release FFA and ATP through the GSDMD-mediate pyroptosis to induce chemoresistance and macrophages infiltration resulting the poor prognosis in advanced-stage OC. Inhibition of adipocyte pyroptosis may be a potential therapeutic modality in advanced-stage OC with omentum metastasis.

Recent advances in understanding the immune microenvironment in ovarian cancer.

The occurrence of ovarian cancer (OC) is a major factor in women's mortality rates. Despite progress in medical treatments, like new drugs targeting homologous recombination deficiency, survival rates for OC patients are still not ideal. The tumor microenvironment (TME) includes cancer cells, fibroblasts linked to cancer (CAFs), immune-inflammatory cells, and the substances these cells secrete, along with non-cellular components in the extracellular matrix (ECM). First, the TME mainly plays a role in inhibiting tumor growth and protecting normal cell survival. As tumors progress, the TME gradually becomes a place to promote tumor cell progression. Immune cells in the TME have attracted much attention as targets for immunotherapy. Immune checkpoint inhibitor (ICI) therapy has the potential to regulate the TME, suppressing factors that facilitate tumor advancement, reactivating immune cells, managing tumor growth, and extending the survival of patients with advanced cancer. This review presents an outline of current studies on the distinct cellular elements within the OC TME, detailing their main functions and possible signaling pathways. Additionally, we examine immunotherapy rechallenge in OC, with a specific emphasis on the biological reasons behind resistance to ICIs.

The Gut Microbiome in Aging and Ovarian Cancer.

The gut microbiome changes with age and affects regions beyond the gut, including the ovarian cancer tumor microenvironment. In this review summarizing the literature on the gut microbiome in ovarian cancer and in aging, we note trends in the microbiota composition common to both phenomena and trends that are distinctly opposite. Both ovarian cancer and aging are characterized by an increase in proinflammatory bacterial species, particularly those belonging to phylum Proteobacteria and genus Escherichia, and a decrease in short chain fatty acid producers, particularly those in Clostridium cluster XIVa (family Lachnospiraceae) and the Actinobacteria genus Bifidobacterium. However, while beneficial bacteria from family Porphyromonadaceae and genus Akkermansia tend to increase with normal, healthy aging, these bacteria tend to decrease in ovarian cancer, similar to what is observed in obesity or unhealthy aging. We also note a lack in the current literature of research demonstrating causal relationships between the gut microbiome and ovarian cancer outcomes and research on the gut microbiome in ovarian cancer in the context of aging, both of which could lead to improvements to ovarian cancer diagnosis and treatment.

Aberrant angiogenic signaling pathways: Accomplices in ovarian cancer progression and treatment

Ovarian cancer is one of the most common malignant tumors in women, and treatment options are limited. Despite efforts to adjust cancer treatment models and develop new methods, including tumor microenvironment (TME) therapy, more theoretical support is needed. Increasing attention is being given to antiangiogenic measures for TME treatment. Another important concept in ovarian cancer TME is angiogenesis, where tumor cells obtain nutrients and oxygen from surrounding tissues through blood vessels to support further expansion and metastasis. Many neovascularization signaling pathways become imbalanced and hyperactive during this process. Inhibiting these abnormal pathways can yield ideal therapeutic effects in patients, even by reversing drug resistance. However, these deep TME signaling pathways often exhibit crosstalk and correlation. Understanding these interactions may be an important strategy for further treating ovarian cancer. This review summarizes the latest progress and therapeutic strategies for these angiogenic signaling pathways in ovarian cancer.

Complement signaling as a T-cell checkpoint in the ovarian cancer (OC) tumor microenvironment (TME)

Abstract T cell checkpoint pathways are barriers to anti-tumor immunity and critical therapeutic targets. We use ascites fluid supernatants (ASC) from patients with newly diagnosed OC as an authentic component of the TME. We previously showed that complement activation is increased in ASC versus paired serum and induces a suppressor phenotype in normal neutrophils characterized by inhibition of critical proliferative, signaling and metabolic pathways in normal human T cells. ASC by itself dramatically inhibited CD3/CD28-stimulated T cell cytokine production (IL-2, IFN-γ, and IL-10) and glucose consumption, but not proliferation. We hypothesized that complement activation in the TME delivers an inhibitory stimulus to T cells that suppresses stimulated cytokine responses required for activation and expansion. Peptide C3 inhibitors (compstatins) restored CD3/CD28-stimulated T cell cytokine responses and glucose consumption. Inhibition of Factor B, a component of the alternative pathway (AP), also rescued cytokine responses, while inhibition of C3aR, C5aR, and C7 had no significant effect. C3b/iC3b deposition on T cells was increased with ASC exposure and reduced by C3 inhibition. Addition of recombinant complement receptor-1 (CR-1) as a decoy of C3b binding to membrane-bound CR-1 fully abrogated the suppressive effect of ASC, while recombinant CD46 had variable effects among different ASC samples. Our results show a novel AP- and CR1-dependent checkpoint of T cell activation in the TME.

ALKBH5 modulates macrophages polarization in tumor microenvironment of ovarian cancer

BackgroundMacrophages play an essential role in regulating ovarian cancer immune microenvironment. Studies have shown that m6A methylation could influence immune microenvironment in cancer. In this study, we investigated the roles of m6A demethylase ALKBH5 and m6A recognition protein IGF2BP2 played in regulating macrophages polarization in ovarian cancer.MethodsIn this study, we first explored the differentially expressed m6A methylation enzymes in M0 and M2 macrophages according to two independent GEO datasets. TIMER2.0 and GSCA database were used to explore the immune analysis of ALKBH5 and IGF2BP2 in ovarian cancer. K-M plotter and TIMER2.0 databases were used to evaluate the prognostic role of ALKBH5 and IGF2BP2 in ovarian cancer. For CNV mutation analysis of ALKBH5 and IGF2BP2, cBioPortal and GSCA databases were used. For single-cell analysis, sc-TIME and HPA softwares were used to analyze the roles of ALKBH5 and IGF2BP2 played in immune cells in ovarian cancer. To identify the role of ALKBH5 played in macrophage polarization, RT-PCR was used to verify the macrophage polarization related markers in vitro study. The function of ALKBH5 played in ovarian cancer was further analyzed through GO and KEGG analysis.FindingsIn this study, we found that ALKBH5 and IGF2BP2 were up-regulated in M2 macrophages, which showed closely correlation with immune cells expressions in ovarian cancer, especially with macrophages. Ovarian cancer patients with higher expression of ALKBH5 and IGF2BP2 showed worse prognosis, possibly because of their close correlation with immune response. ALKBH5 also correlated with macrophage phenotypes in single-cell levels analysis. However, the expression level of IGF2BP2 in ovarian cancer immune microenvironment was very low. The results of RT-PCR indicated the potential role of ALKBH5 in M2 polarization of macrophages.InterpretationALKBH5 participated in regulating macrophage M2 polarization in ovarian cancer immune microenvironment.

Abstract 2894: Targeting NOS2 and the tumor microenvironment in endometrioid ovarian cancer: Implications for treatment and survival

Abstract Purpose of the study: The final aim of the study is to disrupt the deleterious effect of the tumor microenvironment by targeting inducible Nitric Oxide Synthase 2 (NOS2). NOS2 has a role in cancer cell proliferation and immune cells inactivation. It is upregulated by cytokines released from the tumor proinflammatory environment and the adipose tissue. By targeting and reverting NOS2 functions, we want to develop a therapeutic modality that inhibits endometrioid ovarian cancer (EOC) progression and reactivates the immune response in the ovarian cancer tumor microenvironment (OC-TME). Experimental procedures: We analyzed ovarian cancer patients’ cohorts using TNMPlot, Kaplan-Meier Plotter and The Human Protein Atlas. We simulated the tumor proinflammatory microenvironment by administering Interferon-gamma (IFN-ϒ, 25 ng/mL), Interleukin 1 Beta (IL1-β, 10 ng/mL), Tumor Necrosis Factor-alpha (TNF-α, 10 ng/mL) on A2780 EOC human cell line. We also administered conditioned media (CM) from breast fat adipose tissue to assess the influence of lipid components on cytokines release. After upregulating NOS2, we tested the efficacy of Cisplatin in combination with L-NMMA, a NOS2 inhibitor, on A2780 cells through proliferation assays using RealTime-Glo™ MT Cell Viability Assay (Promega®). In addition, to assess the immune landscape in ovarian cancer, we developed a syngeneic model administering ID8 cells (murine OC cell line) through intraperitoneal injection (IP) in C57BL/6 mice. Results: The findings indicated higher NOS2 RNA expression levels in tumors than in normal samples according to TNMPlot (p = 6.84e-01). KMPlotter analysis revealed that patients with high NOS2 expression treated with Cisplatin had a significantly shorter survival rate (10.64 months) compared to those with lower NOS2 expression (25 months, p = 0.0088). Patients untreated with Cisplatin and exhibiting high NOS2 expression also showed lower survival rates (11 months) than those with lower NOS2 expression (34 months, p = 0.056). Furthermore, cytokine treatments increased NOS2 protein levels in A2780 cells after 48 hours, and CM similarly elevated NOS2 protein levels. Subsequent administration of Cisplatin and Cisplatin + L-NMMA to cytokine-treated A2780 cells notably reduced cell proliferation by 85% and 89%, respectively. Conclusion: Taken together, our results showed the importance of a proinflammatory environment and adipose tissue in NOS2 upregulation. These findings show a potential correlation between endometriosis, obesity, and endometrioid ovarian cancer. With the development of an in vivo and in vitro model, we can gain a better understanding of the role of NOS2 in immune inactivation as well as in cell proliferation in EOC. Citation Format: Gianmarco Melone, Mailin Li, Ivonne Uzair, Maria Chervo, Karina Ortega, Wei Qian, Jianying Zhou, Liliana Guzman, Sean Hynes, Sharon Glynn, Christoforos Thomas, Polina Matre, Lewis Francis, Jenny Chang. Targeting NOS2 and the tumor microenvironment in endometrioid ovarian cancer: Implications for treatment and survival [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 2894.

Abstract 7551: Combined epigenetic therapy and immune checkpoint blockade drive reshaping of the tumor microenvironment of platinum resistant ovarian cancer

Abstract Introduction: Immunotherapy has demonstrated modest effectiveness for patients with platinum-resistant ovarian cancer, which is in part related to the immunosuppressive tumor microenvironment (TME) of ovarian cancers. Epigenetic modulators in combination with immune checkpoint inhibitors (ICIs) may represent a potential way to sensitize ovarian cancer to immunotherapy by TME reprogramming. Methods: We evaluated the effect of epigenetic modulation in combination with ICI by comprehensive transcriptomic analyses of serially biopsied platinum-resistant/refractory ovarian cancer. Leveraging serial tumor samples from 30 patients that received oral azacitidine and pembrolizumab in the TRIO026 phase II clinical trial (NCT02900560), we performed bulk RNA sequencing and direct digital counting of 770 target genes using molecular barcodes (nCounter, NanoString) for 72 serial tumor samples, prior to treatment initiation and 6 weeks on-therapy. RNA sequencing of ~200 million reads for each sample was used for differential gene expression and gene set enrichment analysis (GSEA) and cell type deconvolution. Target gene expression data was normalized and analyzed for differential gene and pathway expression. Results: Differential gene expression analyses revealed an upregulation of inflammatory and cytolytic genes (IFNG, GZMA and GZMH; FDR-adjusted p=0.0184) as well as the co-inhibitory molecule CTLA-4 (FDR-adjusted p=0.0184). GSEA revealed an enrichment of IFNG response, natural killer cell mediated cytotoxicity, neutrophil degranulation, inflammatory response, and antigen processing/presentation related gene sets on-therapy (FDR-adjusted p &amp;lt; 0.001). Target gene expression analyses orthogonally confirmed these findings, which were also concordant with pathway scoring analyses showing an increased score for immune inflammatory pathways such as INFG signaling (Wilcoxon rank sum test p=0.01). Immune cell subset deconvolution suggested TME reshaping driven by a greater density of intra-tumoral T cells on-therapy (Wilcoxon rank sum test p=0.012). Higher IFNG, CXCL13, CXCR5 expression, CD8 T cell and NK cell density were associated with longer time on treatment, both at baseline and at the on-therapy timepoint. GSEA revealed an upregulation of interferon gamma, adaptive immunity, antigen presentation as well as conserved immune response gene sets on-therapy, for the patients attaining longer time on-treatment. Conclusion: Combination epigenetic and immunotherapy induces an inflammatory response and reshaping of the tumor microenvironment that may enhance their clinical efficacy, highlighting the therapeutic potential of utilizing epigenetic modulators as a way to sensitize platinum-resistant ovarian cancer to immune checkpoint inhibition. Citation Format: Blair V. Landon, Guneet Kaleka, Archana Balan, Julia L. Boland, Christopher Cherry, Gavin Pereira, Cynthia Zahnow, Boris Winterhoff, Stephen Baylin, Victor E. Velculescu, Gottfried E. Konecny, John A. Glaspy, Valsamo Anagnostou. Combined epigenetic therapy and immune checkpoint blockade drive reshaping of the tumor microenvironment of platinum resistant ovarian cancer [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 7551.

Abstract 3590: Tumor intrinsic production of glucocorticoids in ovarian carcinoma

Abstract Background: Cortisol is a glucocorticoid (GC) known to be produced by the hypothalamic-pituitary-adrenal (HPA) axis. Dysregulation of the HPA axis is prevalent in patients with ovarian cancer. These patients have elevated nighttime levels of cortisol and increased tumoral expression of the glucocorticoid receptor (GR); both of which correlate with shortened overall survival (OS). We hypothesize that outside the HPA axis, ovarian cancer may have tumor-intrinsic production of GC. In this study, we examined whether 11 beta-hydroxysteroid dehydrogenase 1 (11β-HSD1; enzyme which activates cortisone to cortisol) has steroidogenic activity in the ovarian cancer tumor microenvironment (TME). Methods: We used the Genotype-Tissue Expression (GTex) Portal and the Cancer Cell Line Encyclopedia (CCLE) to determine expression of steroidogenic enzymes in the ovary and ovarian cancer cell lines. We further performed UMAP analysis of a single-cell RNA-seq dataset of tumor samples from patients with ovarian carcinoma to cluster for cell type. ELISAs were performed to quantify cortisol production and secretion by cells stimulated with metabolite, cortisone. Subcellular fractionation and Western blotting were performed to measure protein expression of 11β-HSD1, GR, and phospho-Ser 211 GR. Results: HSD11B1 mRNA (which encodes 11β-HSD1) is detectable in bulk ovarian tissue at a median expression of 78.19 transcripts per million (TPM) as measured in the GTex data. In vitro, primary human ovarian fibroblasts express functional 11β-HSD1 and produce and secrete cortisol upon cortisone stimulation in a time-dependent manner, with a mean total secretion of 17.91 ± 0.21 ng/ml at 12 hours. Analysis of scRNA-seq data identifies that cancer-associated fibroblasts (CAFs) may be a source of HSD11B1 in the ovarian TME, with roughly 9% of CAFs expressing HSD11B1. In HeyA8 ovarian cancer cells (positive for 11β-HSD1), cortisone stimulation in vitro results in cortisol production with a mean total secretion of 22.04 ± 5.46 ng/ml at 12 hours. Moreover, this cortisol results in GR activation in an 11β-HSD1-dependent manner. Discussion: We identify 11β-HSD1 as a potential mechanism of GC production within ovarian cancer, with CAFs being a putative source. We further show that 11β-HSD1 activity can lead to activation of the GR within cancer cells. Citation Format: Pahul Hanjra, Elaine Stur, Steven Cole, Susan Lutgendorf, Anil Sood. Tumor intrinsic production of glucocorticoids in ovarian carcinoma [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 3590.

Abstract 639: Evaluation of individual drug response in tumor microenvironment to cisplatin treatment in precision-cut tumor slices of ovarian cancer

Abstract Ovarian cancer is a complex and heterogeneous disease and the major cause of death among women with gynecological cancers. While patients usually respond well to the platinum-based first-line chemotherapy, the disease often becomes increasingly resistant to the treatment. The tumor microenvironment (TME) plays an important role in tumor development and drug resistance. Characterizing the TME of ovarian cancer after drug treatment is essential to identify molecular mechanisms that allow residual tumor cells to survive chemotherapy in ovarian cancer. We have established a preclinical model using precision-cut tumor slices (PCTS) with 250 µm thickness which preserves the TME of solid tumors with their heterogeneous cell composition during cultivation. The tumor morphology, viability and heterogeneity in terms of tumor and stromal cells as well as the immune compartment are preserved within the system. Based on the PCTS model, we also established a method to perform single-cell RNA sequencing (scRNA-seq) of the PCTS after cultivation and drug treatment to characterize the cellular features and dynamic relationships of different cell populations in the TME after drug treatment. Over 20 cell subtypes including different clusters of epithelial cells, immune cells and fibroblasts can be identified in PCTS after cisplatin treatment through the scRNA-seq analysis. This enables further deeper analysis of each cell subgroup in the TME after drug treatment. In response to cisplatin treatment, patients PCTS showed an individual induction of PD-L1 in different cell types. Additionally, multiplex immunofluorescence (mIF) stainings of the PCTS were performed to spatially trace the response of the TME to drug treatment. The mIF staining allows the simultaneous detection of up to 6 different markers on one FFPE tissue section. Images were analyzed using a machine-learning based workflow, which allows the comparison of biomarker expression and immune cell spatial distribution in the PCTS stromal and tumor areas before and after treatment. Combining the PCTS as a preclinical model with scRNA-seq and mIF staining analysis allows us to bridge the cellular characteristics and cellular spatial distribution with treatment response in the TME of solid tumors. It enables the systematic analysis of residual cancer populations after cisplatin treatment within the complex TME and further identification of predictive markers and targets for an individualized combination of chemotherapy with compounds targeting these mechanisms. The individual drug response of patients can be deeply evaluated and efficacious therapies can be further developed. Citation Format: Julia Thiel, Adrian Kneer, Weimeng Yu, Bernd Winkler, Georg Sauer, German Ott, Walter E. Aulitzky, Thomas E. Mürdter, Matthias Schwab, Chunguang Liang, Meng Dong. Evaluation of individual drug response in tumor microenvironment to cisplatin treatment in precision-cut tumor slices of ovarian cancer [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 639.

Abstract 5274: Targeting retinoblastoma protein in tumor-associated macrophages suppresses ovarian cancer progression

Abstract Ovarian cancer is characterized by an immunosuppressive tumor microenvironment (TME) maintained by tumor-associated M2-like macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. An effective approach to target TAMs in ovarian cancer treatment is currently not available, but would be expected to release immunosuppressive pressure, enable a robust T cell response and improve immunotherapy outcomes. Retinoblastoma protein (Rb) is a well-known tumor suppressor and regulator of tumor cell proliferation. Accumulating evidence demonstrates its role in immune cells, in myeloid cells, in particular. However, these mechanisms remain poorly understood. Based on our previous data on Rb role in myeloid cell viability, we decided to test the effects of Rb modulation in macrophages. We used the small molecule, AP-3-84 compound, and newly developed analogs which bind the LxCxE motif of Rb and disrupt Rb interaction with its binding adaptor proteins. AP-3-84 induced cell death preferentially in macrophages, but not in T cells or tumor cells (without effects on cell proliferation). Gene and protein expression analysis revealed that Rb targeting induced major intracellular stress response programs and p53/mitochondria-related cell death pathways in TAMs. Moreover, we found that M2 type polarized macrophages expressed higher levels of Rb compared to M1 macrophages. In agreement with that, M2 type macrophages were significantly more sensitive to AP-3-84 treatment than M1 polarized cells. Next, we demonstrated that low dose therapeutic use of AP-3-84 in mice bearing ovarian cancer significantly re-shaped the immune composition of the TME by depleting TAMs and inducing remarkable T cell infiltration. Importantly, these AP-3-84 effects were immune-mediated, since identical AP-3-84 treatment in NSG immunodeficient mice had no effect on tumor growth. Similarly, anti-CD4/CD8 depletion also reverted AP-3-84 effects on cancer inhibition. An analysis of activation and differentiation markers in myeloid and T cell subsets in the TME showed a shift of the remaining macrophages towards an M1-like cell type accompanied by activation of T cells. Using ovalbumin-overexpressing ovarian cancer cell line, we documented the accumulation of antigen-specific T cells in the TME associated with a substantial delay of ovarian cancer upon AP-3-84 therapeutic Rb targeting. Ex vivo, we observed an analogous cell death induction by AP-3-84 treatment in TAMs from post-surgery ascites from ovarian cancer patients. Using available datasets, we further documented that an increase in Rb expression in the TME myeloid cells is associated with poorer prognosis. Overall, our data supports that therapeutic targeting of the Rb LxCxE motif is a promising approach for ovarian cancer treatment due to depletion of TAMs and re-shaping the ovarian cancer TME. Citation Format: Evgenii N. Tcyganov, Taekyoung Kwak, Xue Yang, Adi N. Poli, Colin Hart, Avishek Bhuniya, Joel Cassel, Andrew Kossenkov, Noam Auslander, Paridhima Sharma, Mark G. Cadungog, Stephanie Jean, Sudeshna Chatterjee-Paer, David Weiner, Laxminarasimha Donthireddy, Bryan Bristow, Rugang Zhang, Joseph M. Salvino, Luis J. Montaner. Targeting retinoblastoma protein in tumor-associated macrophages suppresses ovarian cancer progression [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 5274.

Ovarian cancer ascites confers platinum chemoresistance to ovarian cancer cells

Ovarian cancer (OC), the second most common form of gynecologic malignancy, has a poor prognosis and is often discovered in the late stages. Platinum-based chemotherapy is the first line of therapy. Nevertheless, treatment OC has proven challenging due to toxicity and the development of acquired resistance to therapy. Tumor microenvironment (TME) has been associated with platinum chemoresistance.Malignant ascites has been used as OC tumor microenvironment and its ability to induce platinum chemoresistance has been investigated.Our results suggest that exposure to OC ascites induces platinum chemoresistance in 11 of 13 cases (85 %) on OC cells. In contrast, 75 % of cirrhotic ascites (3 of 4) failed to confer platinum chemoresistance to OC cells. Cytokine array analysis revealed that IL -6 and to a lesser extent HGF were enriched in OC ascites, whereas IL -22 was enriched in cirrhotic ascites. Pharmaceutical inhibitors targeting the IL -6/ JAK pathway were mildly effective in overcoming platinum chemoresistance induced by malignant ascites. In contrast, crizotinib, an HGF/c- MET inhibitor, and 2-hydroxyestradiol (2HE2) were effective in restoring platinum chemosensitivity to OC.Our results demonstrate the importance of OC ascites in supporting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity of OC cells.

Abstract PR-009: Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer using human multicellular in vitro models

Abstract Effective treatment of high grade serous ovarian cancer (HGSOC) still represents a clinical challenge. This is partially due to poor understanding of the complexity of this disease and the highly immunosuppressive tumor microenvironment (TME) that makes HGSOC resistant to most of the newly discovered therapies. The lack of relevant and physiological human in vitro models represents a major issue as the development of new treatments are mostly based on mouse models that do not fully recapitulate the human disease. In this study, we developed 3-dimensional (3D) human multicellular in vitro models using as a template and validation, data collected in a previous study that ‘deconstructs’ the metastatic TME of HGSOC. Our human 3D in vitro pentaculture (five cell types) model is composed of patient-derived primary adipocytes, fibroblasts and mesothelial cells isolated from macroscopic normal omentum – the primary site of metastasis in HGSOC – of women undergoing a gynaeco-oncology surgery, combined with different high grade serous malignant cell lines and monocytes isolated from healthy individuals. We found that the peripheral blood monocytes were able to differentiate and polarize to macrophages in our model in a similar way that observed in human HGSOC omental metastasis. Interestingly, our pentaculture models self-rearrange at the cellular level in a different way depending on the malignant cell line used in the culture. RNAseq analysis of the pentacultures showed that the different cultures clustered according to the malignant cell line used suggesting that malignant cells have a major influence on the transcriptome of the TME. Moreover, we demonstrated that our pentacultures partially replicated human biopsies: some of the macrophage and fibroblast clusters identified in a previous scRNAseq experiment on human HGSOC biopsies were also found in the pentacultures. We then confirmed the presence of some these macrophages clusters at the protein level. Finally, these pentacultures reproduce some of the actions of macrophage modifying drugs that have previously been shown in vivo. In conclusion, the study of the immunosuppressive TME of HGSOC in these human 3D in vitro models allows us to identify the contribution of different cell types to tumor growth and spread and provides us a semi-high throughput platform for therapeutic screening. Citation Format: Beatrice Malacrida, Samar Elorbany, Eleni Maniati, Rachel Bryan-Ravenscroft, Sophie L.P. Skingsley, Florian Laforets, Ranjit Manchanda, Frances R. Balkwill. Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer using human multicellular in vitro models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-009.