Ovarian Tumor Microenvironment Research Articles

Abstract IA006: Friend or foe: Reevaluating the therapeutic potential of monocytes and tumor-associated macrophages in ovarian cancer

Abstract The hostile tumor microenvironment in ovarian cancer is an active deterrent to the efficacy of immunotherapeutic agents such as immune checkpoint inhibitors, Chimeric Antigen Receptor (CAR) T-cells, and Bispecific T-cell Engagers (BiTEs). Tumor-associated macrophages (TAMs) and Myeloid-Derived Suppressor Cells (MDSCs) have been shown to promote an immunosuppressive metabolic and cytokine tumor microenvironment that is detrimental to cytotoxic effector T-cell activity in ovarian cancer. These immunosuppressive cells are also associated with poor prognostic outcomes. The ideal approach to modulating TAMs and MDSCs is unknown. Data from preclinical models support reprogramming these cells to a more inflammatory phenotype or removing them from the tumor microenvironment entirely. However, neither approach has translated to clinical benefits. We have previously demonstrated that CAR-T cells modified to secrete IL-18 exerted their therapeutic effects partly via polarization of bone marrow macrophages towards an M1 phenotype. We also found that the depletion of TAMs improved CAR-T cell efficiency, even when these CAR-T cells were co-modified to secrete IL-12. Because of the propensity for monocytes to be recruited to the ovarian cancer tumor microenvironment, we evaluated the efficacy of monocytes genetically modified to secrete BiTEs. First, we designed and evaluated BiTEs that recognize the retained portion of MUC16 (Muc16-ecto) and CD3+ T-cells and showed that these BiTEs were efficacious in vitro and in vivo in a xenograft model of ovarian cancer. We validated that monocytes transduced to secrete Muc16-specific BiTEs (Mono-BiTEs) could effectively lyse tumor cells in vitro and suppress xenograft ovarian cancer cells in vivo. Adding IFN-alpha and IFN-gamma to Mono-BiTEs significantly improved Mono-BiTEs antitumor efficacy over BiTE therapy or unmodified monocyte therapy alone. Adoptive cell therapy with genetically engineered autologous monocytes could provide an alternative approach to reprograming or eliminating myeloid-derived cells in ovarian cancer. Citation Format: Oladapo Yeku. Friend or foe: Reevaluating the therapeutic potential of monocytes and tumor-associated macrophages in ovarian cancer [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 IA006.

Abstract B080: AB-1015, an Integrated Circuit T (ICT) cell therapy containing an ALPG/MSLN logic gate and FAS/PTPN2 shRNA-miR, for the treatment of ovarian cancer

Abstract B080: AB-1015, an Integrated Circuit T (ICT) cell therapy containing an ALPG/MSLN logic gate and FAS/PTPN2 shRNA-miR, for the treatment of ovarian cancer

Mechanical activation and expression of HSP27 in epithelial ovarian cancer

Understanding the complex biomechanical tumor microenvironment (TME) is of critical importance in developing the next generation of anti-cancer treatment strategies. This is especially true in epithelial ovarian cancer (EOC), the deadliest of the gynecologic cancers due to recurrent disease or chemoresistance. However, current models of EOC progression provide little control or ability to monitor how changes in biomechanical parameters alter EOC cell behaviors. In this study, we present a microfluidic device designed to permit biomechanical investigations of the ovarian TME. Using this microtissue system, we describe how biomechanical stimulation in the form of tensile strains upregulate phosphorylation of HSP27, a heat shock protein implicated in ovarian cancer chemoresistance. Furthermore, EOC cells treated with strain demonstrate decreased response to paclitaxel in the in vitro vascularized TME model. The results provide a direct link to biomechanical regulation of HSP27 as a mediator of EOC chemoresistance, possibly explaining the failure of such therapies in some patients. The work presented here lays a foundation to elucidating mechanobiological regulation of EOC progression, including chemoresistance and could provide novel targets for anti-cancer therapeutics.

Exploring the tumor micro-environment in primary and metastatic tumors of different ovarian cancer histotypes.

Ovarian cancer is a highly heterogeneous disease consisting of at least five different histological subtypes with varying clinical features, cells of origin, molecular composition, risk factors, and treatments. While most single-cell studies have focused on High grade serous ovarian cancer, a comprehensive landscape of the constituent cell types and their interactions within the tumor microenvironment are yet to be established in the different ovarian cancer histotypes. Further characterization of tumor progression, metastasis, and various histotypes are also needed to connect molecular signatures to pathological grading for personalized diagnosis and tailored treatment. In this study, we leveraged high-resolution single-cell RNA sequencing technology to elucidate the cellular compositions on 21 solid tumor samples collected from 12 patients with six ovarian cancer histotypes and both primary (ovaries) and metastatic (omentum, rectum) sites. The diverse collection allowed us to deconstruct the histotypes and tumor site-specific expression patterns of cells in the tumor, and identify key marker genes and ligand-receptor pairs that are active in the ovarian tumor microenvironment. Our findings can be used in improving precision disease stratification and optimizing treatment options.

Single-cell RNA sequencing in ovarian cancer: revealing new perspectives in the tumor microenvironment.

Single-cell sequencing technology has emerged as a pivotal tool for unraveling the complexities of the ovarian tumor microenvironment (TME), which is characterized by its cellular heterogeneity and intricate cell-to-cell interactions. Ovarian cancer (OC), known for its high lethality among gynecologic malignancies, presents significant challenges in treatment and diagnosis, partly due to the complexity of its TME. The application of single-cell sequencing in ovarian cancer research has enabled the detailed characterization of gene expression profiles at the single-cell level, shedding light on the diverse cell populations within the TME, including cancer cells, stromal cells, and immune cells. This high-resolution mapping has been instrumental in understanding the roles of these cells in tumor progression, invasion, metastasis, and drug resistance. By providing insight into the signaling pathways and cell-to-cell communication mechanisms, single-cell sequencing facilitates the identification of novel therapeutic targets and the development of personalized medicine approaches. This review summarizes the advancement and application of single-cell sequencing in studying the stromal components and the broader TME in OC, highlighting its implications for improving diagnosis, treatment strategies, and understanding of the disease's underlying biology.

Adipose-derived exosomal miR-421 targets CBX7 and promotes metastatic potential in ovarian cancer cells

BackgroundChromobox protein homolog 7 (CBX7), a member of the Polycomb repressor complex, is a potent epigenetic regulator and gene silencer. Our group has previously reported that CBX7 functions as a tumor suppressor in ovarian cancer cells and its loss accelerated formation of carcinomatosis and drove tumor progression in an ovarian cancer mouse model. The goal of this study is to identify specific signaling pathways in the ovarian tumor microenvironment that down-regulate CBX7. Given that adipocytes are an integral component of the peritoneal cavity and the ovarian tumor microenvironment, we hypothesize that the adipose microenvironment is an important regulator of CBX7 expression.ResultsUsing conditioned media from human omental explants, we found that adipose-derived exosomes mediate CBX7 downregulation and enhance migratory potential of human ovarian cancer cells. Further, we identified adipose-derived exosomal miR-421 as a novel regulator of CBX7 expression and the main effector that downregulates CBX7.ConclusionIn this study, we identified miR-421 as a specific signaling pathway in the ovarian tumor microenvironment that can downregulate CBX7 to induce epigenetic change in OC cells, which can drive disease progression. These findings suggest that targeting exosomal miR-421 may curtail ovarian cancer progression.

IFN-γ in ovarian tumor microenvironment upregulates HLA-E expression and predicts a poor prognosis

BackgroundInflammation and immunity are two main characteristics of tumor microenvironment (TME). Interferon-gamma (IFN-γ) is generally considered as a pro-inflammatory cytokine which mediates anti-tumor immune response. Recently, IFN-γ was also reported to play a protumorigenic role. However, the mechanisms of tumor-promoting effect induced by IFN-γ remain unclear.MethodsThe expression of leukocyte antigen-E (HLA-E), IFN-γ, CD3 and CD56 in clinical samples of ovarian cancer was detected by mutiplexed immunohistochemistry. The mechanism to induce HLA-E overexpression by IFN-γ was explored using human ovarian cancer cell lines through western blot and flow cytometry. We further clarify the role of overexpressed-HLA-E on natural killer (NK)-mediated cell lysis.ResultsWe found that IFN-γ could upregulate HLA-E protein expression through activating of JAK/STAT1 signaling pathway, and increase cell surface HLA-E level through enhancing proteasome activity. We also observed that only high levels of membrane HLA-E expression contributed to the inhibition of NK-mediated cytotoxicity. We showed that progression-free survival (PFS) of ovarian cancer patients was negatively correlated with IFN-γ expression in their tumor tissues, due to more tumor infiltrating NK cells compared with T lymphocytes.ConclusionsOur study revealed the protumorigenic role of IFN-γ by upregulation of HLA-E expression and rendering tumors less susceptible to immune attack. We also provided a novel insight into the relationship between tumor microenvironment and immune evasion.

Combining Mass Cytometry Data by CyTOFmerge Reveals Additional Cell Phenotypes in the Heterogeneous Ovarian Cancer Tumor Microenvironment: A Pilot Study.

The prognosis of high-grade serous ovarian carcinoma (HGSOC) is poor, and treatment selection is challenging. A heterogeneous tumor microenvironment (TME) characterizes HGSOC and influences tumor growth, progression, and therapy response. Better characterization with multidimensional approaches for simultaneous identification and categorization of the various cell populations is needed to map the TME complexity. While mass cytometry allows the simultaneous detection of around 40 proteins, the CyTOFmerge MATLAB algorithm integrates data sets and extends the phenotyping. This pilot study explored the potential of combining two datasets for improved TME phenotyping by profiling single-cell suspensions from ten chemo-naïve HGSOC tumors by mass cytometry. A 35-marker pan-tumor dataset and a 34-marker pan-immune dataset were analyzed separately and combined with the CyTOFmerge, merging 18 shared markers. While the merged analysis confirmed heterogeneity across patients, it also identified a main tumor cell subset, additionally to the nine identified by the pan-tumor panel. Furthermore, the expression of traditional immune cell markers on tumor and stromal cells was revealed, as were marker combinations that have rarely been examined on individual cells. This study demonstrates the potential of merging mass cytometry data to generate new hypotheses on tumor biology and predictive biomarker research in HGSOC that could improve treatment effectiveness.

Specific Tumor Localization of Immunogenic Lipid-Coated Mesoporous Silica Nanoparticles following Intraperitoneal Administration in a Mouse Model of Serous Epithelial Ovarian Cancer.

Immunogenic lipid-coated mesoporous silica nanoparticles (ILM) present pathogen-associated molecular patterns (PAMPs) on the nanoparticle surface to engage pathogen-associated receptors on immune cells. The mesoporous core is capable of loading additional immunogens, antigens or drugs. In this study, the impact of lipid composition, surface potential and intercalation of lipophilic monophosphoryl lipid A (MPL-A) in the lipid coat on nanoparticle properties and cellular interactions is presented. Loading and retention of the model antigen ovalbumin into the mesoporous silica core were found to be similar for all nanoparticle formulations, with presentation of ova peptide (SIINFEKL) by major histocompatibility complex (MHC) evaluated to facilitate the selection of an anionic nanoparticle composition. ILM were able to induce lysosomal tubulation and streaming of lysosomes towards the cell surface in dendritic cells, leading to an enhanced surface presentation of MHC. Myeloid cells robustly internalized all ILM formulations; however, non-myeloid cells selectively internalized cationic ILM in vitro in the presence of 20% serum. Interestingly, ILM administration to the peritoneal cavity of mice with disseminated ovarian cancer resulted in selective accumulation of ILM in tumor-associated tissues (>80%), regardless of nanoparticle surface charge or the presence of MPL-A. Immunofluorescence analysis of the omental tumor showed that ILMs, regardless of surface charge, were localized within clusters of CD11b+ myeloid cells 24 h post administration. Selective uptake of ILMs by myeloid cells in vivo indicates that these cells outcompete other cell populations in the ovarian tumor microenvironment, making them a strong target for therapeutic interventions.

Lymphatic vasculature in ovarian cancer.

Ovarian cancer (OVCA) is the second most common gynecological cancer and one of the leading causes of cancer related mortality among women. Recent studies suggest that among ovarian cancer patients at least 70% of the cases experience the involvement of lymph nodes and metastases through lymphatic vascular network. However, the impact of lymphatic system in the growth, spread and the evolution of ovarian cancer, its contribution towards the landscape of ovarian tissue resident immune cells and their metabolic responses is still a major knowledge gap. In this review first we present the epidemiological aspect of the OVCA, the lymphatic architecture of the ovary, we discuss the role of lymphatic circulation in regulation of ovarian tumor microenvironment, metabolic basis of the upregulation of lymphangiogenesis which is often observed during progression of ovarian metastasis and ascites development. Further we describe the implication of several mediators which influence both lymphatic vasculature as well as ovarian tumor microenvironment and conclude with several therapeutic strategies for targeting lymphatic vasculature in ovarian cancer progression in present day.

CXCL8 Promotes Endothelial-to-Mesenchymal Transition of Endothelial Cells and Protects Cells from Erastin-Induced Ferroptosis via CXCR2-Mediated Activation of the NF-κB Signaling Pathway.

CXCL8-CXCR1/CXCR2 signaling pathways might form complex crosstalk among different cell types within the ovarian tumor microenvironment, thereby modulating the behaviors of different cells. This study aimed to investigate the expression pattern of CXCL8 in the ovarian tumor microenvironment and its impact on both endothelial-to-mesenchymal transition (EndMT) and ferroptosis of endothelial cells. The human monocytic cell line THP-1 and the human umbilical vein endothelial cell line PUMC-HUVEC-T1 were used to conduct in vitro studies. Erastin was used to induce ferroptosis. Results showed that tumor-associated macrophages are the major source of CXCL8 in the tumor microenvironment. CXCL8 treatment promoted the nucleus entrance of NF-κB p65 and p65 phosphorylation via CXCR2 in endothelial cells, suggesting activated NF-κB signaling. Via the NF-κB signaling pathway, CXCL8 enhanced TGF-β1-induced EndMT of PUMC-HUVEC-T1 cells and elevated their expression of SLC7A11 and GPX4. These trends were drastically weakened in groups with CXCR2 knockdown or SB225002 treatment. TPCA-1 reversed CXCL8-induced upregulation of SLC7A11 and GPX4. CXCL8 protected endothelial cells from erastin-induced ferroptosis. However, these protective effects were largely canceled when CXCR2 was knocked down. In summary, CXCL8 can activate the NF-κB signaling pathway in endothelial cells in a CXCR2-dependent manner. The CXCL8-CXCR2/NF-κB axis can enhance EndMT and activate SLC7A11 and GPX4 expression, protecting endothelial cells from ferroptosis.

A novel hypoxia-stimulated lncRNA HIF1A-AS3 binds with YBX1 to promote ovarian cancer tumorigenesis by suppressing p21 and AJAP1 transcription.

Hypoxia is characteristic of the ovarian tumor (OC) microenvironment and profoundly affects tumorigenesis and therapeutic response. Long noncoding RNAs (lncRNAs) play various roles in tumor progression; however, the characteristics of lncRNAs in pathological responses of the OC microenvironment are not entirely understood. Through high-throughput sequencing, lncRNA expression in hypoxia (1% O2 ) and normoxia (21% O2 ) SKOV3 cells was explored and analyzed. The 5'- and 3'-rapid amplification of complementary DNA ends was used to detect the full length of the novel HIF1A-AS3 transcript. Real-time quantitative polymerase chain reaction was used to assess HIF1A-AS3 expression in OC cells and tissues. In vitro and in vivo evaluations of the biological functions of hypoxic HIF1A-AS3 were conducted. To clarify the underlying mechanisms of HIF1A-AS3 in hypoxic OC, a dual-luciferase assay, chromatin immunoprecipitation, RNA pull-down, RNA immunoprecipitation, and RNA-sequencing were used. We used high-throughput sequencing to investigate a novel lncRNA, HIF1A-AS3, as a hypoxic candidate significantly elevated in OC cells/tissues. HIF1A-AS3 was predominantly localized in the nucleus and promotedin vitro and in vivo OC growth and tumorigenesis. Hypoxia-inducible factor 1α bound to hypoxia response elements in the HIF1A-AS3 promoter region and stimulated its expression in hypoxia. Under hypoxia, HIF1A-AS3 directly integrated withY-Box binding protein 1 and inhibited its ability to bind to the promoters of p21 and AJAP1 to repress their transcriptional activity, thereby promoting hypoxic OC progression. Our results revealed the crucial role and mechanism of the novel hypoxic HIF1A-AS3 in the oncogenesis of OC. The novel HIF1A-AS3 could be a crucial biomarker and therapeutic target for future OC treatments.

Insights into the tumor-stromal-immune cell metabolism cross talk in ovarian cancer.

The ovarian cancer tumor microenvironment (TME) consists of a constellation of abundant cellular components, extracellular matrix, and soluble factors. Soluble factors, such as cytokines, chemokines, structural proteins, extracellular vesicles, and metabolites, are critical means of noncontact cellular communication acting as messengers to convey pro- or antitumorigenic signals. Vast advancements have been made in our understanding of how cancer cells adapt their metabolism to meet environmental demands and utilize these adaptations to promote survival, metastasis, and therapeutic resistance. The stromal TME contribution to this metabolic rewiring has been relatively underexplored, particularly in ovarian cancer. Thus, metabolic activity alterations in the TME hold promise for further study and potential therapeutic exploitation. In this review, we focus on the cellular components of the TME with emphasis on 1) metabolic signatures of ovarian cancer; 2) understanding the stromal cell network and their metabolic cross talk with tumor cells; and 3) how stromal and tumor cell metabolites alter intratumoral immune cell metabolism and function. Together, these elements provide insight into the metabolic influence of the TME and emphasize the importance of understanding how metabolic performance drives cancer progression.

Biomaterial considerations for ovarian cancer models

Ovarian cancer is the 5th most common and the deadliest gynecological cancer, with a 5-year survival rate of less than 50 percent. Most deaths due to ovarian cancer are caused by recurrent disease, which typically corresponds to an increase in chemoresistance of the tumor cells. However, little is known about how ovarian tumor chemoresponse changes and if such changes are regulated by the tumor microenvironment (TME). Moreover, the ovarian TME, including the tissue compositions and biomechanical features, is not well-characterized primarily due to a lack of optimal models. To more effectively characterize the TME of ovarian cancer, which may help develop innovative treatment strategies, appropriate models are desperately needed. The most utilized models include mouse models with both patient-derived xenografts and mouse or human tumor cell line derivatives, and more recently microphysiological systems (MPS). While mouse models provide high levels of physiological complexity, there is virtually no control over the TME components after tumor initiation or implantation. On the other hand, MPS or organoid models permit high levels of control of initial composition but lack many features of in vivo models. Selection of appropriate components to create a TME model is paramount for generating a physiologically relevant in vitro and ex vivo systems. The importance of biomaterial or matrix selection in ovarian TME models lies in the role of these components to activate oncogenic signaling pathways either through receptor-ligand interactions or mechanotransduction. Recent studies suggest that off-target or post-target effects of chemotherapies may interfere with mechanotransductive pathways. In ovarian cancer, changes in fibrous proteins, adhesive glycoproteins, and glycosaminoglycans can remodel the mechanical environment, further altering mechanotransductive pathways. Therefore, the next-generation of ovarian tumor models should incorporate relevant biomaterials including hyaluronic acid (HA), collagens, fibrinogen, and fibronectin to investigate the link between matrix properties and mechanobiology with metastasis and chemoresistance.

P3 Macrophage activation in the ovarian cancer tumor microenvironment and design of a novel immunotherapy

P3 Macrophage activation in the ovarian cancer tumor microenvironment and design of a novel immunotherapy

Comprehensive Analysis Identifies the PPAR-Targeted Genes Associated with Ovarian Cancer Prognosis and Tumor Microenvironment.

There is a significant role for peroxisome proliferator-activated receptors (PPARs) in the development of cancer. Nevertheless, the role of PPARs-related genes in ovarian cancer (OC) remains unclear. The open-accessed data used for analysis were downloaded from The Cancer Genome Atlas database, which was analyzed using the R software. In our study, we comprehensively investigated the PPAR target genes in OC, including their biological role. Meanwhile, a prognosis signature consisting of eight PPAR target genes was established, including apolipoprotein A-V, UDP glucuronosyltransferase 2 family, polypeptide B4, TSC22 domain family, member 1, growth hormone inducible transmembrane protein, renin, dedicator of cytokinesis 4, enoyl CoA hydratase 1, peroxisomal (ECH1), and angiopoietin-like 4, which showed a good prediction efficiency. A nomogram was constructed by combining the clinical feature and risk score. Immune infiltration and biological enrichment analysis were applied to investigate the difference between high- and low-risk patients. Immunotherapy analysis indicated that low-risk patients might respond better to immunotherapy. Drug sensitivity analysis indicated that high-risk patients might respond better to bleomycin, nilotinib, pazopanib, pyrimethamine, and vinorelbine, yet worse to cisplatin and gefitinib. Furthermore, the gene ECH1 was selected for further analysis. Our study identified a prognosis signature that could effectively indicates patients survival. Meanwhile, our study can provide the direction for future studies focused on the PPARs in OC.

Abstract 3653: Oncostatin m receptor signaling reprograms tumor microenvironment for chemoresistance

Abstract Introduction: High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy and accounts for ~70% of tumor recurrences after standard chemotherapy and immunotherapy. Ovarian tumor microenvironment (TME) is undeniably complex which presents considerable challenge in understanding the immunotherapy treatment responses. Our previous study has revealed that Oncostatin M receptor (OSMR) is highly expressed in ovarian cancer cells and cancer associated fibroblasts (CAFs), whereas its ligand OSM is predominantly expressed in tumor associated macrophages (TAMs). This study aims to investigate how the OSM-OSMR paracrine signaling between tumor cells, CAFs and TAMs reprograms the TME to influence therapeutic responses in ovarian cancer. Methods: We employed a 3D-3-cell co-culture method in vitro to understand how OSM/OSMR signaling modulates the interactions between ovarian cancer cells, CAFs and TAMs, that can impact therapy response of cisplatin resistant cancer cells in the ovarian TME. We performed RNA sequencing to study the differential gene signatures of each cell component in a 3-cell co-culture with their monolayer counterparts. This system was also challenged with chemotherapy-cisplatin and immunotherapy such as our newly developed anti-OSMR monoclonal antibodies to assess the therapy response of each cell component. Results: Using single-cell and single-nuclear RNA seq dataset analyses of 17 human HGSOC patient ascites, we found that CAFs and TAMs are the predominant stromal or immune cell population enriched in ovarian cancer tissues. Using immunofluorescence, we identified that enrichment of CAFs along with extracellular matrix proteins surrounding tumor cells as an indication of heavily desmoplastic tumors. Using tumor-infiltrating immune cells dataset-TIMER, we found that high OSMR in cancer cells or CAFs along with high OSM in TAMs are associated with poor survival in ovarian cancer patients. Further, RNA sequencing data of 3-cell coculture models revealed an invasive, desmoplastic and immune-suppressive gene signature compared to independent culture conditions. Notably, CAFs and ovarian cancer cells co-cultured with TAMs exhibited an improved activation of JAK/STAT3 and PI3K-AKT oncogenic pathways for chemoresistance in ovarian cancer cells. Conclusions: Our preliminary findings uncovered a potential mechanism of action of OSM-OSMR signaling in reprogramming ovarian cancer cells and stromal fibroblasts in ovarian cancer TME. Importantly, we highlight the importance of crosstalk between CAFs, ovarian cancer cells and TAMs for harnessing OSM/OSMR signaling for chemoresistance in cancer cells. Our findings also demonstrated the potential of anti-OSMR antibody therapy to target cancer cells and CAFs in the tumor microenvironment for abrogating desmoplasia; thus, improving chemosensitivity. Citation Format: Anjali Geethadevi, Shirng-Wern Tsaih, Ajay Nair, Zhiqiang Ku, Ishaque Pulikkal Kadamberi, Deepak Parashar, Prachi Gupta, Sudhir Kumar, Jasmine George, Sonam Mittal, William Bradley, Robert F. Schwabe, Zhiqiang An, Ningyan Zhang, Sunila Pradeep, Pradeep Chaluvally Raghavan. Oncostatin m receptor signaling reprograms tumor microenvironment for chemoresistance. [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 3653.

Abstract 5702: Ovarian cancer tumor microenvironment and atezolizumab (atezo) clinical activity: IMagyn050 sub-study

Abstract Background: Tumor biomarkers such as CD8 density and location (i.e., immune inflamed phenotype) and immune rich molecular subtype have been linked to immune checkpoint blockade (ICB) overall survival (OS) in different cancers. The IMagyn050 trial (NCT03038100), which evaluated the efficacy of Atezo vs placebo (Pla) with carboplatin, paclitaxel and bevacizumab (CPB) in front line ovarian cancer patient (pts), did not meet its co-primary endpoints of PFS in ITT or PD-L1+ (Moore et al. JCO 2021). In the current IMagyn050 substudy we assessed potential predictive tumor immune biomarkers for Atezo clinical benefit. Methods: FFPE tumors from the biomarker evaluable population were tested for PD-L1 IHC, CD8/PanCK IHC (total CD8 T cells and immune location phenotypes [inflamed, excluded, desert]) and RNA-seq (to derive molecular subtypes, biological pathways and cellular components [xCELL]) in tissue from baseline (n=860), on-treatment (OT, 9 weeks, n=233), intra- (n=8) and inter-lesion (n=12) matched samples. Hazard ratio (HR) interaction test from multivariate adjusted Cox-regression analysis for PFS and OS was performed to test predictive biomarkers. Results: While tumors with CD8 T cells, immunoreactive molecular subtypes or immune inflamed phenotype were enriched for PD-L1+, only pts with immune inflamed tumors showed improved OS Atezo benefit (HR 0.67). Improved Atezo PFS/OS benefit was also observed in pts with whose tumors had high oxidative phosphorylation (OXPHOS, HR: 0.72/0.65) and UV Response (UV, HR: 0.64/0.58) but not IFNγ response. Plasma B cells were linked to improved OS Atezo benefit vs Pla (HR 0.53). We leveraged OT samples from pts in the neoadjuvant cohort to assess treatment effect on the tumor microenvironment. Analyses showed that CPB reduced tumor proliferation and increased tumor immune inflammation (CD8 T cells, PD-L1 and IFNα/IFNγ response), further increased by Atezo. Immune inflammation is challenging in ovarian cancer due to extensive tumor heterogeneity. Prevalence of tumor biomarkers varied by anatomic locations: total CD8, CD8 localization and molecular subtypes. Inter- and intra-lesion biomarker status within the same pt showed PD-L1 and plasma B cells as most consistent. Molecular subtypes and immune phenotypes had moderate intra-lesion agreement but discordant between lesions. PD-L1 and OXPHOS were the only biomarkers linked to Atezo benefit regardless of anatomical location. Conclusion: This comprehensive exploratory study suggests that DNA damage, OXPHOS, plasma B cells and immune inflamed tumors, but not molecular subtypes or total CD8 T cells, may predict Atezo + CPB OS. This treatment promotes immune inflammation in OC tumors. Notably, we found that several biomarkers are highly heterogeneous. Our findings highlight the challenges of achieving durable clinical benefit from targeted immunotherapy in ovarian cancer pts. Citation Format: Venkatesh Krishnan, Ching-Wei Chang, Habib Hamidi, Michael A. Bookman, Charles Landen, Tashanna Myers, Hiroaki Kajiyama, Sakari Hietanen, Lyndsay Willmott, Premal Thaker, Cagatay Taskiran, Jalid Sehouli, Victor Khor, Yvonne Lin Liu, Sandro Pignata, Kathleen Moore, Luciana Molinero. Ovarian cancer tumor microenvironment and atezolizumab (atezo) clinical activity: IMagyn050 sub-study. [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 5702.

Abstract 3642: SPHK1 mediated regulation of antitumor immunity in ovarian cancer

Abstract Introduction: Sphingosine kinase 1 (SPHK1) is an enzyme that helps to synthesize biolipid sphingosine-1-phosphate (S1P). We showed extracellular vesicles facilitate an increased accumulation of S1P in the ovarian tumor microenvironment (TME). S1P enhances the exhaustion of CD8 T cells, thus downregulating anti-tumor immunity. Furthermore, we also found less ovarian tumor burden in SPHK1−/− mice compared to WT mice. Based on our preliminary data, we hypothesized that the SPHK1 depleted tumor microenvironment increases the infiltration of the immune cells and its activity downregulates the tumor progression. Methods: SPHK1 global knockout mice and WT C57BL/6 mice were used in the study. Tumor cells (ID8 P53−/− BRCA−/−) were injected, and tumor progression was studied. Immunophenotyping was performed by flow cytometry on ascites and tumor samples. Activation of CD8 T cells, B cells, and dentritic cells were studied by flow cytometry. Bulk RNA sequencing was performed on SPHK1−/− and WT tumors. Results: SPHK1−/− mice showed regression in tumor growth in multiple tumor models. Tumor tissues from SPHK1−/− mice enhanced infiltration of CD4 and CD8 T cells, B cells, and dentritic cells compared to WT mice. Furthermore, infiltrated immune cells were active in SPHK1−/− mice. Bulk RNA sequencing also showed enhanced immune response in SPHK1−/− tumors compared to WT tumors. Conclusion: SPHK1−/- environment decreased the tumor growth by enhancing the infiltration and activation of immune cells. We are currently focusing on identifying the immune cells which are critical players in this process. We will further implement these findings in human immune cells for clinical benefits for ovarian cancer patients. Citation Format: Prachi Gupta, Sudhir Kumar, Ishaque Pulikkal Kadamberi, Sonam Mittal, Jasmine George, Anjali Geethadevi, Pradeep Chaluvally-Raghavan, Sunila Pradeep. SPHK1 mediated regulation of antitumor immunity 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 3642.

Abstract 3655: Exploring the role of FasL in the ovarian cancer tumor microenvironment

Abstract Introduction: High-grade serous ovarian cancer (OvCa) remains the deadliest gynecologic cancer in the United States because it often presents at an advanced stage when the disease is incurable. Despite the impressive clinical success of cancer immunotherapy in patients with lung cancer and melanoma, it is largely ineffective in OvCa patients. Resistance to cancer immunotherapy is mediated by the apoptosis of T-cells, which can be prevented by blocking Fas ligand (FasL), a death gene. However, little is known about the expression and function of FasL in OvCa. Therefore, the aims of this study were to identify the cells in the OvCa tumor microenvironment that produce and secrete FasL, and to delineate the functional role of FasL in T-cell apoptosis within the OvCa tumor microenvironment. Methods: FasL protein localization and levels were evaluated in OvCa tumors by immunohistochemistry (IHC) using a FasL antibody on tissue microarrays (TMAs) containing primary and matching metastases from 72 OvCa patients. In-situ hybridization (ISH) using a FasL specific probe was performed on omental OvCa tumors from pre- (n=12) and post-chemotherapy (n=12) patients to localize FasL mRNA. To define the role of OvCa and endothelial cell derived FasL on T cell apoptosis, co-culture experiments were completed. First, FasL was knocked down with a FasL siRNA or neutralized with a FasL antibody in OvCa (OVCAR8 and OVCAR5) cells or endothelial (HUVEC) cells. Next, the OvCa or endothelial cells were co-cultured with primary human blood mononuclear cell-derived T cells. Subsequently, the level of FasL secreted was measured using a FasL specific enzyme-linked immunoassay, and early (4h; cleaved-caspase 3) and late (24h; TUNEL assay) T cell apoptosis were analyzed by flow cytometry. Results: FasL protein and mRNA are present in OvCa and endothelial cells in the tumor microenvironment of OvCa patients. Higher levels of FasL are detected in the endothelial cells when compared to the OvCa cells in these OvCa tumors. These results were confirmed in vitro as primary human endothelial cells secrete higher levels of FasL when compared to the OVCAR8 and OVCAR5 ovarian cancer cell lines. Functionally, both OvCa and endothelial cells stimulate early and late apoptosis in primary human blood mononuclear cell-derived T cells after a four hour co-culture. Knockdown or neutralization of FasL significantly inhibits the ability of OvCa or endothelial cells to induce early and late T cell apoptosis. In summary, FasL is localized to OvCa and endothelial cells in the OvCa tumor microenvironment, and both OvCa and endothelial cell derived FasL induce T cell apoptosis. Discussion: These findings indicate that blocking FasL may be an effective approach for reducing T-cell apoptosis and improving cancer immunotherapy response in OvCa patients. Citation Format: Grace Keegan, Hilary Kenny, Agnes Bilecz, Ricardo Lastra, Ernst Lengyel. Exploring the role of FasL in the ovarian cancer tumor microenvironment. [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 3655.