Ovarian Cancer Tumor Microenvironment Research Articles

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 < 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.

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 A103: Improved cell phenotyping of ovarian cancer tumor microenvironment

Abstract Introduction: Multiplexed imaging at single-cell resolution is becoming widely used to decipher the role of the cellular microenvironment in cancer and other complex diseases. To identify spatial patterns of single cells on a tissue, accurate cell-type phenotyping is a crucial step. We present Tribus, an interactive, semi-supervised classifier that avoids thresholds and manual labeling, with user-friendly visualization of the results and automated quality control reports. Materials and Methods: We developed Tribus, an open-source Python 3 software utilizing self-organizing maps for data clustering with an integrated Napari plug-in for fast visualization. It assigns the cell phenotypes based on user-defined label description tables and median marker expressions, following a hierarchical structure to minimize overspill bias. It has a modular architecture and can be run via command line, Jupyter Notebook, or the Napari interface. For exploring the Tribus performance, we used 1) manually expert annotated datasets with different tissue origins 2) dataset with cluster-based annotation 3) previously unpublished dataset. To ensure usability, we utilized datasets generated by using different multiplexed imaging technologies, and of variable size including large tissue microarrays and whole-slide images. Results: Using Tribus we accurately annotated the cell phenotypes in an expert-labeled Co-detection by indexing (CODEX) dataset, consisting of 8 regions of tissue samples from a single donor, including both healthy colon and small bowel sections. Tribus analysis of a high-grade ovarian cancer (HGSC) tumor microarray dataset generated by tCyCIF revealed a subset of lymphoid cells previously mislabelled using a cluster annotation approach. Finally, we utilized Tribus for deep cell phenotyping on a large whole-slide image tCyCIF dataset consisting of over a million cells from five matched HGSC samples collected before and after neoadjuvant chemotherapy (NACT). In the paired samples we successfully identified cell subtypes crucial for immune surveillance. Our analysis accurately differentiated M1 and M2 macrophage subpopulations. In addition, the analyzed post-NACT samples showed a higher proportion of tumor-infiltrating CD8+ T-cells, interestingly displaying occasional spatial clusters by the tumor borders. Therefore, Tribus has shown a great potential to study the spatial patterns and cell-cell interactions of relevant immune cells in response to neoadjuvant chemotherapy treatment. Conclusion: Overall, Tribus is an accurate, fast and efficient way to analyze different sizes of datasets across different technologies, resulting in accelerated analysis of the spatial tumor microenvironment (TME). The accurate cell-type phenotyping with Tribus uncovered high-resolution cellular phenotypes and their spatial patterns in the TME in ovarian cancer after neoadjuvant chemotherapy. Citation Format: Ziqi Kang, Teodora Farago, Angela Szabo, Inga-Maria Launonen, Fernando Perez, Ella Anttila, Kevin Elias, Julia Casado, Peter Sorger, Anniina Färkkilä. Improved cell phenotyping of ovarian cancer tumor microenvironment [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 A103.

Abstract A080: CD8+T-cell interplay with myeloid cells as spatial determinant of chemotherapy response in the tumor microenvironment of metastatic high-grade serous ovarian cancer

Abstract Our goal was to characterize the effects of chemotherapy on the tumor microenvironment of high-grade serous ovarian cancer (HGSC) and suggest effective combination treatment strategies. We employed comprehensive multiomic spatial and single-cell profiling techniques, including tissue cyclic immunofluorescence t-CycIF, scRNA sequencing, and spatial transcriptomics (GeoMx) to characterize the tumor microenvironment of a total of 70 HGSC samples obtained from 41 patients from our prospective, observational trials (DECIDER, ONCOSYS-OVA). The longitudinal samples were collected before and after neoadjuvant chemotherapy (NACT), and they predominantly originated from intra-abdominal metastases. Utilizing cell type calls from whole slide t-CycIF high-dimensional marker expression data, we observed a significant increase in antigen presentation in IBA1+CD11c+ macrophages and CD11c+ myeloid cells interacting with CD4+ and interferon activated CD8+T-cells following NACT. These interactions formed spatial gradients within the tissues. To further investigate spatial heterogeneity, we identified recurring cellular neighborhoods using spatial clustering of cell types across all samples. These multicellular structures exhibited varying proportions of the 18 different cell types. Four out of five immune hotspot neighborhoods primarily comprised different myeloid cells. Interestingly, while the expression of the terminal exhaustion marker TIM3 did not differ in CD8+T-cells overall between the pre- and post-chemotherapy samples, TIM3 expression was specifically higher in CD8+T-cells in myeloid cell-rich neighborhoods from the post-chemotherapy samples, highlighting spatially restricted exhaustion patterns in HGSC. Notably, the most significant differences in overall cell type composition and cell-cell interaction patterns in samples collected before and after NACT were observed in the neighborhood corresponding to the tumor-stromal interface. CD8+T-cells exhibited higher-than-expected interactions with various myeloid cells, while their interactions with tumor cells were lower-than-expected, suggesting potential immune modulation following chemotherapy. Ligand-receptor analysis using paired site-matched scRNAseq data revealed upregulation of chemokines CXCL5 and CCL7 in macrophages after NACT, potentially induced by TNF from tumor cells. Spatial transcriptomics analysis provided further insights into the gene expression programs occurring between macrophages, CD8+T-cells, and tumor cells localized at the tumor-stromal interface. The prognostic power of the main findings were tested in bulk RNA-seq data from 33 patients treated with NACT. In conclusion, our findings indicate that the tumor microenvironment undergoes alterations following NACT, characterized by increased spatially coordinated interactions between CD8+T-cells and myeloid cells, particularly at the tumor-stromal interface. These insights contribute to a better understanding of the complex dynamics within the tumor microenvironment, facilitating the development of novel treatment strategies for HGSC. Citation Format: Inga-Maria P. Launonen, Erdogan P. Erkan, Iga Niemiec, Ada Junquera Mencía, Angela Szabo, Fernando Perez, Essi Kahelin, Sampsa Hautaniemi, Jaana Oikkonen, Johanna Hynninen, Anni Virtanen, Tuulia Vallius, Ajit J. Nirmal, Peter Sorger, Anna Vähärautio, Anniina Färkkilä. CD8+T-cell interplay with myeloid cells as spatial determinant of chemotherapy response in the tumor microenvironment of metastatic high-grade serous 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 A080.

Abstract A072: Altering the ovarian cancer tumor microenvironment using car t cell therapy

Abstract Ovarian cancer accounts for 12,810 deaths per year in the USA, making it the deadliest gynecologic malignancy. Unfortunately, patients who are resistant to standard of care treatments have very limited effective therapeutic options. Immunotherapies are an emerging class of treatments that utilize a patient’s immune system to recognize and destroy tumor cells and have resulted in dramatic responses in many hematologic malignancies. In particular, reprograming patient T cells to recognize specific surface antigen(s) known as chimeric antigen receptor (CAR) T cell therapy, has provided both pediatric and adult patients with relapsed or treatment-unresponsive hematologic malignancies novel and potentially curative treatment options. CAR T cell therapy received FDA approval in 2018 for the treatment of B cell leukemia, however despite significant efforts, has been largely ineffective in solid malignancies. This is due in part to the complex solid tumor immune microenvironment (TME) and how it may change in response to therapies. For example, there is evidence that an FDA approved treatment for ovarian cancer called poly(ADP) ribose polymerase inhibitors (PARP; PARPi) leads to increased immune cell infiltration and cancer sensitivity to immunotherapies. This proposal will delineate the immune composition of the ovarian cancer TME using mouse models that closely recapitulate ovarian cancer progression and examine the impact of CAR T cells on the TME. We will utilize mouse ovarian cancer models that are sensitive and resistant to PARP inhibition. The information derived from these studies will provide a better understanding of the evolution of ovarian cancer TME and provide insight on how CAR T cells may alter the TME. Ultimately, we hope this work will lead to the development of safer and more effective therapeutic options for ovarian cancer patients. Citation Format: Alexandra N. McMellen, Michael S. Leibowitz. Altering the ovarian cancer tumor microenvironment using car t cell therapy [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 A072.

INHBA(+) cancer-associated fibroblasts generate an immunosuppressive tumor microenvironment in ovarian cancer

Effective targeting of cancer-associated fibroblasts (CAFs) is hindered by the lack of specific biomarkers and a poor understanding of the mechanisms by which different populations of CAFs contribute to cancer progression. While the role of TGFβ in CAFs is well-studied, less attention has been focused on a structurally and functionally similar protein, Activin A (encoded by INHBA). Here, we identified INHBA(+) CAFs as key players in tumor promotion and immunosuppression. Spatiotemporal analyses of patient-matched primary, metastatic, and recurrent ovarian carcinomas revealed that aggressive metastatic tumors enriched in INHBA(+) CAFs were also enriched in regulatory T cells (Tregs). In ovarian cancer mouse models, intraperitoneal injection of the Activin A neutralizing antibody attenuated tumor progression and infiltration with pro-tumorigenic subsets of myofibroblasts and macrophages. Downregulation of INHBA in human ovarian CAFs inhibited pro-tumorigenic CAF functions. Co-culture of human ovarian CAFs and T cells revealed the dependence of Treg differentiation on direct contact with INHBA(+) CAFs. Mechanistically, INHBA/recombinant Activin A in CAFs induced the autocrine expression of PD-L1 through SMAD2-dependent signaling, which promoted Treg differentiation. Collectively, our study identified an INHBA(+) subset of immunomodulatory pro-tumoral CAFs as a potential therapeutic target in advanced ovarian cancers which typically show a poor response to immunotherapy.

NLRC5 overexpression in ovarian tumors remodels the tumor microenvironment and increases T-cell reactivity toward autologous tumor-associated antigens.

Epithelial ovarian cancer (OC) stands as one of the deadliest gynecologic malignancies, urgently necessitating novel therapeutic strategies. Approximately 60% of ovarian tumors exhibit reduced expression of major histocompatibility complex class I (MHC I), intensifying immune evasion mechanisms and rendering immunotherapies ineffective. NOD-like receptor CARD domain containing 5 (NLRC5) transcriptionally regulates MHC I genes and many antigen presentation machinery components. We therefore explored the therapeutic potential of NLRC5 in OC. We generated OC cells overexpressing NLRC5 to rescue MHC I expression and antigen presentation and then assessed their capability to respond to PD-L1 blockade and an infected cell vaccine. Analysis of microarray datasets revealed a correlation between elevated NLRC5 expression and extended survival in OC patients; however, NLRC5 was scarcely detected in the OC tumor microenvironment. OC cells overexpressing NLRC5 exhibited slower tumor growth and resulted in higher recruitment of leukocytes in the TME with lower CD4/CD8 T-cell ratios and increased activation of T cells. Immune cells from peripheral blood, spleen, and ascites from these mice displayed heightened activation and interferon-gamma production when exposed to autologous tumor-associated antigens. Finally, as a proof of concept, NLRC5 overexpression within an infected cell vaccine platform enhanced responses and prolonged survival in comparison with control groups when challenged with parental tumors. These findings provide a compelling rationale for utilizing NLRC5 overexpression in "cold" tumor models to enhance tumor susceptibility to T-cell recognition and elimination by boosting the presentation of endogenous tumor antigens. This approach holds promise for improving antitumoral immune responses in OC.

Targeting the immune microenvironment for ovarian cancer therapy.

Ovarian cancer (OC) is an aggressive malignancy characterized by a complex immunosuppressive tumor microenvironment (TME). Immune checkpoint inhibitors have emerged as a breakthrough in cancer therapy by reactivating the antitumor immune response suppressed by tumor cells. However, in the case of OC, these inhibitors have failed to demonstrate significant improvements in patient outcomes, and existing biomarkers have not yet identified promising subgroups. Consequently, there remains a pressing need to understand the interplay between OC tumor cells and their surrounding microenvironment to develop effective immunotherapeutic approaches. This review aims to provide an overview of the OC TME and explore its potential as a therapeutic strategy. Tumor-infiltrating lymphocytes (TILs) are major actors in OC TME. Evidence has been accumulating regarding the spontaneous TILS response against OC antigens. Activated T-helpers secrete a wide range of inflammatory cytokines with a supportive action on cytotoxic T-cells. Simultaneously, mature B-cells are recruited and play a significant antitumor role through opsonization of target antigens and T-cell recruitment. Macrophages also form an important subset of innate immunity (M1-macrophages) while participating in the immune-stimulation context. Finally, OC has shown to engage a significant natural-killer-cells immune response, exerting direct cytotoxicity without prior sensitization. Despite this initial cytotoxicity, OC cells develop various strategies to induce an immune-tolerant state. To this end, multiple immunosuppressive molecules are secreted to impair cytotoxic cells, recruit regulatory cells, alter antigen presentation, and effectively evade immune response. Consequently, OC TME is predominantly infiltrated by immunosuppressive cells such as FOXP3+ regulatory T-cells, M2-polarized macrophages and myeloid-derived suppressor cells. Despite this strong immunosuppressive state, PD-1/PD-L1 inhibitors have failed to improve outcomes. Beyond PD-1/PD-L1, OC expresses multiple other immune checkpoints that contribute to immune evasion, and each representing potential immune targets. Novel immunotherapies are attempting to overcome the immunosuppressive state and induce specific immune responses using antibodies adoptive cell therapy or vaccines. Overall, the OC TME presents both opportunities and obstacles. Immunotherapeutic approaches continue to show promise, and next-generation inhibitors offer exciting opportunities. However, tailoring therapies to individual immune characteristics will be critical for the success of these treatments.

Mitophagy genes in ovarian cancer: a comprehensive analysis for improved immunotherapy

BackgroundMitophagy is a process of selectively degrading damaged mitochondria, which has been found to be related to immunity, tumorigenesis, tumor progression, and metastasis. However, the role of mitophagy-related genes (MRGs) in the tumor microenvironment (TME) of ovarian cancer (OV) remains largely unexplored.MethodsWe analyzed the expression, prognosis, and genetic alterations of 29 MRGs in 480 OV samples. Unsupervised clustering was used to classify OV into two subtypes (clusters A and B) based on MRG changes. We compared the clinical features, differential expressed genes (DEGs), pathways, and immune cell infiltration between the two clusters. We constructed a mitophagy scoring system (MRG_score) based on the DEGs and validated its ability to predict overall survival of OV patients.ResultsWe found that patients with high MRG_scores had better survival status and increased infiltration by immune cells. Further analysis showed that these patients may be more sensitive to immune checkpoint inhibitor (ICI) treatment. Additionally, the MRG_score significantly correlated with the sensitivity of chemotherapeutic drugs and targeted inhibitors.ConclusionOur comprehensive analysis of MRGs in the TME, clinical features, and patient prognosis revealed that the MRG_score is a potentially effective prognostic biomarker and predictor of treatment. This study provides new insights into the role of MRGs in OV and identifies patients who may benefit from ICI treatment, chemotherapy, or targeted treatment.

Deciphering tumor immune microenvironment differences between high-grade serous and endometrioid ovarian cancer to investigate their potential in indicating immunotherapy response

BackgroundOvarian cancer is a significant public health concern with a poor prognosis for epithelial ovarian cancer. To explore the potential of immunotherapy in treating epithelial ovarian cancer, we investigated the immune microenvironments of 52 patients with epithelial ovarian cancer, including 43 with high-grade serous ovarian cancer and 9 with endometrioid ovarian cancer.ResultsFresh tumor tissue was analyzed for genetic mutations and various parameters related to immune evasion and infiltration. The mean stromal score (stromal cell infiltration) in high-grade serous ovarian cancer was higher than in endometrioid ovarian cancer. The infiltration of CD8 T cells and exhausted CD8 T cells were found to be more extensive in high-grade serous ovarian cancer. Tumor Immune Dysfunction and Exclusion scores, T cell exclusion scores, and cancer-associated fibroblasts (CAF) scores were also higher in the high-grade serous ovarian cancer group, suggesting that the number of cytotoxic lymphocytes in the tumor microenvironment of high-grade serous ovarian cancer is likely lower compared to endometrioid ovarian cancer.ConclusionsThe high mean stromal score and more extensive infiltration and exhaustion of CD8 T cells in high-grade serous ovarian cancer indicate that high-grade serous ovarian cancer exhibits a higher level of cytotoxic T cell infiltration, yet these T cells tend to be in a dysfunctional state. Higher Tumor Immune Dysfunction and Exclusion scores, T cell exclusion scores, and CAF scores in high-grade serous ovarian cancers suggest that immune escape is more likely to occur in high-grade serous ovarian cancer, thus endometrioid ovarian cancer may be more conducive to immunotherapy. Therefore, it is crucial to design immunotherapy clinical trials for ovarian cancer to distinguish between high-grade serous and endometrioid ovarian cancer from the outset. This distinction will help optimize treatment strategies and improve outcomes for patients with different subtypes.

Methodical Manipulation of the TME in Ovarian Cancer.

The complex interplay between ovarian cancer cells and the tumor microenvironment (TME) modulates progression, with dynamic cellular interactions influenced by external modulators, including neoadjuvant chemotherapy (NACT). A recent article described the alterations within the TME following NACT, either with or without bevacizumab, in ovarian cancer. See related article by Tavira et al., p. 176.

In situ profiling reveals metabolic alterations in the tumor microenvironment of ovarian cancer after chemotherapy

In this study, we investigated the metabolic alterations associated with clinical response to chemotherapy in patients with ovarian cancer. Pre- and post-neoadjuvant chemotherapy (NACT) tissues from patients with high-grade serous ovarian cancer (HGSC) who had poor response (PR) or excellent response (ER) to NACT were examined. Desorption electrospray ionization mass spectrometry (DESI-MS) was performed on sections of HGSC tissues collected according to a rigorous laparoscopic triage algorithm. Quantitative MS-based proteomics and phosphoproteomics were performed on a subgroup of pre-NACT samples. Highly abundant metabolites in the pre-NACT PR tumors were related to pyrimidine metabolism in the epithelial regions and oxygen-dependent proline hydroxylation of hypoxia-inducible factor alpha in the stromal regions. Metabolites more abundant in the epithelial regions of post-NACT PR tumors were involved in the metabolism of nucleotides, and metabolites more abundant in the stromal regions of post-NACT PR tumors were related to aspartate and asparagine metabolism, phenylalanine and tyrosine metabolism, nucleotide biosynthesis, and the urea cycle. A predictive model built on ions with differential abundances allowed the classification of patients’ tumor responses as ER or PR with 75% accuracy (10-fold cross-validation ridge regression model). These findings offer new insights related to differential responses to chemotherapy and could lead to novel actionable targets.

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.