High-grade Serous Ovarian Cancer Cell Lines Research Articles

Oncolytic adenovirus MEM-288 encoding membrane-stable CD40L and IFNβ induces an anti-tumor immune response in high grade serous ovarian cancer

Single agent immune checkpoint inhibitors have been ineffective for patients with advanced stage and recurrent high grade serous ovarian cancer (HGSOC). Using pre-clinical models of HGSOC, we evaluated the anti-tumor and immune stimulatory effects of an oncolytic adenovirus, MEM-288. This conditionally replicative virus encodes a modified membrane stable CD40L and IFNβ. We demonstrated this virus successfully infects HGSOC cell lines and primary human ascites samples in vitro. We evaluated the anti-tumor and immunostimulatory activity in vivo in immune competent mouse models. Intraperitoneal delivery of MEM-288 decreased ascites and solid tumor burden compared to controls, and treatment generated a systemic anti-tumor immune response. The tumor microenvironment had a higher proportion of anti-tumor macrophages and decreased markers of angiogenesis. MEM-288 is a promising immunotherapy agent in HGSOC, with further pre-clinical studies required to understand the mechanism of action in the peritoneal microenvironment and clinical activity in combination with other therapies.

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, demonstrates in vitro and in vivo antitumor activity against primary and metastatic ovarian tumors overexpressing HER2.

High-grade serous ovarian cancer (HGSOC) and ovarian clear cell carcinoma (CC), are biologically aggressive tumors endowed with the ability to rapidly metastasize to the abdominal cavity and distant organs. About 10% of HGSOC and 30% of CC demonstrate HER2 IHC 3 + receptor over-expression. We evaluated the efficacy of trastuzumab deruxtecan (T-DXd; DS-8201a), a novel HER2-targeting antibody-drug conjugate (ADC) to an ADC isotype control (CTL ADC) against multiple HGSOC and CC tumor models. Eleven ovarian cancer cell lines including a matched primary and metastatic cell line established from the same patient, were evaluated for HER2 expression by immunohistochemistry and flow cytometry, and gene amplification by fluorescence in situ hybridization assays. In vitro experiments demonstrated T-DXd to be significantly more effective against HER2 3 + HGSOC and CC cell lines when compared to CTL ADC (p < 0.0001). T-DXd induced efficient bystander killing of HER2 non-expressing tumor cells when admixed with HER2 3 + cells. In vivo activity of T-DXd was studied in HER2 IHC 3 + HGSOC and CC mouse xenograft models. We found T-DXd to be significantly more effective than CTL ADC against HER2 3 + HGSOC (KR(CH)31) and CC (OVA10) xenografts with a significant difference in tumor growth starting at day 8 (p = 0.0003 for KR(CH)31, p < 0.0001 for OVA10). T-DXd also conferred a survival advantage in both xenograft models. T-DXd may represent an effective ADC against primary and metastatic HER2-overexpressing HGSOC and CC.

Unraveling the role of ubiquitin C-terminal hydrolase L1 in high grade serous ovarian cancer pathogenesis.

e15078 Background: High grade serous ovarian cancer (HGSOC) is the most lethal of all gynecologic malignancies, due to the fact that the majority of patients are diagnosed at an advanced stage and eventually develop chemoresistant disease 12-18 months following completion of frontline therapy. Therefore, novel targeted therapies are urgently needed to combat chemoresistance and improve patient clinical outcomes. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that plays a vital role in protein homeostasis and is highly overexpressed in HGSOC. It was previously uncovered that targeting UCHL1 with small molecule inhibition reduced cell viability in a chemoresistant ovarian cancer cell line. Methods: Comparative-label free proteomic analysis was used to uncover significant changes in HGSOC cell line OVCAR8WT following treatment with UCHL1 small molecule inhibitor, LDN-5744 or corresponding DMSO control. Significant pathway changes were determined by KEGG analysis. Matched chemosensitive and resistant HGSOC cells PEA1 and PEA2 were treated in combination with LDN-5744 and carboplatin, with cell viability was determined by an MTS assay. Furthermore, western blot analysis was employed to compare common cell growth pathway changes as a result of UCHL1 inhibition in HGSOC cells. Results: Proteomic analysis revealed a significant (p&lt;0.05) upregulation in methyl phosphate capping enzyme (MEPCE) (4.9-fold), asparagine synthetase (ASNS) (2.5-fold), Phosphoserine aminotransferase 1 (PSAT1) (1.7-fold), and downregulation in centrosomal protein 55 (CEP55) (-7.2-fold), These identified proteomic changes were confirmed by western blot. KEGG pathways analysis using all significantly (p&lt;0.05) differentially expressed proteins showcased significant (p&lt;0.05) enrichment in amino acid biosynthesis, and metabolism, and chemical carcinogenesis-reactive oxygen species. Chemoresistant PEA2 cells treated in combination with LDN-5744 and carboplatin demonstrated a 59% reduction in cell viability, significantly (p&lt;0.001) lower than the 13% and 22%, that was observed from UCHL1 and carboplatin treatment alone, respectively. Conversely, PEA1, the chemosensitive counterpart to PEA2 demonstrated a significant (p&lt;0.001) 37% increase in cell viability upon combinatorial treatment compared with carboplatin alone, indicating that UCHL1 inhibition rescues the cells from chemotherapy induced cell death. Finally, LDN-5744 treatment in PEA2 led to a reduction in levels of phospho-(p-)AKT, p-STAT3, and p-ERK, while conversely in PEA1 cells levels of these proteins were increase or unchanged. Conclusions: Our findings demonstrate that targeting UCHL1 results in prominent metabolic changes in HGSOC cells and that the efficacy of UCHL1 inhibition is heavily dependent upon platinum status. Overall, this study highlights UCHL1’s novel therapeutic role in the recurrent HGSOC.

Enhanced amphiregulin exposure promotes modulation of the high grade serous ovarian cancer tumor immune microenvironment.

High grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Furthermore, HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies. Amphiregulin (AREG), a low affinity epidermal growth factor receptor ligand is known to be significantly upregulated in HGSOC patient tumors following neoadjuvant chemotherapy exposure. While much is known about AREG's role in oncogenesis and classical immunity, it is function in tumor immunology has been comparatively understudied. Therefore, the objective of this present study was to elucidate how increased AREG exposure impacts the ovarian tumor immune microenvironment (OTIME). Using NanoString IO 360 and protein analysis, it was revealed that treatment with recombinant AREG led to prominent upregulation of genes associated with ovarian pathogenesis and immune evasion (CXCL8, CXCL1, CXCL2) along with increased STAT3 activation in HGSOC cells. In vitro co-culture assays consisting of HGSOC cells and peripheral blood mononuclear cells (PBMCs) stimulated with recombinant AREG (rAREG) led to significantly enhanced tumor cell viability. Moreover, PBMCs stimulated with rAREG exhibited significantly lower levels of IFNy and IL-2. In vivo rAREG treatment promoted significant reductions in circulating levels of IL-2 and IL-5. Intratumoral analysis of rAREG treated mice revealed a significant reduction in CD8+ T cells coupled with an upregulation of PD-L1. Finally, combinatorial treatment with an AREG neutralizing antibody and carboplatin led to a synergistic reduction of cell viability in HGSOC cell lines OVCAR8 and PEA2. Overall, this study demonstrates AREG's ability to modulate cytotoxic responses within the OTIME and highlights its role as a novel HGSOC immune target.

N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

High-grade serous ovarian cancer (HGSC) is a challenging disease, especially for patients with immunologically "cold" tumors devoid of tumor-infiltrating lymphocytes (TILs). We found that HGSC exhibits among the highest levels of MYCN expression and transcriptional signature across human cancers, which is strongly linked to diminished features of antitumor immunity. N-MYC repressed basal and induced IFN type I signaling in HGSC cell lines, leading to decreased chemokine expression and T cell chemoattraction. N-MYC inhibited the induction of IFN type I by suppressing tumor cell-intrinsic STING signaling via reduced STING oligomerization, and by blunting RIG-I-like receptor signaling through inhibition of MAVS aggregation and localization in the mitochondria. Single-cell RNA sequencing of human clinical HGSC samples revealed a strong negative association between cancer cell-intrinsic MYCN transcriptional program and type I IFN signaling. Thus, N-MYC inhibits tumor cell-intrinsic innate immune signaling in HGSC, making it a compelling target for immunotherapy of cold tumors.

ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

MicroRNA Expression Profiles in Human Samples and Cell Lines Revealed Nine miRNAs Associated with Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

Metastasis and drug resistance are major contributors to cancer-related fatalities worldwide. In ovarian cancer (OC), a staggering 70% develop resistance to the front-line therapy, cisplatin. Despite proposed mechanisms, the molecular events driving cisplatin resistance remain unclear. Dysregulated microRNAs (miRNAs) play a role in OC initiation, progression, and chemoresistance, yet few studies have compared miRNA expression in OC samples and cell lines. This study aimed to identify key miRNAs involved in the cisplatin resistance of high-grade-serous-ovarian-cancer (HGSOC), the most common gynecological malignancy. MiRNA expression profiles were conducted on RNA isolated from formalin-fixed-paraffin-embedded human ovarian tumor samples and HGSOC cell lines. Nine miRNAs were identified in both sample types. Targeting these with oligonucleotide miRNA inhibitors (OMIs) reduced proliferation by more than 50% for miR-203a, miR-96-5p, miR-10a-5p, miR-141-3p, miR-200c-3p, miR-182-5p, miR-183-5p, and miR-1206. OMIs significantly reduced migration for miR-183-5p, miR-203a, miR-296-5p, and miR-1206. Molecular pathway analysis revealed that the nine miRNAs regulate pathways associated with proliferation, invasion, and chemoresistance through PTEN, ZEB1, FOXO1, and SNAI2. High expression of miR-1206, miR-10a-5p, miR-141-3p, and miR-96-5p correlated with poor prognosis in OC patients according to the KM plotter database. These nine miRNAs could be used as targets for therapy and as markers of cisplatin response.

Abstract 4538: Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous 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 4538.

Abstract 182: Cyclooxygenase-2 blockade is crucial to restore natural killer cell activity before anti-CTLA-4 therapy against high-grade serous ovarian cancer

Abstract Objective: Here our goal was to evaluate the impact of the immunosuppressive enzyme cyclooxygenase-2 (COX-2) expression on the immune effector capacity against high-grade serous ovarian cancer (HGSOC) tumor cells through bioinformatic tools and in vitro validation. Methods: We performed an integrated bioinformatics analysis from transcriptomic data based on RNAseq (TCGA-OV, n=368; Australian cohort AOCS, n=80) and RNA microarrays (GSE26193, n=62; GSE30161, n=45). We estimated the proportion and characteristics of immune cells employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms. Survival curves and Cox-regression models were built up according to known risk factors and immune scores predicted by in-silico analysis. Afterward, we validated these results using flow cytometry and cytotoxicity assays on circulating NK cells collected from advanced HGSOC patients treated at our institution (n=5). After three days of incubation, isolated NK cell cultures were stimulated with three pulses of a selective COX-2 inhibitor (celecoxib) or MOCK, daily. An anti-CTLA-4 monoclonal antibody (ipilimumab) or MOCK was added at the third pulse. Two ovarian cancer cell lines (SKOV3 and HeyA8) were treated similarly. Next, NK cells were co-cultured with SKOV3 or HeyA8 and cytotoxicity on these cells was examined using lactate dehydrogenase (LDH) cell viability assay. Results: Transcriptomic data evidenced a non-linear relationship between COX-2 gene, PTGS2, and CTLA4 in advanced stages of HGSOC. Hence, PTGS2/CTLA4 ratio allows us to investigate how COX-2 influences changes in the immune profile of these patients. Based on bioinformatics, a high PTGS2/CTLA4 ratio determines particularly dysfunctional NK cell states, along with the reduction of NK gene score associated with infiltration, cytotoxicity, and survival in HGSOC. Additionally, selective COX-2 blockade improves NK cell cytotoxicity against HGSOC cell lines independently of the CTLA-4 signaling. In fact, ipilimumab effect was almost absent in those cases with high COX2 activity. Interestingly, previous treatment with celecoxib on NK cells mainly explains the difference in cytotoxicity (p= 0.0253, two-way ANOVA test with Tukey’s multiple comparisons). Conclusions: This work represents the first evidence of the influence of COX-2 on NK cell function as a crucial factor for the efficacy of anti-CTLA-4 therapy in HGSOC patients who exhibited high tumoral levels of CTLA-4. HGSOC patients present dysfunctional circulating NK cells preferentially due to the strong CO-2 expression. Last, we emphasize the modulation of chronic inflammation to direct an immune pattern with greater antitumor capacity in HGSOC. (Funding: Fondecyt 1201083 and FONDAP 152220002 [MAC], CONICYT/Doctorado Nacional 2019-Folio 21190421 [FGV], CONICYT FONDAP 15130011 [MAC, IW]). Citation Format: Fernan Gomez-Valenzuela, Ignacio Wichmann, Felipe Suárez, Sumie Kato, Enrique Ossandón, Marcela Hermoso, Elmer Fernández, Mauricio A. Cuello. Cyclooxygenase-2 blockade is crucial to restore natural killer cell activity before anti-CTLA-4 therapy against high-grade serous 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 182.

Abstract 278: Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian cancer

Abstract Chemoresistance remains a major clinical challenge for the treatment of high-grade serous ovarian cancer (HGSC), the most lethal gynecologic malignancy. The majority of patients who initially respond to first-line platinum/taxane-based chemotherapy eventually relapse with chemoresistant disease, leading to poor survival outcomes. It is increasingly recognized that cancer cells can enter a reversible drug-tolerant persister (DTP) state through non-genetic mechanisms to evade chemotherapy-induced cytotoxicity prior to the development of chemoresistance. However, how environmental signals and extracellular factors from the tumor microenvironment drive the process remains poorly understood. Cancer-associated fibroblasts (CAFs) are a functionally heterogeneous population of activated fibroblasts. CAFs play a critical role in shaping the tumor microenvironment and tumor behaviors, including promoting tumor growth and mediating therapy resistance. Here, we demonstrated that HGSC cell lines co-cultured with patient-derived FAP-high CAFs, a subpopulation of CAFs previously identified in our lab to be tumor-promoting, significantly increase the formation of DTPs upon prolonged carboplatin treatment compared to the mono-cultured condition. Interestingly, the extracellular matrix (ECM) organization signature is enriched in FAP-high CAFs compared to their FAP-low counterparts, and we found that ECM deposited by FAP-high CAFs also facilitates the emergence of DTPs in HGSC cells. Future work will focus on elucidating the mechanisms by which FAP-high CAFs and the associated ECM promote DTP formation in HGSC cells through transcriptomic and proteomic profiling. This will aid the identification of potential therapeutic vulnerabilities to target cancer cells in the DTP state in FAP-high HGSC patients. Citation Format: Angel S. Ng, Ngoc Bui, Laurie Ailles. Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in 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 278.

Abstract B019: Investigating Cannabis sativa bioactive compounds as an anti-cancer treatment in high-grade serous ovarian cancer

Abstract B019: Investigating <i>Cannabis sativa</i> bioactive compounds as an anti-cancer treatment in high-grade serous ovarian cancer

Abstract A112: Mechanisms underlying platinum-induced ovarian cancer stem cell plasticity

Abstract Most women diagnosed with late-stage high grade serous ovarian cancer (HGSOC) develop recurrent, platinum-resistant tumors. Ovarian cancer stem cells (OCSCs) are hypothesized to contribute to the emergence of these resistant tumors. CSCs have been postulated to reside in a plastic state, which can allow for the conversion of non-CSC to CSC. This process of dedifferentiation continues during tumor development and chemotherapeutic agents like platinum can exaggerate CSC plasticity. We have previously demonstrated that acute platinum treatment enriched for OCSCs. However, whether platinum transforms non-OCSCs into OCSCs to contribute to this subpopulation of cells remains unclear, and the underlying mechanism remains incompletely understood. To examine OCSC plasticity, aldehyde dehydrogenase (ALDH; functional marker) and fluorescence activated cell sorting were used to isolate OCSCs (ALDH+) and non-OCSCs (ALDH-) from HGSOC cell lines, OVCAR5 and OVCAR3. To determine the stability of the non-OCSC phenotype, ALDH- cells were cultured for 3 and 5 days and ALDH activity was measured using flow cytometry. At both timepoints examined, ALDH- cells remained ALDH-, with approximately less than 1% being ALDH+ (p&amp;lt;0.05). To determine if platinum can induce conversion of non-OCSC to OCSC, ALDH- cells were treated with cisplatin (12µM or 15 µM for 16h), and the percent of ALDH+ cells was measured using flow cytometry. Treatment of ALDH- cells with cisplatin resulted in conversion of approximately 4% and 10% of ALDH- cells into ALDH+ cells (p&amp;lt;0.05) in OVCAR5 and OVCAR3 cells, respectively. Furthermore, both cell lines displayed increased expression (p&amp;lt;0.05) of stemness genes BMI1, NANOG, OCT4, and SOX2 and spheroid formation was observed in converted OCSCs compared to parental ALDH+ and whole cell populations treated with cisplatin, suggesting that platinum induced the observed differences in the stemness phenotype. With the goal of targeting key genes and pathways to inhibit platinum-induced OCSC conversion, RNA-sequencing was performed using converted OCSCs. Activated pathways in converted OCSCs were identified, including protein kinase signaling, inhibition of matrix metalloproteases, SPINK1 pathway, and VDR/RXR activation. We are further investigating if these pathways contribute to the mechanism involved in OCSC plasticity and whether targeting these pathways can reduce the OCSC population, which is critical to blocking the persistence of OCSCs and ultimately reduce mortality in patients. Citation Format: Tara X. Metcalfe, Shu Zhang, Christiane Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Mechanisms underlying platinum-induced ovarian cancer stem cell plasticity [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 A112.

Abstract A069: High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants

Abstract Introduction: As high-grade serous ovarian cancer (HGSOC) rarely shows symptoms, most patients present with metastases. Tumor heterogeneity between metastases may impede a patient’s response to therapy and thus impact survival. We aim to decipher the heterobiology of HGSOC lesions using high-resolution imaging of patient-derived explants (PDEs), to determine the differential treatment response between primary and metastatic tumors. Methods: PAX8, CK7, RAD51, γH2AX, Ki67, cPARP, and CD3 were chosen to enumerate tumor and T cells, and their activity. Antibodies were optimized in monolayer and spheroid cultures of HGSOC cell lines, COV318 and OVCAR4. CD3 was optimized in FFPE human tonsillitis tissue. Primary and metastatic HGSOC explants were cultured for 48h followed by 24h fixation. Several immunostaining and optical clearing protocols were tested on PDEs, which were imaged using a fully automated confocal high-content screening system. Results: Immunostaining and automated image analysis facilitated the identification of individual nuclei within PDEs without Refractive Index (RI) matching or optical clearing. However, high background impeded further image analysis, preventing the quantification of certain antibodies. Glycerol-based RI matching and ScaleS tissue clearing were implemented to try and overcome this. However, neither generated sufficiently high-quality images for analysis. A novel Clearing-enhanced 3D (Ce3D) immunostaining system recently developed for high-quality multiplex fluorescence microscopy of large tissue volumes was tested. While individual nuclei were recognized, as well as RAD51 foci and CK7 cytoskeleton staining, image analysis was still impeded due to high background. Conclusions: Protocols without RI matching and optical clearing can stain large, complex structures such as PDEs. However, high background impedes image analysis suggesting the need for optical clearing and RI matching. The inclusion of various RI matching and/or optical clearing steps failed to overcome high background staining. Further work is ongoing to refine immunostaining, RI matching, optical clearing, and mounting of PDEs to allow the quantification of all antibodies. Citation Format: Adele E. Connor, Joanne Lysaght, Donal J. Brennan, Jeremy C. Simpson, Antoinette S. Perry. High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants [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 A069.

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

MiR-451a suppresses the proliferation and migration of high-grade serous ovarian cancer by targeting RAB5A through the Ras/Raf/MEK/ERK pathway.

Ovarian cancer is one of the most common cancers in women. Profiles changes of microRNAs (miRNAs) are closely linked to malignant tumors. In the present study, we investigated expression of miR-451a in high-grade serous ovarian cancer (HGSOC). We also investigated the potential pathological roles and the likely mechanism of miR-451a in the development of HGSOC using animal models and cell lines. Using bioinformatics techniques and a real-time PCR, we analyzed differently expressed miRNAs in HGSOC compared to normal tissue. MTT (i.e. 3-[4, 5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide), EDU (i.e. 5-ethynyl-2'-deoxyuridine) and transwell assays were performed to investigate the effect of miR-451a on the proliferation and migration of HGSOC SKOV-3 cells. A dual luciferase reporter assay was performed to verify the targeting relationship of miR-451 and RAB5A (one of the Rab GTPase proteins that regulates endocytosis and vesicle transport). Also, we analyzed levels of the RAB5A mRNA and protein by real-time PCR, western blotting and immunohistochemistry assays in HGSOC cells and tissues. Finally, we performed in vivo experiments using HGSOC mice. miR-451a was substantially upregulated in HGSOC and associated with favorable clinical characteristics. miR-451a knockdown significantly increased growth and metastasis of HGSOC cell line SKOV-3 through Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. In addition, RAB5A, an early endosome marker, was shown to be a direct target of miR-451a. Moreover, RAB5A is correlated with unfavorable clinical features and shows independent prognostic significance in HGSOC. We found that the miR-451a/RAB5A axis is associated with tumorigenesis and progression through the Ras/Raf/MEK/ERK pathway, providing prognostic indicators and therapeutic targets for patients with HGSOC.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4–CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Cyclooxygenase-2 Blockade Is Crucial to Restore Natural Killer Cell Activity before Anti-CTLA-4 Therapy against High-Grade Serous Ovarian Cancer.

Chronic inflammation influences the tumor immune microenvironment (TIME) in high-grade serous ovarian cancer (HGSOC). Specifically, cyclooxygenase-2 (COX-2) overexpression promotes cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression. Notably, elevated COX-2 levels in the TIME have been associated with reduced response to anti-CTLA-4 immunotherapy. However, the precise impact of COX-2, encoded by PTGS2, on the immune profile remains unknown. To address this, we performed an integrated bioinformatics analysis using data from the HGSOC cohorts (TCGA-OV, n = 368; Australian cohort AOCS, n = 80; GSE26193, n = 62; and GSE30161, n = 45). Employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms, we concluded that COX-2 was linked to immune cell ecosystems associated with shorter survival, cell dysfunction and lower NK cell effector cytotoxicity capacity. Next, we validated these results by characterizing circulating NK cells from HGSOC patients through flow cytometry and cytotoxic assays while undergoing COX-2 and CTLA-4 blockade. The blockade of COX-2 improved the cytotoxic capacity of NK cells against HGSOC cell lines. Our findings underscore the relevance of COX-2 in shaping the TIME and suggest its potential as a prognostic indicator and therapeutic target. Increased COX-2 expression may hamper the effectivity of immunotherapies that require NK cell effector function. These results provide a foundation for experimental validation and clinical trials investigating combined therapies targeting COX-2 and CTLA-4 in HGSOC.

PH-sensitive niosomes for ATRA delivery: A promising approach to inhibit Pin1 in high-grade serous ovarian cancer

The peptidyl-prolyl cis/trans isomerase Pin1 positively regulates numerous cancer-driving pathways, and it is overexpressed in several malignancies, including high-grade serous ovarian cancer (HGSOC). The findings that all-trans retinoic acid (ATRA) induces Pin1 degradation strongly support that ATRA treatment might be a promising approach for HGSOC targeted therapy. Nevertheless, repurposing ATRA into the clinics for the treatment of solid tumors remains an unmet need mainly due to the insurgence of resistance and its ineffective delivery. In the present study, niosomes have been employed for improving ATRA delivery in HGSOC cell lines. Characterization of niosomes including hydrodynamic diameter, ζ-potential, morphology, entrapment efficiency and stability over time and in culture media was performed. Furthermore, pH-sensitiveness and ATRA release profile were investigated to demonstrate the capability of these vesicles to release ATRA in a stimuli-responsive manner. Obtained results documented a nanometric and monodispersed samples with negative ζ-potential. ATRA was efficiently entrapped, and a substantial release was observed in the presence of acidic pH (pH 5.5). Finally, unloaded niosomes showed good biocompatibility while ATRA-loaded niosomes significantly increased ATRA Pin1 inhibitory activity, which was consistent with cell growth inhibition. Taken together, ATRA-loaded niosomes might represent an appealing therapeutic strategy for HGSOC therapy.

Targeting Ovarian Cancer with Chalcone Derivatives: Cytotoxicity and Apoptosis Induction in HGSOC Cells

Ovarian cancer ranks as the eighth most prevalent form of cancer in women across the globe and stands as the third most frequent gynecological cancer, following cervical and endometrial cancers. Given its resistance to standard chemotherapy and high recurrence rates, there is an urgent imperative to discover novel compounds with potential as chemotherapeutic agents for treating ovarian cancer. Chalcones exhibit a wide array of biological properties, with a particular focus on their anti-cancer activities. In this research, we documented the synthesis and in vitro study of a small library of chalcone derivatives designed for use against high-grade serous ovarian cancer (HGSOC) cell lines, specifically OVCAR-3, OVSAHO, and KURAMOCHI. Our findings revealed that three of these compounds exhibited cytotoxic and anti-proliferative effects against all the tested HGSOC cell lines, achieving IC50 concentrations lower than 25 µM. Further investigations disclosed that these chalcones prompted an increase in the subG1 phase cell cycle and induced apoptosis in OVCAR-3 cells. In summary, our study underscores the potential of chalcones as promising agents for the treatment of ovarian cancer.

The role and impact of alternative polyadenylation and miRNA regulation on the expression of the multidrug resistance-associated protein 1 (MRP-1/ABCC1) in epithelial ovarian cancer

The ATP-binding cassette transporter (ABCC1) is associated with poor survival and chemotherapy drug resistance in high grade serous ovarian cancer (HGSOC). The mechanisms driving ABCC1 expression are poorly understood. Alternative polyadenylation (APA) can give rise to ABCC1 mRNAs which differ only in the length of their 3′untranslated regions (3′UTRs) in a process known as 3′UTR-APA. Like other ABC transporters, shortening of the 3′UTR of ABCC1 through 3′UTR-APA would eliminate microRNA binding sites found within the longer 3′UTRs, hence eliminating miRNA regulation and altering gene expression. We found that the HGSOC cell lines Caov-3 and Ovcar-3 express higher levels of ABCC1 protein than normal cells. APA of ABCC1 occurs in all three cell lines resulting in mRNAs with both short and long 3′UTRs. In Ovcar-3, mRNAs with shorter 3′UTRs dominate resulting in a six-fold increase in protein expression. We were able to show that miR-185-5p and miR-326 both target the ABCC1 3′UTR. Hence, 3′UTR-APA should be considered as an important regulator of ABCC1 expression in HGSOC. Both HGSOC cell lines are cisplatin resistant, and we used erastin to induce ferroptosis, an alternative form of cell death. We showed that we could induce ferroptosis and sensitize the cisplatin resistant cells to cisplatin by using erastin. Knocking down ABCC1 resulted in decreased cell viability, but did not contribute to erastin induced ferroptosis.