OVCAR-3 Cells Research Articles

BPA exposure enhances the metastatic aggression of ovarian cancer through the ERα/AKT/mTOR/HIF-1α signaling axis.

Long-term exposure to bisphenol A (BPA) in humans may promote ovarian cancer development. In present study, the mechanisms by which BPA mediates the aggression metastatic behavior of ovarian cancer were investigated in vitro/in vivo. The results showed that BPA (10μM) significantly promoted the proliferation, migration and invasion of human ovarian cancer cells (ES-2 and OVCAR-3cells); moreover, it promoted ES-2 and OVCAR-3cell glucose uptake, lactic acid release and intracellular ATP synthesis. After administration of 5μg/kg/day BPA, tumor volume was increased compared with that in control group. KEGG and GO enrichment analyses showed that the genes from ES-2cell in 10μM BPA-treated group were enriched mainly in central carbon metabolism and PI3K-AKT signaling pathway. Then, qRT‒PCR and western blotting results showed that BPA (10μM) increased the mRNA and protein expression levels of glycolysis-related genes and mTOR, p-AKT HIF-1α and ERα in vitro/vivo; whereas this effect was reduced after treatment with the ERα inhibitor methyl-piperidino-pyrazole. Furthermore, coimmunoprecipitation and mass spectrometry showed that BPA promoted the direct interaction of ERα with lactate dehydrogenase A. These results show that BPA directly promoted the proliferation, migration and invasion of ovarian cancer cells through the ERα/AKT/mTOR/HIF-1α signaling axis to enhance glycolysis.

Albumin nanoparticles with tunable ultraviolet-to-red autofluorescence for label-free cell imaging and selective biosensing of copper ion

Early and simple detection of aberrant cooper metabolism in diseases with neurological-manifestations and several other conditions, including cancer, becomes an urgent necessity. Instrumental methods used today are limited to high-cost equipment and reagents and demand highly qualified personnel. In this work, we report easy-to-use and cost-effective nano-sized sensors for the selective and quantitative detection of copper ion based on fluorescence quenching. Glutaraldehyde cross-linked albumin nanoparticles with tunable ultraviolet-to-red autofluorescence emissions are developed as dual-agents for sensing and imaging. These albumin nanoparticles show great selectivity towards copper ion when tested against a selection of biochemical components and other metal ions, and a limit of detection as low as 1.9 μM, relevant for sensing in clinical diagnosis, was determined. In addition, a lack of toxicity and good cellular uptake were observed and the ultraviolet-to-red intrinsic fluorescence of the albumin nanoparticles was preserved when tested in vitro on NIH:OVCAR3 cell line. Preliminary studies confirm the albumin nanoparticles' ability to detect Cu2+in vitro and establishes their potential for future practical use.

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models.

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

TEAD4 predicts poor prognosis and transcriptionally targets PLAGL2 in serous ovarian cancer.

The oncogenic function of TEA domain transcription factor 4 (TEAD4) has been confirmed in multiple human malignancies, while its potential role and regulatory mechanism in serous ovarian cancer progression are left unknown. By the gene expression analyses from Gene Expression Profiling Interactive Analysis (GEPIA) database, TEAD4 expression is shown to be up-regulated in serous ovarian cancer samples. Here, we confirmed the high expression of TEAD4 in clinical serous ovarian cancer specimens. In the following functional experiments, we found that TEAD4 overexpression promoted serous ovarian cancer malignant phenotypes, including proliferation, migration and invasion in serous ovarian cancer SK-OV-3 and OVCAR-3 cells, while TEAD4 knockout exerted the opposite function. The tumor growth inhibition of TEAD4 depletion was also affirmed by a Xenograft model in mice. In addition, this phenotypic deterioration induced by TEAD4 overexpression was diminished by PLAG1 like zinc finger 2 (PLAGL2) silencing. More importantly, combined with the results of the dual-luciferase assay, the transcriptional regulation of TEAD4 on PLAGL2 promoter was evidenced. Our results showed that the cancer-promoting gene TEAD4 was involved in serous ovarian cancer progression via targeting PLAGL2 at the transcriptional level.

Copper(I)-Thiosemicarbazone Complexes with Dual Anticancer and Antiparasitic Activity.

Four new Cu(I) complexes of the general formula [Cu(PP)(LL)][BF4 ], in which PP is a phosphane ligand (triphenylphosphane or 1,2-bis(diphenylphosphano)ethane (dppe)) and LL is a bioactive thiosemicarbazone ligand (4-(methyl)-1-(5-nitrofurfurylidene)thiosemicarbazone) or 4-(ethyl)-1-(5-nitrofurfurylidene)thiosemicarbazone) were synthesized and fully characterized by classical analytical and spectroscopic methods. The anti-trypanosome and anticancer activities were investigated in vitro on Trypanosoma cruzi and in two human cancer cell lines (ovarian OVCAR3 and prostate PC3). To test the selectivity toward parasites and cancer cells, the cytotoxicity on normal monkey kidney VERO and human dermal fibroblasts HDF cells was also evaluated. The new heteroleptic complexes were more cytotoxic on T. cruzi and chemoresistant prostate PC3 cells than the benchmark drugs nifurtimox and cisplatin. The compounds also showed a high level of cellular internalization by the OVCAR3 cells and, in particular, those containing the dppe phosphane showed activation of the cell death mechanism via apoptosis. On the other hand, the production of reactive oxygen species induced by these complexes was not evident.

LncRNA AC005224.4/miR-140-3p/SNAI2 regulating axis facilitates the invasion and metastasis of ovarian cancer through epithelial-mesenchymal transition.

Ovarian cancer is one of the most widespread malignant diseases of the female reproductive system worldwide. The plurality of ovarian cancer is diagnosed with metastasis in the abdominal cavity. Epithelial-mesenchymal transition (EMT) exerts a vital role in tumor cell metastasis. However, it remains unclear whether long non-coding RNA (lncRNA) are implicated in EMT and influence ovarian cancer cell invasion and metastasis. This study was designed to investigate the impacts of lncRNA AC005224.4 on ovarian cancer. LncRNA AC005224.4, miR-140-3p, and snail family transcriptional repressor 2 ( SNAI2 ) expression levels in ovarian cancer and normal ovarian tissues were determined using real-time quantitative polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) and Transwell (migration and invasion) assays were conducted to measure SKOV3 and CAOV-3 cell proliferation and metastasis. E-cadherin, N-cadherin, Snail, and Vimentin contents were detected using Western blot. Nude mouse xenograft assay was utilized to validate AC005224.4 effects in vivo . Dual-luciferase reporter gene assay confirmed the targeted relationship between miR-140-3p and AC005224.4 or SNAI2 . AC005224.4 and SNAI2 upregulation and miR-140-3p downregulation were observed in ovarian cancer tissues and cells. Silencing of AC005224.4 observably moderated SKOV3 and CAOV-3 cell proliferation, migration, invasion, and EMT process in vitro and impaired the tumorigenesis in vivo . miR-140-3p was a target of AC005224.4 and its reduced expression level was mediated by AC005224.4. miR-140-3p mimics decreased the proliferation, migration, and invasion of ovarian cancer cells. SNAI2 was identified as a novel target of miR-140-3p and its expression level was promoted by either AC005224.4 overexpression or miR-140-3p knockdown. Overexpression of SNAI2 also facilitated ovarian cancer cell viability and metastasis. AC005224.4 was confirmed as an oncogene via sponging miR-140-3p and promoted SNAI2 expression, contributing to better understanding of ovarian cancer pathogenesis and shedding light on exploiting the novel lncRNA-directed therapy against ovarian cancer.

Porphin e6 complex loaded with gold nanorod mesoporous silica enhances photodynamic therapy in ovarian cancer cells in vitro.

A growing amount of experimental evidence has proven that the application of gold nanorods (AuNRs) in photodynamic therapy (PDT) can significantly enhance its therapeutic efficacy. The aim of this study was to establish a protocol for investigating the effect of gold nanorods loaded with the photosensitizer chlorin e6 (Ce6) on photodynamic therapy in the OVCAR3 human ovarian cancer cell line in vitro and to determine whether the PDT effect was different from that of Ce6 alone. OVCAR3 cells were randomly divided into three groups: the control group, Ce6-PDT group, and AuNRs@SiO2@Ce6-PDT group. Cell viability was measured by MTT assay. The generation of reactive oxygen species (ROS) was measured by a fluorescence microplate reader. Cell apoptosis was detected by flow cytometry. The expression of apoptotic proteins was detected by immunofluorescence and western blotting. The results showed that compared with that of the Ce6-PDT group, the cell viability of the AuNRs@SiO2@Ce6-PDT group was significantly decreased (P < 0.05) in a dose-dependent manner, and ROS production increased significantly (P < 0.05). The flow cytometry results showed that the proportion of apoptotic cells in the AuNRs@SiO2@Ce6-PDT group was significantly higher than that in the Ce6-PDT group (P < 0.05). Immunofluorescence and western blot results showed that the protein expression levels of cleaved caspase-9, cleaved caspase-3, cleaved PARP, and Bax in the AuNRs@SiO2@Ce6-PDT-treated-OVCAR3 cells were higher than those in the Ce6-PDT-treated cells (P < 0.05), and the protein expression levels of caspase-3, caspase-9, PARP, and Bcl-2 were slightly lower than those in the Ce6-PDT group (P < 0.05). In summary, our results show that AuNRs@SiO2@Ce6-PDT has a significantly stronger effect on OVCAR3 cells than the effect of Ce6-PDT alone. The mechanism may be related to the expression of Bcl-2 family and caspase family in the mitochondrial pathway.

IFNα primes cancer cells for Fusicoccin-induced cell death via 14-3-3 PPI stabilization

The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well aspathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.

Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites: An Experimental Study.

This study aimed to explore the value of M701, targeting epithelial cell adhesion molecule (EpCAM) and CD3, in the immunotherapy of ovarian cancer ascites by the in vitro assay. The expression of EpCAM in ovarian cancer tissues was analyzed by databases. The EpCAM expression and immune cell infiltration in different foci of ovarian cancer were detected by 8-channel flow cytometry. The toxic effect of M701 on OVCAR3 was tested using the in vitro cytotoxicity assay. The 3D cell culture and drug intervention experiments were performed to evaluate the therapeutic effect of M701 in ovarian cancer specimens. Flow cytometry was used to examine the effect of M701 on the binding of immune cells to tumor cells and the activation capacity of T cells. The results of the bioinformatic analysis showed that the expression of EpCAM in ovarian cancer tissue was significantly higher than that in normal ovarian tissue. The 8-channel flow cytometry of clinical samples showed that the EpCAM expression and lymphocyte infiltration were significantly heterogeneous among ovarian cancer patients and lesions at different sites. The in vitro experiment results showed that M701 had a significant killing effect on OVCAR3 cells. M701 also obviously killed primary tumor cells derived from some patients with ovarian cancer ascites. M701 could mediate the binding of CD3+ T cells to EpCAM+ tumor cells and induce T cell activation in a dose-dependent manner. M701 showed significant inhibitory activity on tumor cells derived from ovarian cancer ascites, which had a promising application in immunotherapy for patients with ovarian cancer ascites.

Effects of leptin on the viability of human ovarian cancer cells and changes in cytokine expression levels.

Obesity is associated with increased mortality among ovarian cancer and is a poor prognostic factor. There are significant links between the leptin hormone, a product of the obesity gene, and the development of ovarian cancer. Leptin is a vital hormone-like cytokine secreted from adipose tissue and is mainly involved in the maintenance of energy homeostasis. It regulates several intracellular signaling pathways and also interacts with various hormones and energy regulators. It acts as a growth factor by stimulating cell proliferation and differentiation and in this way contributes to cancer cell development. The aim of the study was to investigate the effects of leptin on human ovarian cancer cells. In this study, the effects of increasing the concentration of leptin were investigated on the cell viability of OVCAR-3 and MDAH-2774 ovarian cancer lines by MTT assay. Moreover, to elucidate the molecular mechanisms of leptin in ovarian cancer cells, changes in the expression levels of 80 cytokines were evaluated after leptin treatment via a human cytokine antibody array. Leptin increases the proliferation of both ovarian cancer cell lines. IL-1 level was increased in OVCAR-3 cells and TGF-β level was increased in MDAH-2774 cells after leptin treatment. A decrease in IL-2, MCP-2/CCL8 and MCP-3/CCL7 levels was detected in both ovarian cancer cell lines with leptin administration. An increase in IL-3 and IL-10 expressions, insulin-like growth factor binding proteins (IGFBP) IGFBP-1, IGFBP-2 and IGFBP-3 levels were detected in both ovarian cancer cell lines with leptin administration. In conclusion; leptin has a proliferative effect on human ovarian cancer cell lines and affects different cytokines in different types of ovarian cancer cells.

Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Label-Free Quantification Mass Spectrometry Identifies Protein Markers of Chemotherapy Response in High-Grade Serous Ovarian Cancer

Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography-mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan-Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785.

Eriodictyol regulated ferroptosis, mitochondrial dysfunction, and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer cells.

This study aimed to investigate the antitumor effect and the underlying molecular mechanism of eriodictyol on ovarian cancer cells.CaoV3 and A2780 were exposed to eriodictyol at different concentrations of 0-800 μM. Cell apoptosis and viability were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and Cell Counting Kit-8 (CCK-8) assay, respectively. Mitochondrial membrane potentialwas evaluated by flow cytometers with a JC-1 detection kit. Fe2+ content was evaluated using an iron assay kit. The section of tumor tissues was observed using hematoxylin-eosin (H&E) staining and nuclear factor erythroid 2-related factor 2 (Nrf2) expression was detected by immunohistochemistry (IHC) staining.Eriodictyol suppressed cell viability and induced cell apoptosis of CaoV3 and A2780 cells. Half maximal inhibitory concentration (IC50 ) value of CaoV3 at 24and 48 h was (229.74 ± 5.13) μM and (38.44 ± 4.68) μM, and IC50 value of A2780 at 24and 48 h was (248.32 ± 2.54) μM and (64.28 ± 3.19) μM. Fe2+ content and reactive oxygen species production were increased and protein levels of SLC7A11 and GPX4 were decreased by eriodictyol. Besides, eriodictyol reduced the ratio of JC-1 fluorescence ratio, glutathione and malondialdehyde contents but elevated Cytochrome C level. Nrf2 phosphorylation were obviously downregulated by eriodictyol. Finally, eriodictyol suppressed tumor growth, aggravated mitochondrial dysfunction and downregulated Nrf2 expression in tumor tissue in mice.Eriodictyol regulated ferroptosis, mitochondrial dysfunction and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer.

Abstract 2499: Complement activation at the interface between cancer cells and adipocytes

Abstract Introduction: Ovarian cancer (OC) preferred site of metastasis is the fat-rich omentum. The interaction between cancer cells and adipocytes induces genotypical and phenotypical changes favoring increased invasiveness and metastatic potential of OC cells. In this study we stablish a novel in-vitro direct co-culture model by using immortalized human visceral pre-adipocytes and OC cells to understand the genotypical and phenotypical changes that adipocytes coculture generates over OC cells. Materials and Methods: OVCAR5 or OVCAR8 OC cells expressing either GFP or RFP were co-cultured with matured primary immortalized human visceral pre-adipocyte (VNPAD). The cells were seeded together in direct monolayer co-culture. After co-culture, OC cells were sorted by FACS and used to evaluate cell proliferation, resistance to cisplatin and transcriptomic changes. Results: Direct co-culture (3 days) of adipocytes and OC cells increased proliferation (p &amp;lt; 0.001), invasiveness (p &amp;lt; 0.005), and induced epithelial to mesenchymal transition (EMT) of OC cells relative to cells cultured alone. Co-culture with adipocytes also increased resistance to platinum (cisplatin IC50 from 3.90 µM to 7.07 µM for OVCAR5, and from 7.54 µM to 9.35 µM for OVCAR8 cells). RNAseq of co-cultured OVCAR5 cells detected 7783 differentially expressed genes (FDR&amp;lt;0.05, fold change &amp;gt;2). The most affected pathways included the PI3K/AKT pathway, inflammatory response, complement activation, and lipid localization. Activation of the complement (C) system in cells co-cultured with adipocytes was associated with upregulation of both C3 and C5 which was confirmed by quantitative RT-PCR, western blot and ELISA. Treatment of primary OC cells with media conditioned by adipocytes increased the expression of C3 and C5 (P&amp;lt;0.05). Similarly, treatment of OVCAR5 cells with a lipid mixture increased expression of C3 and C5 (mRNA and protein). The co-culture of OC cells with adipocytes and the treatment with lipids activated the PI3K pathway and the integrated stress response (ISR) pathway mediated by ATF4. shRNA knockdown of C3 and a chemical inhibitor for the C3 and C5 receptor (sb290157) reverted the increased proliferative phenotype and activation of PI3K in cocultured OC cells. Stable knockdown of C3 with shRNA in OVCAR5 cells reduced tumor volume and tumor weight in an intraperitoneal OC xenograft model (p&amp;lt;0.05). IHC of paired primary tumors and metastatic implants in the omentum showed increased C3 expression adjacent to fat (p&amp;lt;0.05). Conclusion: Co-culture of adipocytes and cancer cells drives changes in proliferation and cisplatin responsiveness in OC cells. These changes are induced by transfer of lipids from adipocytes to cancer cells and subsequent activation of the complement proteins C3 and C5 through the ISR pathway and ATF4. Citation Format: Andres Felipe Valdivia, Hao Huang, Horacio Cardenas, Aloysius Klingelhutz, Daniela Matei. Complement activation at the interface between cancer cells and adipocytes [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 2499.

FOXN3 inhibits the progression of ovarian cancer through negatively regulating the expression of RPS15A.

Ovarian cancer is the second most common cause of gynecological cancer death and has a high recurrence rate. FOXN3, a transcription inhibitor belonging to FOX family, has anti-tumor effects on several cancers. Bioinformatics analysis revealed that the expression of FOXN3 was downregulated in ovarian cancer specimens. However, the role of FOXN3 in ovarian cancer remains unclear. Herein, we investigated the role of FOXN3 in ovarian cancer using OVCAR3 and A2780 cells. Flow cytometry and CCK-8 analysis showed that overexpression of FOXN3 inhibited the proliferation and cell cycle progression of OVCAR3 cells. Cell invasion and migration abilities were decreased by FOXN3 according to transwell and wound healing assays. The suppression of FOXN3 on angiogenesis in OVCAR3 cells was evidenced by reduced vessel formation and VEGFA protein expression. Taken together, FOXN3 had an inhibitory effect on the proliferation, migration, invasion and angiogenesis of OVCAR3 cells, while its knockdown exhibited an opposite effect in A2780 cells. By inoculation of FOXN3-overexpressing cells into nude mice, tumorigenesis assay demonstrated that FOXN3 could delay the growth of ovarian cancer cells in vivo. The interaction between FOXN3 and RPS15A was preliminarily explored via dual-luciferases assay and ChIP. FOXN3 was confirmed to bind to the promoter (at - 1588/- 1581 and - 1476/- 1467) of gene RPS15A and inhibit its transcriptional expression. We further found that overexpression of RPS15A diminished the inhibition of FOXN3 on ovarian cancer cell malignant behaviors. These findings indicate that FOXN3 negatively regulates the expression of RPS15A and thus suppresses the progression of ovarian cancer.

Abstract 3181: A novel memory-like NK cell CAR targeting the proximal mesothelin domain shows promising pre-clinical activity in ovarian cancer using cell lines, patient-derived organoid tumors, and xenograft mice

Abstract Epithelial ovarian cancer (OVC) is the leading cause of death among gynecological malignancies. Natural killer (NK) cells are an attractive platform for immunotherapy owing to their superior safety, multiple mechanisms of cytotoxicity, and reduced risk of alloreactivity. Recently, paradigm-shifting studies have shown that brief activation of NK cells with inflammatory cytokines induces differentiation into cytokine induced memory-like (CIML) feature. CIML NK cells have demonstrated potent activity in preclinical and in recent clinical trial settings, suggesting CIML NK cells as a novel platform for genetically modified chimeric antigen receptor (CAR) cells for OVC therapy. Based on patient samples and patient-derived organoids (PDOs), we confirmed mesothelin (MSLN) as an attractive candidate for NK cells. Here, we designed and tested aMSLN CAR CIML NK cells for their functionality against OVC cell lines, PDOs, and xenograft mice. We designed the aMSLN CAR using the ScFv sequence against the proximal domain of MSLN with 4-1BB and CD3z as co-stimulatory domains. Primary NK cells were purified from peripheral blood, activated using IL-12, IL-18, and IL-15 resulting in CIML NK cells. The CAR gene was transduced into CIML NK cells via our optimized BaEV lentiviral system to achieve high transduction efficiency of 40-60%. The direct cytotoxic effect of NK cells was tested against OVC cell lines where the cells were co-cultured with NK cells for 6 hours and analyzed using apoptotic markers. Compared to CIML NK cells, aMSLN CAR CIML NK cells showed enhanced cytotoxicity against OVCAR3 (47.9% vs 14.8%), SKOV3 (29.3% vs 8.0%) and OVCAR8 (60.7% vs 39.8%) cells at a 10:1 ratio. In addition, the aMSLN CAR CIML NK cells exhibit long-term cytotoxicity when evaluated over a period of 3 days; killing efficiency of 71.7% vs 37.6% and 54.6% vs 32.8% for aMSLN CAR CIML NK cells compared to CIML NK cells in OVCAR8 cells and SKOV3 cells, respectively. To test the therapeutic potential of NK cells in a relevant in vitro model, we used PDOs. After 18-hour co-culture, the cytotoxicity was determined by evaluating apoptotic EpCAM+ cancer cells amongst the organoid cell population. Results showed that aMSLN CAR CIML NK cells caused increased apoptotic cells compared to CIML NK cells; 58.2% vs 11.0%, 4.5% vs 61.0%, and 10.5% vs 32.0% in three different PDOs. Finally, we tested the in vivo therapeutic efficacy of aMSLN CAR CIML NK cells against intraperitoneal OVCAR8-mcLuc model wherein we observed significant tumor control, with 80.86% tumor growth inhibition as measured by BLI after 2 doses of 5 million/animal NK cells. We are currently evaluating the functionality of aMSLN CAR CIML NK cells after their exposure to ascites from OVC patients as well as developing methods to enhance their function in highly immunosuppressive tumor microenvironment. Citation Format: Mubin Tarannum, Khanhlinh Dinh, Juliana Vergara, Oyku Ay, Sarah Hill, Rizwan Romee. A novel memory-like NK cell CAR targeting the proximal mesothelin domain shows promising pre-clinical activity in ovarian cancer using cell lines, patient-derived organoid tumors, and xenograft mice [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 3181.

Abstract 2005: FSHR-mediated targeted delivery of paclitaxel immunoliposomes in receptor overexpressing ovarian cancer cells

Abstract The purpose of this study was to explore the Fabʹ fragment of anti-FSHR (Follicle Stimulating Hormone Receptor) antibody as a targeting ligand to graft on the surface of paclitaxel (PTX) liposomes (PLs) for site-specific drug delivery in ovarian cancer (OC) cells. Anti-FSHR antibody Fab’ fragment conjugated PTX encapsulated immunoliposomes (ILs) were prepared, characterized, and evaluated to determine their in vitro efficacy in FSHR receptor positive (Caov3) and negative (SKOV3) OC cells. FSHR expression was determined by confocal microscopy and flow cytometry. The results confirmed that FSHR expression levels of Caov3 were higher and no expression was detected in SKOV3. ILs were prepared by covalent conjugation of Fab’ fragments to functionalized PLs via thioether linkage which was confirmed by SDS-PAGE analysis. Immunoreactivity of the prepared ILs was determined against Fabʹ in Caov3 cells using flow cytometry and it was observed that after conjugation, Fabʹ remained intact and also maintained the binding characteristic to the receptor. In MTT assay at the end of 48 h, the IC50 value for ILs was found to be 10.8 and 3.5 folds lower than Taxol® and PLs respectively in Caov3. However, being receptor negative, there was no significant difference noticed for IC50 when PLs and ILs were compared for SKOV3. To prove, whether immune tagging improves cell-specific uptake of ILs in FSHR-expressing cells, cell uptake studies were carried out using confocal microscopy and flow cytometry which showed higher uptake of ILs in Caov3 but not in SKOV3. For assessment of anti-angiogenic activity, a wound scratch assay was performed on Caov3 cells. At an equimolar concentration of treatment given to the cells by PLs and ILs, the % recovery of the wound was 1.3 and 2.3 times lower than Taxol® respectively indicating the superior performance of ILs in inhibiting cell proliferation. The quantitation of cell death after treatment was carried out by flow cytometry after staining with propidium iodide and the highest amount of cell death was seen in ILs group for Caov3 cells. Evaluation of cell cycle in the Caov3 indicated superior results of arrest of cells in G2/M phase. Further, an increase in cell death was observed as indicated by the increase in % of cells in subG1 stage. Performance of PLs was similar in Caov3 and SKOV3 leading to almost around 22% of arrest in G2/M stage, however, for ILs, the number of cells in G2/M was 28% for Caov3 cells at the end of 24 h indicating that the targeted formulation was able to better get internalized in the cells due to receptor-mediated uptake. The results highlight the importance of FSHR as a prominent target for OC therapy and the potential of anti-FSHR Antibody Fab' conjugated nanocarriers as a site-specific delivery system to reduce the limitations associated with OC chemotherapy. Citation Format: Priyanka Bhatt. FSHR-mediated targeted delivery of paclitaxel immunoliposomes in receptor overexpressing ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2005.

Abstract 686: Immunostimulatory gene therapy with oncolytic viruses potentiates the effect of paclitaxel and cisplatin in ovarian cancer

Abstract Ovarian cancer is commonly treated with debulking surgery, followed by a combination of a taxane and a platinum-based chemotherapy. However, &amp;gt;70% of patients relapse within 3 years with tumors resistant to platinum-based agents. 5-year survival is ranging from 30-40%. Thus, delaying recurrence of disease is a high unmet need in ovarian cancer. Combining the chemotherapy regime with immunostimulatory gene therapy utilizing oncolytic viruses as gene vehicles is an attractive treatment option targeting alternative pathways to induce tumor cell death, control tumor growth and prevent development of metastasis. LOAd703 is an oncolytic adenovirus derived from the LOAd platform of replication-restricted Ad5/35 adenoviruses. LOAd703 encodes for the human immunostimulatory genes trimerized, membrane-bound CD40L (TMZ-CD40L) and 4-1BBL, expressed under a CMV promotor which enables gene expression in both tumor cells and tumor stroma while replication is restricted to tumor cells. Herein, we investigated the effect of combining LOAd703 with paclitaxel and cisplatin in vitro and in vivo. Four human ovarian cancer cell lines (CAOV3, COV362, OVCAR3 and PEA2) were treated with LOAd703 alone, in combination with paclitaxel and/or cisplatin or were left untreated. LOAd703 efficiently killed the ovarian cancer cell lines by oncolysis, as evaluated by an MTS cell viability assay. This effect was further improved when LOAd703 was given in combination with paclitaxel and/or cisplatin. Flow cytometry revealed that the ability of LOAd703 to induce expression of the immunostimulatory transgenes TMZ-CD40L and 4-1BBL, was not negatively affected by the combination with paclitaxel and/or cisplatin. The combination treatment was further tested in an in vivo model where BALB/c nu/nu mice were xenografted with 5 million OVCAR3 cells. The mice were subsequently treated with LOAd703 (1 × 10e9 FFU/mouse), both alone as well as in combination with paclitaxel (6mg/kg) and cisplatin (2.5mg/mg). The treatments were given over the course of 4 weeks where LOAd703 was administered intratumorally twice per week while chemotherapy was given intraperitoneally once per week. The results showed that the combination of LOAd703 and the chemotherapeutics controlled tumor growth significantly better than either LOAd703 alone or the standard treatment with paclitaxel and cisplatin. In conclusion, LOAd703 efficiently killed ovarian cancer cell lines in vitro, and could potentiate the effect of standard-of-care chemotherapy. This effect was confirmed in vivo, in which the combination therapy had a better tumor control than the other treatment groups. A clinical trial is ongoing to confirm the effect of adding LOAd703 to different treatment regimes in ovarian cancer (NCT03225989). Citation Format: Sedigheh Naseri, Jonas Härdin, Emma Eriksson, Tanja Lövgren, Gustav Ullenhag, Angelica Loskog. Immunostimulatory gene therapy with oncolytic viruses potentiates the effect of paclitaxel and cisplatin 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 686.

Abstract 6182: Tumor treating fields (TTFields) concomitant with PARP inhibitors or carboplatin for treatment of ovarian cancer cell lines

Abstract Purpose/Objectives: Ovarian cancer continues to be the leading cause of death among gynecological malignancies. Platinum-based chemotherapy is recommended after surgery for most patients with ovarian cancer; and PARP inhibitors (PARPi) are suggested as maintenance therapy. Both therapies induce DNA damage that require the proper activity of the Fanconi Anemia (FA)- BRCA pathway for its resolution, and hence are most beneficial in cancers with a BRCA mutation. However, 75% of patients with ovarian cancer do not harbor BRCA mutations, and thus may experience limited benefit from these treatments. TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cancer cell death. Recently, TTFields have been shown to induce a state of BRCAness in various cancer types, and to be effective in ovarian cancer pre-clinical models. The objective of the current study was to examine the effect of TTFields concomitant with carboplatin or PARPi on ovarian cancer cell lines. Materials/Methods: A2780 (BRCA wild type) and OVCAR3 (BRCA mutated) ovarian carcinoma cells were treated with TTFields (72 h, 1 V/cm RMS, 200 kHz), alone or with concomitant application of carboplatin or the PARP inhibitors olaparib or niraparib. Efficacy was examined via measurements of cell count, colony formation, and induction of apoptosis. The overall effect was calculated by multiplying cell count with colony formation. Results: Application of TTFields to A2780 or OVCAR3 cells resulted in reduced cell count, increased overall effect, and elevated apoptosis. Carboplatin, olaparib, and niraparib each displayed dose dependent effects in both cell lines, with higher sensitivity demonstrated in the BRCA mutated cells. Concomitant application of TTFields with any of these drugs displayed a synergistic interaction in the BRCA wild type A2780 cells and an additive effect in the BRCA mutant OVCAR-3 cells. Conclusions: The data suggest potential benefits for TTFields concomitant with platinum-based chemotherapy and PARPi in ovarian cancer, even in the absence of background BRCA mutations, in accordance with the BRCAness state induced by TTFields. As such, TTFields may enhance the efficacy of treatment for ovarian cancer in both the adjuvant and maintenance settings. Citation Format: Antonia Martinez-Conde, Hila Ene, Roni Frechtel-Gerzi, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) concomitant with PARP inhibitors or carboplatin for treatment of ovarian cancer cell lines. [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 6182.

Abstract 2965: LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16

Abstract Background: MUC16 is a large glycoprotein overexpressed on cell membrane of solid tumors, especially in almost 80% of ovarian cancer. The extracellular part of MUC16 could be cleaved as CA125, which is well known as a diagnosis biomarker for ovarian cancer. Despite recent advances in therapies such as PARP inhibitors, advanced ovarian cancer remains a disease of high unmet need. Herein a novel bispecific antibody targeting MUC16 and CD3, LBL-033, is developed to specifically kill MUC16 positive tumor cells by engaging T cells. Methods: LBL-033 was constructed using our unique T cell engager platform, which is a 2:1 format bispecific antibody with two arms targeting MUC16 membrane-proximal domain with high affinity and one arm targeting CD3 with fine-tuned affinity. The binding affinity of LBL-033 to MUC16 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-033 was investigated in C57BL/6-huCD3e mice implanted with MC38-huMUC16 cells. GLP-compliant repeat-dose toxicologic study was evaluated in cynomolgus monkeys. Results: The binding affinity of LBL-033 to MUC16 and CD3 protein was 2.67 nM and 29.6 nM, respectively. The epitope that anti-MUC16 binds was identified at the C-terminal of MUC16 extracellular domain; and the activity was not affected in the presence of soluble CA-125. In CD3 reporter gene assay, LBL-033 could activate the NFAT reporter element through MUC16 antigen-dependent CD3 cross-linking with an EC50 value of 1.068 nM. LBL-033 was shown a potent TDCC activity in OVCAR3 cells with T cell activation. In C57BL/6-huCD3e transgenic mice implanted with MC38-huMUC16 tumor cells, LBL-033 was shown a dose dependent anti-tumor activity. In addition, LBL-033 had a significant synergistic effect, when combined with anti-PD-1. LBL-033 was shown favorable pharmacokinetic properties with a good safety profile in cynomolgus monkeys. Conclusion: LBL-033, a novel bispecific antibody targeting CD3 and MUC16 with a unique epitope, which is located at the MUC16 membrane-proximal domain, with affinity differentiation between anti-MUC16 and anti-CD3. It is shown a great anti-tumor efficacy in animal models, with a good safety profile in monkeys. These data support LBL-033 as a novel therapeutic bispecific antibody for ovarian cancer and other MUC16 positive tumors. A dose escalation study in humans will begin in Q1 2023. Citation Format: Jianming Sun, Xiao Huang, Yurong Qin, XingXing Fang, Min Chen, Huan Lin, Jing Guan, Jing Huang, Tongjun Liu, Baohui Wang, Guojin Wu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16 [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 2965.