Chiara Battistini

Navitoclax, a Bcl-2/xL Inhibitor, and YM155, a Survivin Inhibitor, in Combination with Carboplatin, Effectively Inhibit Ovarian Cancer Tumor Growth

Abstract High-grade serous ovarian cancer is generally treated with upfront chemotherapy, including carboplatin. The persistence of platinum-resistant cells drives recurrent disease. A high-throughput screen using a 3D organotypic culture assembled with extracellular matrix, primary human fibroblasts, and mesothelial cells was established and validated. Using a library of FDA-approved drugs, the 3D high-throughput screen was performed with the goal of identifying a combination of drugs that synergistically target two populations of ovarian cancer: aldehyde dehydrogenase (ALDH) high (ALDHhi) and ALDH low (ALDHlo) enzyme activity cells, which are less sensitive to carboplatin treatment than the bulk ovarian cancer cells. Initial results showed that omipalisib, verteporfin, CA3, mitoxantrone, navitoclax, venetoclax, and YM155 had significant single-drug activity in either the ALDHlo or both the ALDHlo/ALDHhi cell populations. Synergistic drug activity was identified with three drug combinations: navitoclax/omipalisib, navitoclax/YM155, and YM155/omipalisib. In vitro, the combination of navitoclax/YM155 was most efficient at blocking primary human ovarian cancer sphere formation and the proliferation of four different ovarian cancer cell lines in the 3D organotypic culture. In vivo, the combination of navitoclax/YM155/carboplatin decreased ovarian cancer metastasis, decreased the percentage of ALDHhi ovarian cancer cells in tumors, and increased survival when compared with carboplatin treatment alone in xenograft models. Our results suggest that the combination of navitoclax/YM155/carboplatin has promise as a therapy for treating ovarian cancer.

Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness

AbstractIn ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Ovarian Cancer Cells in Ascites Form Aggregates That Display a Hybrid Epithelial-Mesenchymal Phenotype and Allows Survival and Proliferation of Metastasizing Cells

Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Cancer cells float in peritoneal fluid, named ascites, together with a definitely higher number of non neo-neoplastic cells, as single cells or multicellular aggregates. The aim of this work is to uncover the features that make these aggregates the metastasizing units. Immunofluorescence revealed that aggregates are made almost exclusively of ovarian cancer cells expressing the specific nuclear PAX8 protein. The same cells expressed epithelial and mesenchymal markers, such as EPCAM and αSMA, respectively. Expression of fibronectin further supported a hybrid epithelia-mesenchymal phenotype, that is maintained when aggregates are cultivated and proliferate. Hematopoietic cells as well as macrophages are negligible in the aggregates, while abundant in the ascitic fluid confirming their prominent role in establishing an eco-system necessary for the survival of ovarian cancer cells. Using ovarian cancer cell lines, we show that cells forming 3D structures neo-expressed thoroughly fibronectin and αSMA. Functional assays showed that αSMA and fibronectin are necessary for the compaction and survival of 3D structures. Altogether these data show that metastasizing units display a hybrid phenotype that allows maintenance of the 3D structures and the plasticity necessary for implant and seeding into peritoneal lining.

Quantitative Proteomics and Phosphoproteomics Analysis of Patient-Derived Ovarian Cancer Stem Cells

High-grade serous ovarian carcinoma (HGSOC) is the deadliest gynecologic cancer. Key to the progression and ultimate lethality of this subtype is the intra-tumoral heterogeneity, which is defined as the coexistence of different cell types and populations within a single tumor. Among those, ovarian cancer stem cells (OCSCs) are a distinct subpopulation of tumor cells endowed with stem-like properties, which can survive current standard therapies, resulting in tumor recurrence. Here, we generated ex vivo primary OCSC-enriched three-dimensional (3D) spheres from 10 distinct treatment naive patient-derived adherent (2D) cultures. We used state-of-the-art quantitative mass spectrometry to characterize the molecular events associated with OCSCs by analyzing their proteome and phosphoproteome. Our data revealed a stemness-related protein signature, shared within a heterogeneous patient cohort, which correlates with chemo-refractoriness in a clinical proteomics dataset. Moreover, we identified targetable deregulated kinases and aberrant PDGF receptor activation in OCSCs. Pharmacological inhibition of PDGFR in adherent OC cells reduced the stemness potential, measured by sphere formation assay. Overall, we provide a valuable resource to identify new OCSC markers and putative targets for OCSC-directed therapies.