Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ . Method: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. Conclusion: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype. There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations upregulated several anterior HOX genes and downregulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment upregulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

PIEZO1 and PIEZO2 are mechanosensitive cation channels which are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in multiple organ systems. Within the musculoskeletal system, PIEZO1 functions to maintain muscle and bone mass, sense tendon stretch, and regulate senescence and apoptosis in response to mechanical stimuli within cartilage and the intervertebral disc. PIEZO2 is essential for transducing pain and touch sensations as well as proprioception in the nervous system, which can affect musculoskeletal health. PIEZO1 and PIEZO2 have been shown to act both independently as well as synergistically in different cell types. Conditions that alter PIEZO channel mechanosensitivity, such as inflammation or genetic mutations, can have drastic effects on these functions. For this reason, therapeutic approaches for PIEZO-related disease focus on altering PIEZO1 and/or PIEZO2 activity in a controlled manner, either through inhibition with small molecules, or through dietary control and supplementation to maintain a healthy cell membrane composition. While many opportunities to better understand PIEZO1 and PIEZO2 remain, the studies summarized in this review highlight how crucial PIEZO channels are to musculoskeletal health and point to promising possible avenues for their modulation as a therapeutic target.

Dysregulation of mechano-transduction via chondrocyte pericellular matrix (PCM) has been shown to induce osteoarthritis (OA), a highly prevalent degenerative disease of joint tissues. By performing exome sequencing in symptomatic OA patients, a high impact mutation in COL6A3 encoding one of the monomeric sub-unit-3 of collagen type VI (COLVI) was identified. COLVI is an essential and specific protein functioning in the PCM. To study the downstream effects of the mutation in interaction with hyper-physiologic mechanical loading conditions, genetically COL6A3-edited human induced pluripotent stem cells (hiPSCs) were employed in our established in-vitro cartilage organoid model. We showed mutated COLVI had significant lower binding to fibronectin (FN) and provoked an osteoarthritic chondrocyte state. Moreover, the COL6A3 mutation abolished the initial stress responses of chondrocytes to hyper-physiological mechanical loading conditions. Together, the established sustainable cartilage organoid model provides unique opportunities to study the transduction of mechanical cues to chondrocytes and explore ways to counteract effects. Acquired insights could guide effective treatment development for OA

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype. There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations upregulated several anterior HOX genes and downregulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment upregulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

Introduction: Epithelial ovarian cancer comprises approximately 90% of all cases of ovarian cancer and is the leading gynecologic cause of death in Western societies. Ovarian cancer patients typically respond well to first-line platinum and taxane-based chemotherapies; however, approximately 80% of these patients relapse and succumb to more chemoresistant disease. Relapse and development of chemoresistance is linked to the presence of a rare population of chemoresistant cells, termed cancer stem-like cells (CSCs), which are enriched in spheroids in the malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Here we implement a 3D model of the development of chemoresistance in ovarian cancer to investigate the functional and genetic changes that occur as chemoresistance develops. Using this model to gain insight into the development of chemoresistance in ovarian cancer will facilitate the development of more effective treatments. Methods: To investigate the development of chemoresistance in ovarian cancer, we utilized an in vitro model using the 3D hanging drop spheroid platform with an ovarian cancer cell line, two primary patient samples. In our model, spheroids were serially passaged every 7 days and evaluated for proliferation and response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression CSC markers ALDH and CD133 as well as tumor initiating capacity were analyzed. RNA-sequencing and qRT-PCR was performed on spheroids from early (P0), middle (P3), and late (P6) passages for two patient samples to establish gene signatures associated with the evolution of stemness, tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cell proliferation, enriched CSCs, and emergence of a platinum-resistant phenotype with passaging. Contrarily, serial passaged spheroids were enriched for ALDH and consequently exhibited greater sensitivity to ALDH1A inhibitor. Furthermore, in vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSC proportions, compared to early passage spheroids, validating the increased proliferative capacity predicted by our in vitro serial passage platform. RNA-sequencing revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Finally, our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimental data. Conclusions: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence of chemoresistance in ovarian cancer in a controllable and reproducible format. Citation Format: Pooja Mehta, Micheal Bregenzer, Maria Ward Rashidi, Shreya Raghavan, Elyse Fleck, Eric Horst, Zainab Harissa, Visweswaran Ravikumar, Samuel Brady, Andrea Bild, Arvind Rao, Ronald Buckanovich, Geeta Mehta. Serially passaging ovarian cancer spheroids as an in situ model for emergence of chemoresistance and enrichment of cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A52.

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