Speaker
Description
Introduction
Ovarian cancer (OC) is the leading cause of death among gynecological malignancies, primarily due to its high mortality rate and frequent recurrence. The standard treatment, platinum-based chemotherapy, often becomes ineffective as patients develop platinum resistance, a process associated with enhanced metastatic potential, epithelial-mesenchymal transition (EMT), and angiogenesis. Notch signaling, particularly through Notch3, plays a critical role in the development of chemoresistance in OC, contributing to cancer stemness, EMT, and tumor-stroma interactions. Additionally, Pin1 has been implicated in stabilizing the Notch3 intracellular domain, modulating its processing and degradation, which further promotes chemoresistance.
Methods
To investigate the role of Notch signaling in OC chemoresistance, we developed a 3D biofabricated tumor model that mimics the ovarian cancer microenvironment. Tumoroids were generated from Kuramochi ovarian cancer cells, where Pin1 expression was silenced via targeted knockdown. The cells were bioprinted into GelMA/alginate (5%/4%) constructs using extrusion-based techniques, optimized for stability and cell viability. Western blot analysis confirmed efficient Pin1 silencing at the protein level. The optimal carboplatin concentration was determined using MTT assays to select sublethal doses that induced cytotoxicity without compromising the integrity of the constructs. Immunofluorescence staining was performed to evaluate tumoroids proliferation (Ki67), cytoskeletal organization (phalloidin), ovarian cancer-specific markers (WT-1, PAX-8), and extracellular matrix deposition (fibronectin).
Results
The biofabricated constructs supported the formation of viable, structured tumoroids with sustained proliferation and high-grade serous OC marker expression. Bioprinted constructs properties allowed for long-term culture post-printing. Pin1 knockdown was confirmed by western blotting, and MTT assays demonstrated a dose-dependent response to carboplatin. Stromal components, including mesothelial and endothelial cells, were successfully integrated into the constructs, recapitulating the spatial tumor-stroma organization of OC.
Discussion
This study demonstrates the potential of biofabricated 3D models for replicating the ovarian tumor microenvironment and studying chemoresistance mechanisms. Pin1 silencing allowed for further investigation into its role in stabilizing Notch3 and enhancing invasive tumor behavior. Future work will expand this platform with patient-derived OC organoids and cancer-associated fibroblasts (CAFs) to evaluate combination therapies targeting the Notch-Pin1 axis and to further dissect the molecular mechanisms driving angiogenesis and metastasis in OC. This model could provide a valuable tool for preclinical personalized therapy screening in platinum-resistant ovarian cancer.
References
Giuli MV et al. Notch signaling in female cancers: a node to overcome drug resistance. Cancer Drug Resist. 2021;4(2):327–342.
Franciosa G et al. Pin1 regulates Notch3 expression and T-ALL progression. Oncogene. 2016;35(36):4741–4751. doi:10.1038/onc.2016.5
3.Int J Mol Sci. 2022;23(9):4849.
Lucà R et al. MDM4 inhibition of mTOR reduces ovarian cancer metastasis. Cell Death Dis. 2022;13:367.
Acknowledgments
This research is funded by the Italian National Recovery and Resilience Plan (PNRR), Mission 4, Component 2, Investment 1.5, funded by the European Union – NextGenerationEU.
Disclosure
The authors declare no conflict of interest
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