Construction Of Patient-derived Ovarian Tumor Organoids Growth In Hydrogels

Although treatments for ovarian cancer have improved dramatically over the past few years,the disease remains a major health problem affecting women.According to statistics,～22,000 women worldwide die of ovarian cancer every year.The difficulty in developing treatments for ovarian cancer lies primarily in the inherent dynamics and complexity of tumors,and the lack of in vitro models that can replicate tumor physiology in vivo.Although simplicity,traditional two-dimensional cell culture models have limitations in simu lating the tumor microenvironment and accurately predicting patient response to therapy.Patient-Derived Xenograft(PDX)models can maintain the biology and pathophysiology of parental tumors,but the high cost and long cycle limit the widespread use of PDX models.In recent year,the patient-derived tumor organoid(PTO)model has been developed.PTO is a three-dimensional in vitro model that can better simulate the structure and function of the original tumor tissue.PTO is very promising for drug screening and personalized precision medicine.In addition to tumor epithelial cells,there are cancer associated fibroblasts(CAF)and immune cells in the tumor microenvironment(TME).CAF not only plays a role in remodeling the tumor extracellular matrix,but also secretes growth factors and cytokines to promote the growth of tumor cells.Immune cells,such as T cells,natural killer(NK)cells,and macrophages,can modulate tumor responses to therapy.However,the most PTO contains only tumor epithelial cells,which cannot reflect the diversity and complexity of cells in the TME.The use of co-cultured models of CAF,immune cells,and tumor models can provide insight into the complex interactions in the TME and help to accurately develop new approaches to therapy.The challenge in developing a PTO co-culture model lies in the determination of co-culture conditions and the identification of markers of cell-cell interactions.In this dissertation,we successfully established PTO in vitro in the hydrogels from patient-derived ovarian cancer tissues.The co-cultured models of CAF,NK cells,and tumor cells were established to study the interactions in the TME.The specific content is as follows:In the second chapter,through using three-dimensional culture technology in hydrogels,the primary tumor single cells from patients were cultivated into PTO and the ovarian cancer PTO biobank was established.Comparing PTO with normal ovarian organoids,morphological differences were revealed.Live/Dead staining provides important insight into the viability of PTO,which is well-viable and most cells survive.H&E staining confirmed that PTO had a highly similar histological structure and morphology to parental tumors.Immunohistochemical staining of tumor-related markers(Ki-67,E-cadherin,PAX8,p53,and ca125)showed that the expression of PTO was highly similar to that of the parental tissue.These results confirm that PTO is a reliable in vitro primary tumor model that preserves its original pathological features and heterogeneity between patients.In the third chapter,immunohistochemical staining of CAF biomarker α-SMA was used to identify CAF in tumor tissues.Differences in adhesion were used to separate CAF from tumor cells,and CAF were identified by immunofluorescence staining forα-SMA.The co-culture of tumor cells and CAF in Transwell model showed that CAF can promote the growth of PTO,and the content of cytokines IL-6 and TGF-β1 in the co-culture supernatant was significantly increased.NK cells were added to the coculture environment and CAF was found to reduce the effectiveness of NK cells in mediating tumor killing.The PTO established in this paper provides a reliable in vitro three-dimensional model for basic research on ovarian tumor biology,and provides ideas for cellular immunotherapy and drug screening.