Ex Vivo Culture Of Organoids Derived From Gastric Malignant Ascites And Preliminary Study On The Mechanism Of The Organoids Growth Promotion By Ascites

Background and objectiveGastric cancer is still one of the leading causes of cancer death in China.Peritoneal metastasis and malignant ascites are responsible for high mortality of advanced gastric cancer patients.At present,the relevant mechanism is unknown and the clinical therapeutic effect is poor.One of the reasons is a lack of ex vivo model for understanding mechanisms associated with dissemination of exfoliated cancer cells in ascites.Patient-derived organoids(PDOs),a novel 3-D cultural system in vitro,can recapture the characteristics of parent cancer cells.PDOs derived from multiple kinds of tumors have been established and they are devoted to clinical drug screening.However,there have been no reports on the culture of organoids derived from malignant ascites(MA)yet.Here we established cultures of MA-derived organoids(MADO)for disease modelling and identify organoids in terms of histopathology and genomics.Furthermore,we noticed that supernatant of malignant ascites could enhance the growth of organoids.In order to further understand the molecular biological mechanism of peritoneal metastasis in gastric cancer,we preliminarily explored the possible mechanism of this phenomenon in aspects of exosome and cell signaling pathways.Methods1.Malignant ascites of gastric cancer was collected to generate malignant ascites derived organoids(MADOs).Steps for MADOs culture: ascites supernatant and cell sediment were collected after centrifugation.Every 400 cell clusters or 1000-2000 single cells were resuspended in 50 μl growth factor reduced Matrigel.After the Matrigel was solidified on prewarmed 24-well cell culture plate at 37 ℃,500 μl of complete organoid medium was added to each well and plates transferred to 37℃/5% CO2 incubators.Culture medium was subsequently refreshed every 3 days.2.Identification was conducted after successful cultivation:(1)Histopathology: H&E staining and immunohistochemistry.(2)Genomics: Whole-exome sequencing.3.Exosomes were isolated from the malignant ascites by ultracentrifugation and were identified by Western Blot.Exosomes,ascites supernatant and ascites supernatant without exosomes were used to treat MADOs and the total growth area of MADOs were qualified.Immunofluorescent staining then was used to test non-phosphorylated ?-catenin in MADOs.4.The expression level of STAT3 in SNU5 and SNU16 cells of groups treated with or without ascites supernatant were detected by Real-time PCR.Meanwhile,Western Blot was used to detect expression level of STAT3 and ?-catenin.After SNU16 cells were treated with Stattic,a phosphorylation inhibitor of STAT3,cell cycle was detected by flow cytometry and Western Blot verified that phosphorylated STAT3 were completely blocked.Results1.A total of 36 surgical specimens were collected and 49 MADOs were established with the successful rate of 95%(the sample collection and culture were repeated three times in two patients and two times in nine patients).The results showed good concordance between MADOs and MA tumor cells in histopathology and genomics.2.Adding exosomes derived from malignant ascites did not affect growth of MADOs,while the ascites supernatant without exosomes could promote the growth of MADOs which is similar to complete ascites supernatant.Immunofluorescent staining and Western Blot showed that non-phosphorylated ?-catenin was accumulated in MADOs after being stimulated by ascites supernatant with or without exosomes.3.Real-time PCR results showed that the transcription of STAT3 in SNU5 and SNU16 cells were accelerated when they were treated with ascites supernatant.Meanwhile,Western Blot results showed that expression of both STAT3 and β-catenin was significantly enhanced.Flow cytometry testing showed that severe G2/M phase arrest in SNU16 cells after being treated by Stattic,regardless of being treated by ascites or not.Conclusions1.we successfully established tumor organoids derived from gastric malignant ascites.These models can recapitulate characteristics of MA cancer cells.2.Malignant ascites-derived exosomes are not contributed to promoting the growth of MADOs.3.Certain substances in the ascites supernatant can activate the WNT/ ?-catenin and STAT3 signaling pathways in tumor cells.