The Cellular And Molecular Biological Characteristics Of Cancer Patient-derived Tumor Organoids And Their Possible Implication In Clinic Practice

Part Ⅰ The cellular and molecular characteristics of gastric signet ring cell carcinoma organoids and screening of sensitive chemotherapeutic drugsBackground:Signet-ring cell carcinoma(SRCC)is a special subtype of gastric cancer with significant histological and genetic heterogeneity,and obvious tumor infiltration,prone to lymph node and peritoneal metastasis.Most patients are in the middle and late stages when they were clinically diagnosed.At present,in addition to surgery,adjuvant chemotherapy is the main treatment for SRCC.However,there are many kinds of chemotherapy drugs,and how to choose an appropriate chemotherapy regimen is very important for patients.Therefore,there is an urgent need for reliable tumor models to screen sensitive drugs for personalized treatment strategies.In recent years,the 3D organoid culture technology has attracted much attention,which has gradually transitioned from basic research to clinical application of tumors.Using this technology,tumor tissue can be cultured in three dimensions,and the obtained organoids can maintain the histology and genetic characteristics of the primary tumor for a long time,which made up for the deficiencies of traditional tumor models.Aim:This study aims to construct a gastric cancer organoid model in vitro,to explore the similarities and differences between SRCC and Non-SRCC organoids,and to verify whether gastric cancer organoids can be used as personalized drug screening model.Methods:In this study,a total of 26 samples of gastric cancer patients who were admitted to the hospital for tumor resection from August 2020 to January 2021 in the Department of Pancreatic and Gastric Surgery,Cancer Hospital of the Chinese Academy of Medical Sciences were collected in this study.Collected tissues were cultured in vitro by organoid technology.we compared gastric cancer organoids and matched primary tissues by H&E staining,gastric cancer-related tumor marker expression and whole-exome sequencing.To verify whether SRCC and Non-SRCC organoids retained tumorigenicity by using tumorigenesis assay in mice.Finally,four chemotherapeutic drugs(5-fluorouracil,oxaliplatin,docetaxel and irinotecan)were selected for drug sensitivity experiments of organoids.Results:We successfully cultured 12 gastric cancer organoids using 3D organoid technology,including 4 SRCC organoids and 8 Non-SRCC organoids.Both SRCC and Non-SRCC organoids can be cryopreserved,resuscitated,and cultured for a long time.And their morphological structures remain unchanged.Both kind of organoids were found to reproduce the tissue and genetic properties of the primary tumor through detailed histological,whole-exome,and tumorigenicity analyses.The results of drug susceptibility experiments showed that gastric organoids exhibited heterogeneous responses to conventional chemotherapeutic drugs,and SRCC organoids may be more sensitive to docetaxel.Conclusions:Our data show that SRCC organoids can be successfully constructed in vitro.Both SRCC organoids and Non-SRCC organoids can reproduce the complexity and genetic heterogeneity of the primary tissue,and can be cultured in large quantities for a long time.This drug screening model could be used as an adjunct tool for personalized medicine treatment of gastric cancer in the future.Part Ⅱ Organoid cultures and molecular characteristics of circulating tumor cells in lung cancerBackground:Circulating Tumor Cells(CTCs)are tumor cell with high viability and high metastatic potential that invade blood vessels from primary solid tumors and shed into peripheral blood.The dissemination of CTCs through blood to distant metastases is the main cause of death in most cancer patients.However,due to their small numbers and potential biological differences,current studies are mostly limited to CTC counting.So,the expansion of CTCs is essential to obtain their molecular and functional characterization.3D organoid culture technology is a very useful tool which can mimic the growth environment in vivo and preserve the histological and genetic properties of tumors.If CTC organoids can be successfully cultured,the biogenetic information and tumor invasion mechanisms of CTCs will be gradually discovered,laying a great foundation for the development of anti-metastatic drugs.Aims:To construct a 3D organoid culture system by isolating and enriching CTCs derived from pulmonary venous blood.The histological characteristics of CTC organoids were observed by H&E staining,immunohistochemistry and immunofluorescence.Single-cell sequencing was used to compare the differences between CTC organoids and lung cancer tissue organoids,for exploring the molecular typing and functional characteristics of CTCs.Methods:In this study,106 patients with pulmonary venous blood who underwent lobectomy for lung cancer from August 2020 to January 2022 in the Department of Thoracic Surgery,Cancer Hospital,Chinese Academy of Medical Sciences were collected.And the clinical information was all recorded in detail.CTCs were isolated and enriched by density gradient centrifugation and cultured in by 3D organoids technology.The obtained CTC organoids were verified by H&E staining and expression of lung cancerrelated tumor markers to determine whether the organoid model is suitable for CTC expansion in vitro.Single-cell sequencing of cultured CTC organoids and tissue-derived organoids was performed to analyze their differences and explore the characteristics of CTC organoids at the cellular transcriptional level.Results:We successfully cultured 10 CTC organoids,including 8 cases of lung adenocarcinoma,1 case of lung squamous cell carcinoma,and 1 case of small cell lung cancer.We used H&E,immunohistochemistry,and immunofluorescence techniques to phenotype CTC organoids and found that pulmonary vein blood-derived CTC organoids could reproduce some basic characteristics of lung cancer.After integration of single-cell data and dimensionality reduction clustering annotation,we divided cells into 6 subsets:basal cells,FOXN4+cells,secretory cells,ionocyte cells,ciliated cells and PCDHA+cells.Compared with tissue-derived organoids,the proportion of basal cell in CTC organoids was significantly higher,while secretory cells were significantly lower.Analysis of basal cells revealed that it is closely related to the development and differentiation of epithelial cells.Conclusions:We successfully constructed a 3D organoid culture system of CTCs derived from pulmonary venous blood from patients,which can maintain the characteristics of CTCs and also reflect its heterogeneity.Basal cells subsets of CTCs may be closely related to the metastasis of lung cancer.