Development And Application Of A Hydrogel-Based Model Of Patient Derived Colorectal Cancer Organoids

Colorectal cancer is the fourth leading cause of mortality due to malignancy in the world[1].The molecular targets of colorectal cancer and individualized therapy remain to be explored.Therefore,one of the most important fields of research is the development of patient proximal models for colorectal cancer research.Patient-derived organoid model(PDO)has been gaining increasing attention and traction in the recent years.When compared with the tumor cell lines and tumor xenografts(PDX)models,PDOs retain the tumor heterogeneity,can be derive with high success rates,have a short development cycle,can be used for high-throughput screening and genomic modifications[2]-[4].However,there are some disadvantages of the current PDO models.Current PDO models are almost entirely based on animal-derived 3D matrices,including Matrigel and collagen.These matrices consist of complex and variable compositions,and not conducive to controlled modifications[5]-[6].Moreover,the animal-derived matrix poses a risk of pathogen transfer,which makes them unsuitable for many downstream clinical applications and therapy[7].Another main disadvantage of current PDO is that the organoids are composed solely of epithelial cells without any tumor microenvironment components such as stromal cells,which hinders its application in immune modulation and tumor metastasis.Therefore,it is important of explore the possibilities to use synthetic matrix for organoid culture to greatly expand the applicability of using PDO models in basic and clinical research.Herein,we explore the use of synthetic hydrogel as a 3D matrix to replace animal-derived Matrigel or collagen for organoid culture.We also demonstrate the applicability of the hydrogels in developing a co-culture model for tumor PDO with cancer-associated fibroblasts,and explore its preliminary clinical applications.Methods1.The development of patient derived colorectal cancer organoid modelMorphological observation,Viability measurement and immunofluorescence staining were used to analyze and compare the efficacy,yield,cell activity and proliferation ability of trypsin method,collagenase method and EDTA method to isolate the patient derived colorectal cancer organoids.The effect of cellulose sponge and hydrogel as a 3D matrix for the culture of colorectal cancer PDO was also studied.2.Characterization of the hydrogel-based model of patient derived colorectal cancer organoidsThe hydrogel-based model of patient derived colorectal cancer organoids was characterized by morphological observation and cell viability evaluation;immunofluorescence staining revealed the cell components of the PDO model;3.RNA-Seq and whole exome sequencing was performed to evaluate the gene expression profiling of hydrogel based PDO model with its corresponding tumor tissueTotal RNA and DNA was isolated from the hydrogel based PDO model and also from its corresponding tumor tissue.RNA-Seq and whole exome sequencing was performed to evaluate the gene expression and mutation profiling.Characterization of tumor heterogeneity and variation of genetic mutation profile of the hydrogel based PDO were compared to corresponding tumor tissue to identify the similarities in the gene expression.4.Preliminary study on the use of the PDO model and PDO-cancer associated fibroblasts(CAFs)co-culture model for individualized drug sensitivity testingImmunofluorescence staining and CCK8 experiment were performed to characterize the co-culture model of PDO and CAFs.Preliminary drug testing with PDO model and co-culture model were performed for determining their sensitivity to 5-fluorouracil and irinotecan.Results1.PDO model was developed using collagenase method and hydrogel culture:LIVE/DEAD? Viability/Cytotoxicity experiments and immunofluorescence staining showed that collagenase method is superior than trypsin method and EDTA method to isolate the patient derived colorectal cancer organoids.Hydrogel culture can preserve the morphological characteristics,yield and cell activity of the isolated organoids.2.The synthetic hydrogels can be used as the PDO in vitro culture matrix:The hydrogel-based model of patient derived colorectal cancer organoids preserve the histopathology of the parental tumors and maintain good cell viability and tumor cell proliferation activity.3.The hydrogel based PDO models highly captured the genomic features of its corresponding primary tumor tissues in gene expression and mutation,and much of the genomic diversity of CRC.1)The Pearson correlation coefficienct between matched tissue and PDO for nine cancer-related pathways and the overall cancer pathway was more than 0.8.2)Overlapping of SNP and INDEL located in exons between PDO and matched tissue were 87%and 76%,respectively.4.Synthetic hydrogel can be used as 3D matrix for the co-culture of PDO and CAFs;5-fluorouracil and irinotecan drug testing results demonstrated that the tumor organoids cultured by hydrogel could be used as a preclinical model for drug testing in vitro.Co-culture with CAFs may reduce the sensitivity of tumor cells to certain antitumor drugs.ConclusionIn conclusion,this study explores the use of synthetic hydrogel as a 3D matrix to replace animal derived matrix for the feasibility of the organoid culture.Cell morphology,functional data and gene expression profile were studied,demonstrating the feasibility and utility of hydrogel based organoid technology for investigating the molecular basis of drug response and serves as a preclinical model for colorectal cancer research.Furthermore,this study also establishes a PDO and CAFs co-culture model to better simulate the tumor microenvironment in vivo.We believe that the co-culture model will be important to fully exploit the potential of PDO for predicting patient responses and research on immune modulation and tumor metastasis.