A Research On The Methods Of Three-dimensional Chemosensitivity Test For Personalized Therapy For Liver Cancer

Background:Liver cancer is one of the most common malignant tumors in the world,and it is also the second leading cause of death associated with malignant tumors.Chemotherapy is the most commonly used method for nonsurgical treatment of liver cancer.However,there is still no clear evidence that any chemotherapy regimen is superior to others.Due to the presence of extensive heterogeneity of tumor,clinicians often rely on personal experience in selecting chemotherapy regimens,which has led to uncertainty about the efficacy of chemotherapy for liver cancer.Relevant data shows that the overall response rate of empirical chemotherapy for liver cancer is even less than 30%.Recently,driven by the "precision medicine" model,personalized therapy for liver cancer has attracted increasing attention.A series of clinical studies have confirmed the importance of chemosensitivity test(CST)in guiding personalized therapy for liver cancer.However,in the absence of high-quality evidence,most researchers still remain cautious about the application of chemosensitivity test in clinical practice.Traditional chemosensitivity test for liver cancer mostly use two-dimensional(2D)culture method,which fails to simulate the microenvironment of tumor cells in vivo.Moreover,there is a lack of uniform technical standards for chemosensitivity test of liver cancer,and the criterias for evaluating chemoresponsiveness are various in different institutes.Herein,chemosensitivity test based on three-dimensional(3D)culture system and an accurate and reliable criteria of chemoresponsiveness evaluation may suggest an important avenue of future directions for personalized therapy for liver cancer.Objective:To develop a low-cost,simple and reliable 3D chemosensitivity test,providing important guidance for personalized therapy for liver cancer.Methods:Chapter ? Establishment and evaluation of three-dimensional culture systemTwo commercial 3D culture systems(GravityPLUS and AggreWell)were used as reference to design the system,and the mould of the system was drawn with SOLIDWORKS 2016 software.The 3D culture system was finally constructed by using 3D printing technology and casting method.C3A cells were then cultured in the 3D culture system,compared to traditional 2D culture group.The morphological changes of the two groups were observed dynamically,and the levels of ALB,ALT,AST,BUN and LDH were detected at the same time.Chapter II Three-dimensional chemosensitivity test for primary human hepatoma cellsPrimary human hepatoma cells were isolated and purified by collagenase digestion and percoll density gradient centrifugation.Trypan blue exclusion method and immunofluorescence staining were used to count and identify the cells.Dissociated cells were cultured in the 3D culture system constructed in Chapter I,using traditional 2D culture group as control.The morphologic changes of the two groups were observed dynamically.Then cells were treated with eight chemotherapy regimens respectively,and the viability of the cells was detected by the CCK-8 method.Chemoresponsiveness was subsequently evaluated comprehensively by three indexes:the sensitivity grade,the sensitivity index and the combination index.Results:Chapter ? Establishment and evaluation of the three-dimensional culture systemThe 3D culture system maded by PDMS had good uniformity,and the microwells were spherical,without obvious serrated.C3A cells aggregated into spheroid in three days after inoculation.The diameter of the spheroid was dependent on seeding density,which could finally reach to 400?m and filled the microwells.Secretory function detection of C3 A cells showed that the levels of ALB and BUN of 3D culture group were significantly higher than that of 2D culture group,reflecting synthesis function and detoxification function of the former were better than that of the latter;and as the most sensitive damage indexes of the liver,the levels of ALT,AST and LDH of 3D culture group were lower than that of 2D culture group.Chapter ? Three-dimensional chemosensitivity test for primary human hepatoma cellsThe viability and purity of the two primary human hepatoma cells were more than 90%and 85%,respectively.The two primary human hepatoma cells were able to aggregate into spheroids in three days after inoculation into the 3D culture system,and most of the scattered cells would eventually migrate to the microwells.Chemosensitivity test for one of the primary human hepatoma cells(ID:2652968)showed that 2D culture group was highly sensitive to the combined regimen of ADM plus DDP,while 3D culture group was resistant to all of the chemotherapy regimens.Chemosensitivity test for the other primary human hepatoma cells(ID:2685241)showed that the combined regimen of ADM plus DDP was the most sensitive regimen for 2D culture group,while the combined regimen of GEM plus OXA was the most sensitive regimen for 3D culture group.Conclusion:In this paper,the 3D culture system constructed by using 3D printing technology and casting method was superior to the traditional 2D culture system,which was especially suitable for spheroid culture of human primary hepatoma cells.And combining the CCK-8 method with several chemoresponsiveness indexes including the sensitivity grade,the sensitivity index and the combination index,could successfully evaluate the sensitivity of human primary hepatoma cells to different drugs.In our experiment,there were significant differences in the results of chemosensitivity test for primary human hepatoma cells between 2D culture group and 3D culture group.The sensitivity of 3D culture group to the same chemotherapy regimen was generally lower than that of 2D culture group,which was consistent with the literatures.This method had the advantages of cost-effectiveness,simple operation,good reusability and suitability for high throughput drug screening,with improved accuracy and stability,thus it might provide legitimate information to guide personalized therapy for liver cancer.