Organoid Culture And Chemotherapy Drug Screening For Esophageal Squamous Cell Carcinoma

Objective 1.To understand the growth characteristics of esophageal squamous cell carcinoma cells-derived organoids,and to observe the changes in cell morphological characteristics of the organoids.2.To study the changes of Ki67 expression after treated with albumin bound paclitaxel,paclitaxel,cisplatin and 5-Fu.3.To explore the expression changes of CD44,CD54,CD133,ABCG2,CXCR4,CD271 and CD90 markers in tumor stem cells.4.To explore the changes of m RNA expression of stemness related genes SOX2,OCT4,KLF-4,c-myc,ALDH-1 and LGR5 in tumor cells.Methods Two subtypes of moderately differentiated K140 and highly differentiated K30 squamous cell carcinomas were used for organoid culture in DMEM/F12 medium,and the growth of 3D organoid spheroids and the morphological changes of tumor cells were observed under phase contrast microscope.HE staining was performed to observe cell morphological characteristics of organoids derived from K140 and K30 cells.The IC50 s of albumin bound paclitaxel,paclitaxel,cisplatin and 5-Fu drugs were determined by Cell Titer Glo 3D Kit.By IHC staining,the changes of Ki67 expression before and after drug administration in the organoid tissues were analyzed.Changes in the expression of surface markers CD44,CD54,CD133,CD338,CD184,CD271,and CD90 in tumor cell organoid cultures after chemotherapeutic drug treatment were detected by flow cytometry.The expression changes of stemness genes SOX2,OCT4,KLF-4,C-MYC,ALDH-1 and LGR5 in the tumor cell organoid culture before and after drug administration were detected by q RTPCR experiment.Results 1.Growth characteristics and morphological characteristics of esophageal squamous cell carcinoma cell-derived organoids.Esophageal cancer cell line subtypes K140 and K30 cells were successfully cultured into organoids,and their growth characteristics and growth curves were observed under an inverted microscope.The growth cycle of K140 cell-derived organoids is about 11-12 days,and the growth rate is relatively fast.The growth cycle of K30cell-derived organoids is about 13-14 days,and the growth rate is relatively slow.Tumor cellderived organoids were stained with H&E.Staining of organoids derived from K140 tumor cells showed deep staining of tumor cells and large nuclei.The nuclei of K30 cell-derived organoids were lightly stained,and the nuclei were slightly smaller and mostly mononuclear.3.Viability and IC50 of tumor cells after drug treatment.The organoids were treated by different concentration gradients of drugs,and the ATPase activity in tumor cells was measured by Cell Titer Glo 3D reagent,and the half-inhibitory concentration of various drugs was calculated.Experiments on K140-derived organoids showed that the IC50 results of albumin bound paclitaxel,paclitaxel,cisplatin and 5-Fu chemotherapy drugs were 0.5633(nmol/m L),0.001532(nmol/m L),13.63(nmol/m L),3.421(nmol/m L).Experiments were performed on K30-derived organoids,and the IC50 of each drug were 0.2795(nmol/m L),0.05132(nmol/m L),10.13(nmol/m L),and 26.70(nmol/m L),respectively.4.The effect of drugs on tumor cell proliferation.The expression of Ki67 in the organoid cultures of the two cell lines did not change significantly after cisplatin treatment,while the expression of Ki67 in the organoid cultures was increased after paclitaxel,albumin bound paclitaxel and 5-FU treatment.5.Expression of tumor stem cell surface markers.The expressions of CD44,CD54 and CD133 in the organoid cultures of the two cell lines were significantly increased after treated with chemotherapeutic drugs.The increased expression of CD338,CD271 and CD90 after drug treatment was different between the two cell lines,and the expression of CD184 was not changed.6.The expression of tumor stemness-related gene m RNA.After treatment with four chemotherapeutic drugs,the expression of SOX2,LGR5 and ALDH-1 genes in the organoid cultures of the two cells increased,and the expression of OCT4 decreased.Besides the expression of C-MYC gene decreased after treatment with paclitaxel,and the expressionof KLF4 gene decreased after treatment with albumin bound paclitaxel,the expression of CMYC and KLF4 increased after treated with other drugs.Conclusion In this study,organoid cultures derived form esophageal squamous cell carcinoma cell lines were successfully cultured in vitro.In the organoid cultures of the two cells,the IC50 s of the four commonly used chemotherapeutic drugs were different,and the most different ones were paclitaxel and 5-FU.In the organoid cultures of the two cancer cells,the expression of surface molecular markers varied after treatment with the four commonly used chemotherapeutic drugs,and the increased expressions of CD44,CD54 and CD133 were common.In organoid cultures of the two cancer cells,the expression of resistance-related genes varied after treatment with four commonly used chemotherapeutic drugs.The expression of SOX2,LGR5 and ALDH-1 genes in the organoid cultures of the two cells increased,and the expression of OCT4 decreased.The results of this study will provide preliminary experimental data for the individualized treatment of esophageal squamous cell carcinoma through tumor organoids.