Establishment,Identification And Characterization Of New Chinese Human Intrahepatic Cholangiocarcinoma Cell Lines

Tumor cell lines are widely used in cancer research and drug development.Ethnic differences have a great impact on cancer research.In order to better study Chinese diseases,it is necessary to establish the disease model of Chinese people.With the implementation of the Biosafety Law of the People’s Republic of China,the import and export regulation of human tumor cell lines has become more stringent.From the perspective of biosafety,we also urgently need to establish Chinese human tumor cell lines.In this study,three intrahepatic cholangiocarcinoma cell lines were successfully established through primary culture and subculture,all of which have been stably subcultured for more than 100 generations,and were named ICC-X1,ICC-X2,and ICC-X3,respectively.ICC-X1 cells presents a typical epithelioid appearance,with short fusiform and polygonal shape as the main morphology;Both ICC-X2 and ICCX3 cells are spindle shaped.STR assay showed that they are new human tumor cell lines.The doubling times of the three cell lines were 48 hours,48 hours,and 36 hours,respectively.Karyotype analysis showed that ICC-X1 was mainly subtriploid,while ICC-X2 and ICC-X3 were both subtriploid karyotypes,with significant differences in chromosome number and morphology.All three cell lines were able to form tumorspheres in the ultra-low attachment plate.Organoids can be formed by inoculation into matrigel.Subcutaneously inoculated to BALB/c nude mice,all transplanted tumors can form within 4 weeks,with a tumor formation rate of 100%;The transplanted tumors of ICC-X2 and ICC-X3 had pathological phenomena of tumor budding and metastasis adjacent to the tumor;Pathological phenomena such as nerve invasion,vascular tumor thrombus,and collective metastasis can also be seen in ICCX2 transplanted tumors.Immunohistochemistry showed positive expression of CK7 and CK19 in the three cell lines,with a high proportion of Ki-67 and simultaneous expression of E-cadherin and vimentin.ICC-X1 cells are sensitive to gemcitabine and paclitaxel,but resistant to 5-FU and oxaliplatin;ICC-X2 cells are sensitive to oxaliplatin and paclitaxel,but resistant to gemcitabine and 5-FU;ICC-X3 is sensitive to paclitaxel and resistant to 5-FU,oxaliplatin,and gemcitabine.There was no mutation in TP53 in ICC-X1 cells,while both ICC-X2 and ICC-X3 had TP53 mutations,causing a mutation of proline at position 278 of the amino acid sequence to leucine.Compared to RBE,ICC-X1 has 2824 up-regulated genes and 2770 downregulated genes.Upregulation pathways were involved in phagosomes,protein processing in the endoplasmic reticulum,tumor necrosis factor signaling pathways,NOD-like receptor signaling pathways,and NF-κB signal pathway,interleukin-17 signal pathway,and other signal pathways.Downregulation pathways were involved in endocytosis,tight junction,axonal guidance,Rap1 signaling pathway,Hippo signaling pathway,P53 signaling pathway,Fanconi anemia pathway,homologous recombination,Hedgehog signaling pathway,and other signaling pathways.Compared to RBE,ICCX2 has 2804 upregulated genes and 2729 downregulated genes.Upregulation pathways were involved in the adhesion plaque,lysosomes,steroid biosynthesis pathways,MAPK signaling pathways,relaxin signaling pathways,C-type lectin receptor signaling pathways,Notch signaling pathways,pentose phosphate signaling pathways,and Hippo signaling pathways.Downregulation pathways were involved in cell cycle,platinum resistance,DNA replication,phosphatidylinositol signaling pathway,Fanconi anemia pathway,peroxisome,nucleotide excision repair,mismatch repair,homologous recombination repair,and so on.Compared to RBE,ICC-X3 has2364 up-regulated genes and 2834 down-regulated genes.The up-regulation pathways were mainly enriched in related pathways such as adhesion plaque,ribosomes,NODlike receptor signaling pathways,phagosomes,retrograde endogenous cannabinoid signaling,C-type lectin receptor signaling pathways,Hippo signaling pathways,and steroid biosynthesis.Downregulation pathways were involved in PI3K-Akt signaling pathway,MAPK signaling pathway,axonal guidance,phosphatidylinositol signaling system,peroxisome,inositol phosphate metabolism,multiple amino acid metabolism,and other signaling pathways.Through analysis of transcriptome data,we preliminarily screened three gemcitabine sensitive markers for intrahepatic cholangiocarcinoma: B4GALNT1,RFLNB,and AGTR1.In this study,we established three Chinese human intrahepatic cholangiocarcinoma cell lines,which can serve as effective models for molecular mechanism research and drug development of intrahepatic cholangiocarcinoma in China;The preliminary screening of three gemcitabine sensitive markers for intrahepatic cholangiocarcinoma were expected to provide some help for the precise chemotherapy of patients with intrahepatic cholangiocarcinoma in China.