First report on establishment and characterization of the extrahepatic cholangiocarcinoma sarcoma cell line CBC2T-2

Figure 1 Clinical and pathological profile of CBC2T-2. A and B: Contrast-enhanced computed tomography of the abdomen showing a high-grade occupying lesion of the common bile duct with high level obstruction of the common bile duct (orange arrows); C: Specimen of postoperative carcinosarcoma of the extrahepatic bile duct with thickened duct wall and polyp-like protrusion into the lumen; D and E: Hematoxylin and eosin and immunohistochemistry staining of primary tumor tissue. Scale bars, 200 μm; F: Immunohistochemistry staining of primary tumor tissue. Antibodies against Vimentin, Scale bars, 200 μm; G: Clinicopathological characteristics of patients. AFP: Alpha-fetoprotein; CEA: Carcinoembryonic antigen; CA199: Carbohydrate antigen199.

Figure 2 Morphological features, doubling time and cell cycle of CBC2T-1 cells. A: Bright field morphology of CBC2T-2 cells in the primary,1^st, 10^th, 20^th and 50^th passages (scale bars, 100 μm); B: SEM and TEM electron microscopy were used to observe the cellular ultrastructure (scale bars, 100, 30, 10 μm); C: Flow cytometry of CBC2T-2 cells; D and E: Cumulative growth curve of CBC2T-2 cells.

Figure 3 Characterization of CBC2T-2 cell behavior. A: Representative images of the ability of CBC2T-2 cells to form spheres at days 7 and 14; B and C: Wound healing assay of CBC2T-2 and TFK-1 cells after 24 and 48 h; D: The migratory and invasive abilities of CBC2T-1 and TFK-1 cells were detected by transwell assay; E and F: Representative images showing the colony formation of CBC2T-1 and TFK-1 cells. Scale bars, 100 μm. ^aP < 0.01; ^bP < 0.001.

Figure 4 Cytogenetic analysis and genomic characteristics. A: Chromosome karyotype analysis shows that the cells are triploid and there is variation in the number of structures; B: Cells and tumor tissues have copy number increases and decreases in almost the entire chromosome section; C: Histogram of the distribution of somatic cell InDel annotation results; D: Somatic somatic single-nucleotide variant annotation result distribution map.

Figure 5 Verification the tumorigenic ability of CBC2T-2 cells in non-obese diabetic/severe combined immunodeficient mice. A: Tumor formation in non-obese diabetic/severe combined immunodeficient mice; B: Body weight gain curves in xenograft mice; C: Tumor size over time in three xenograft mice; D: Hematoxylin and eosin staining and immunohistochemical (CK19, CD68, CD117, Vmention, Desmin) results of primary tumors, CBC2T-2 xenografts and CBC2T-2. Scale bars, 50 and 100 μm. HE: Hematoxylin and eosin.

Figure 6 Sensitivity of CBC2T-2 cell line to anticancer drugs. A-E: Sensitivity of CBC2T-2 cells to paclitaxel, gemcitabine, 5-fluorouracll, cisplatin, and oxaliplatin.