Establishment and characterization of a new human ampullary carcinoma cell line, DPC-X1

Abstract

Background: An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have been reported, and a mixed-type ampullary carcinoma cell line has not yet been reported.

Aim: To establish a stable mixed-type ampullary carcinoma cell line originating from Chinese.

Methods: Fresh ampullary cancer tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, short tandem repeat (STR) analysis and transmission electron microscopy. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-FU were evaluated by cell counting kit-8 assay. Subcutaneous injection 1 × 10⁶ cells to three BALB/c nude mice for xenograft studies. The hematoxylin-eosin staining was used to detect the pathological status of the cell line. The expression of biomarkers cytokeratin 7 (CK7), cytokeratin 20 (CK20), cytokeratin low molecular weight (CKL), Ki67 and carcinoembryonic antigen (CEA) were determined by immunocytochemistry assay.

Results: DPC-X1 was continuously cultivated for over a year and stably passaged for more than 80 generations; its population doubling time was 48 h. STR analysis demonstrated that the characteristics of DPC-X1 were highly consistent with those of the patient's primary tumor. Furthermore, karyotype analysis revealed its abnormal sub-tetraploid karyotype. DPC-X1 could efficiently form organoids in suspension culture. Under the transmission electron microscope, microvilli and pseudopods were observed on the cell surface, and desmosomes were visible between the cells. DPC-X1 cells inoculated into BALB/C nude mice quickly formed transplanted tumors, with a tumor formation rate of 100%. Their pathological characteristics were similar to those of the primary tumor. Moreover, DPC-X1 was sensitive to oxaliplatin and paclitaxel and resistant to gemcitabine and 5-FU. Immunohistochemistry showed that the DPC-X1 cells were strongly positive for CK7, CK20, and CKL; the Ki67 was 50%, and CEA was focally expressed.

Conclusion: Here, we have constructed a mixed-type ampullary carcinoma cell line that can be used as an effective model for studying the pathogenesis of ampullary carcinoma and drug development.

Keywords: Ampullary carcinoma; Cell line; Drug resistance; Xenograft.

Figure 1

Clinical data and cell morphology. A: Clinical data of the patient; B: Magnetic resonance cholangiopancreatography shows the expansion of intrahepatic and extrahepatic bile ducts, gallbladder enlargement, and pancreatic duct expansion (arrow); C: In the coronal view of magnetic resonance imaging, soft tissue shadow (arrow) can be seen in the ampulla, and the upper bile duct is dilated; D: General view of the surgical specimen; E: The enlarged appearance of the ampulla tumor shows the gray-white tumor growing around the ampulla (arrow); F: DPC-X1 primary cell morphology; multinucleated cells and megakaryocytes can be seen; G: DPC-X1 cell morphology of the 80^th generation. Scale bars: 100 μm. D: Death; AFP: alpha-fetoprotein; CEA: Carcinoembryonic antigen; CA199: Carbohydrate antigen 199.

Figure 2

DPC-X1 population doubling time, short tandem repeat detection, karyotype analysis and Organoid culture. A: DPC-X1 growth curve; cell doubling time is 48 h; B: The likelihood ratio between the short tandem repeat results of DPC-X1 and the primary tumor tissue is 4.1867 × 1020. The cells are not contaminated by other cell lines; C: Karyotype analysis shows that DPC-X1 cells are mainly sub-tetraploid, with large differences in chromosome number and morphology. The representative karyotype is 80, XX del (2) (q32) del (5) (p12) del (6) (q24) inv (9) del (10) p (13) del (17) p (12); D: Morphology of DPC-X1organoids after one week. The organoids are spherical or cystic in shape; E: Morphology of DPC-X1 organoids after two weeks. The organoids are spore-like or branch-like in shape. Scale bars: 100 μm.

Figure 3

Ultrastructure of DPC-X1 under a transmission electron microscope. A: DPC-X1 has a large and deformed nucleus; it is increased in number. The nucleolus is clustered in the nuclear membrane. There are fewer cytoplasm. Microvilli (yellow arrow) and pseudopodia (blue arrow) were visible on the cell surface; B: DPC-X1 cells are rich in the endoplasmic reticulum (yellow arrow) and ribosome; C: DPC-X1 cells have well-developed Golgi apparatus (yellow arrow), and the size and shape of the mitochondria are different (blue arrow); D: DPC-X1 desmosome structure can be seen between the cells (arrow).

Figure 4

Drug sensitivity test. A: DPC-X1 is sensitive to oxaliplatin; IC50 = 13.26 μmol/L; B: DPC-X1 is sensitive to paclitaxel; IC50 = 0.014 μmol/L; C: DPC-X1 is resistant to fluorouracil; IC50 = 144.9 µmol/L; D: DPC-X1 is resistant to gemcitabine; IC50 > 600 μmol/L. IC50: Half maximal inhibitory concentration; TDC: Test drug concentrations.

Figure 5

Xenograft tumor formation experiment. A: In four weeks, DPC-X1 quickly formed xenograft tumors under the skin of BALB/c nude mice. The tumor formation rate is 100%; B: Anatomically, the xenograft tumor and muscle tissue of the mouse shows invasive growth, and new blood vessels can be seen on the surface of the tumor; C: General view of the xenograft tumor; D: No metastatic lesions were found in the lungs and liver of BALB/c nude mice within four weeks; E: Growth curve of DPC-X1 transplanted tumor; F: Body weight curve of BALB/c nude mice.

Figure 6

Hematoxylin and eosin and immunohistochemical staining of DPC-X1 cells, organoid, xenograft tumor, and primary tumor. A-D: Hematoxylin and eosin staining of DPC-X1 cells, organoids, xenograft tumor, and primary tumor; E-H: Strong cytokeratin (CK)7 positive staining of DPC-X1 cells, organoids, xenograft tumor, and primary tumor; I-L: Strong CK20 positive staining of DPC-X1 cells, organoids, xenograft tumor, and primary tumor; M-P: Strong cytokeratin low molecular weight positive staining of DPC-X1 cells, organoids, xenograft tumor, and primary tumor; Q-T: Ki67 staining of DPC-X1 cells, organoids, xenograft tumor, and primary tumor; U-X: Carcinoembryonic antigen focal staining of DPC-X1 cells, organoids, xenograft tumor, and primary tumor. Scale bars: 100 μm. H&E: Hematoxylin and eosin; CK7: Cytokeratin 7; CK20: Cytokeratin 20; CKL: Cytokeratin low molecular weight; CEA: Carcinoembryonic antigen.