Human biliary cancer cell line DPC-X5 and use thereof

Abstract

The application discloses a human ampullary carcinoma cell line DPC-X5 and an application thereof, and belongs to the field of microbial animal cell lines. The human ampullary carcinoma cell line is named as a human ampullary carcinoma cell line (homo sapiens) DPC-X5, has been preserved in the China Center for Type Culture Collection on October 30, 2025, and the preservation number is CCTCC NO: C202580. The human ampullary carcinoma cell line DPC-X5 can be applied to the establishment of a cell model of ampullary carcinoma occurrence, development or metastasis. The human ampullary carcinoma cell line DPC-X5 can be applied to the research of a cell model of differentiation mechanism, cell morphology and functional abnormality, tumor infiltration and metastasis mechanism of ampullary carcinoma and guidance of clinical comprehensive diagnosis and treatment. The human ampullary carcinoma cell line DPC-X5 can be applied to the research of an occurrence mechanism of ampullary carcinoma and screening of drugs for preventing and treating ampullary carcinoma. The human ampullary carcinoma cell line DPC-X5 can be applied to the establishment of an animal model of ampullary carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of microbial animal cell line technology, specifically relating to a human ampullary cancer cell line DPC-X5 and its applications. Background Technology

[0002] Ampullary carcinoma is a relatively rare tumor, accounting for 0.2% of gastrointestinal cancers but 20% of all periampullary cancers. It can occur at any age, but is more common between 60 and 65 years of age; the male-to-female ratio is approximately 3:2. Due to the early onset of biliary obstruction symptoms, the surgical resection rate of ampullary carcinoma is much higher than other periampullary malignancies, and the prognosis is relatively good. Based on morphological and immunohistochemical characteristics, ampullary carcinoma is classified into intestinal-type, pancreatobiliary-type, and mixed-type ampullary carcinoma. Due to the rarity of the tumor and the lack of clinical trials, standardized adjuvant therapy regimens for ampullary carcinoma remain to be determined.

[0003] Tumor cell lines can reflect the characteristics of tumors to a certain extent and are the most widely used models in tumor research. Tumor cell lines have greatly promoted the understanding of tumor biology and the development of anti-tumor drugs. A comprehensive tumor cell line library should reflect the diversity of tumor phenotypes and provide cell lines with different tumor heterogeneities. Furthermore, due to racial and regional differences, continuously establishing new cell lines with different backgrounds has significant scientific value. However, to date, no Chinese ampullary cancer cell lines have been reported in the literature. Therefore, establishing Chinese Chinese ampullary cancer cell lines is essential. Summary of the Invention

[0004] The purpose of this invention is to address the deficiency in existing technologies regarding the lack of ampullary cancer cell lines by providing a novel human ampullary cancer cell line, DPC-X5, and its applications. This cell line can be used for basic research on ampullary cancer, drug screening, and the construction of animal models.

[0005] The present invention provides a human ampullary cancer cell line named human ampullary cancer cell line (homo sapiens) DPC-X5, accession number: CCTCC NO: C202580.

[0006] The human ampullary cancer cell line DPC-X5 can serve as a cell model for studying the mechanisms of ampullary cancer occurrence, development, or metastasis. The human ampullary cancer cell line DPC-X5 can also be used to establish animal models of ampullary cancer.

[0007] This invention utilizes surgical specimens taken from a 50-year-old female patient. After digestion with a mixture of type II collagenase and neutral protease, primary culture was performed. A human ampullary carcinoma cell line, named homo sapiens (DPC-X5), was then established using cell culture techniques. It was deposited on October 30, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202580.

[0008] This cell line has the following biological characteristics:

[0009] 1. Cells adhere to the wall and grow without contact inhibition, which can lead to superimposed growth.

[0010] 2. The cell doubling time is 85 hours.

[0011] 3. Cell immunohistochemical results showed that DPC-X5 is a moderately or poorly differentiated ampullary cancer cell line.

[0012] 4. Chromosome analysis revealed that 70% of DPC-X5 cells were subtriploid and 30% were subtetraploid, exhibiting complex chromosome number and structural aberrations. The representative karyotypes were 60, XXX der(5), der(8), der(9), and der(21).

[0013] 5. DPC-X5 cells are resistant to gemcitabine, oxaliplatin, paclitaxel, and 5-FU.

[0014] 6. After inoculation into NXG mice, DPC-X5 rapidly formed subcutaneous xenografts with a tumor formation rate of 100%.

[0015] 7. The human ampullary cancer cell line DPC-X5 described in this invention can be used as a cell model for studying the differentiation mechanism, abnormal cell morphology and function, tumor invasion and metastasis mechanism of ampullary cancer, and guiding comprehensive clinical diagnosis and treatment.

[0016] The human ampullary cancer cell line DPC-X5 can be used to establish cell models of ampullary cancer occurrence, development, or metastasis.

[0017] The human ampullary cancer cell line DPC-X5 can be used as a cell model to study the differentiation mechanism, cell morphology and functional abnormalities, tumor invasion and metastasis mechanism, and to guide comprehensive clinical diagnosis and treatment of ampullary cancer.

[0018] The human ampullary cancer cell line DPC-X5 can be used to study the pathogenesis of ampullary cancer and screen drugs for its prevention and treatment.

[0019] The ampullary cancer cell line DPC-X5 can be used to establish an animal model of ampullary cancer.

[0020] This invention establishes a novel human ampullary cancer cell line, DPC-X5, which possesses various biological characteristics suitable for research on ampullary cancer, including its occurrence, development, metastasis mechanisms, and applications in guiding comprehensive clinical diagnosis and treatment. The human ampullary cancer cell line provided by this invention will contribute to advancing research progress in ampullary cancer and offer new ideas and methods for clinical treatment. Attached Figure Description

[0021] Figure 1 Pathological results of tumor tissue derived from DPC-X5 cells (scale bar = 100 μm).

[0022] Figure 2 Morphological observation of DPC-X5 cells under a microscope (scale bar = 50 μm).

[0023] Figure 3 The growth curve for DPC-X5 cells. Figure 3 In the graph, the horizontal axis represents cell culture time, and the vertical axis represents cell number.

[0024] Figure 4 The results are from the immunohistochemistry of DPC-X5 cells. Figure 4 In the diagram, A represents positive expression of CK20 in cells; B represents positive expression of CDX-2 in cells; C represents positive expression of MUC1 in cells; D represents positive expression of E-cadherin in cells; and E represents a positive expression rate of 50% for Ki67 in cells (scale bar = 50 μm).

[0025] Figure 5 Results of chromosome analysis for DPC-X5 cells. Figure 5 The top image shows the chromosome division phase in metaphase cells; the bottom image is an analysis diagram of the chromosome division phases after pairing and sorting using karyotype analysis software; chromosomes highlighted in the analysis diagram represent chromosomes with obvious structural abnormalities or those that do not match their corresponding sequence numbers.

[0026] Figure 6 Results of drug sensitivity in DPC-X5 cells. Figure 6 In the given list, A represents resistance to gemcitabine, B represents resistance to oxaliplatin, C represents resistance to fluorouracil, and D represents resistance to paclitaxel.

[0027] Figure 7 These are the results of an in vivo tumorigenicity experiment in DPC-X5 cell-immunodeficient mice. Figure 7 In the figures, AB shows that DPC-X5 inoculated subcutaneously into NXG mice can form xenografts with a tumor formation rate of 100%; C shows that no metastatic lesions were observed in the lungs and liver of mice after 4 weeks. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments will be used in conjunction with the accompanying drawings to further illustrate the invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Rather, the invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of the invention as defined in the claims.

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0030] I. Establishment of the human ampullary cancer cell line DPC-X5

[0031] Tumor tissue was collected from a patient with ampullary carcinoma, digested with mixed enzymes, and then cultured in primary culture. This successfully established a continuously passaged ampullary carcinoma cell line, which has now reached over 60 passages with stable cell characteristics. The patient's pathological results indicated moderately to poorly differentiated ampullary carcinoma. Figure 1 As shown.

[0032] This invention utilizes surgical specimens from a 50-year-old female patient. After digestion with a mixture of type II collagenase and neutral protease, primary culture was performed. A human ampullary carcinoma cell line, named homo sapiens (DPC-X5), was established using cell culture techniques. It was deposited on October 30, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202580.

[0033] II. Detection of biological characteristics of the human ampullary cancer cell line DPC-X5

[0034] 1. Cell Morphology: After stable cell growth and passage, live cell observation was performed. A monolayer of cells grown on coverslips was fixed with 95% ethanol, stained with hematoxylin and eosin (H&E), and observed under a light microscope. Results showed that under phase-contrast microscopy, the cells exhibited an epithelial-like arrangement, adherent growth, and overlapping growth. (See attached image for DPC-X5 cell morphology under a microscope.) Figure 2 As shown.

[0035] 2. DPC-X5 cells in logarithmic growth phase were collected and dissociated using trypsin (VivaCell). These cells, along with the primary tumor tissue, were sent to Suzhou Jianda Biotechnology Co., Ltd. for STR (short tandem repeat) analysis to clarify the correlation between the cells and the primary tumor tissue. The STR typing results are shown in Table 1.

[0036] Table 1

[0037]

[0038] Note: "-" in the table indicates that the site is not applicable to the matching probability calculation.

[0039] As can be seen from Table 1, the STR typing results of DPC-X5 cells are highly consistent with those of the primary tumor tissue, indicating that the DPC-X5 cell line originated from the tumor tissue and maintained its genetic characteristics during in vitro culture.

[0040] 3. DPC-X5 cells in the logarithmic growth phase were collected and enzymatically dissociated using 0.25% trypsin (VivaCell) to prepare a single-cell suspension. After measuring the density of the cell suspension, 8 × 10⁸ cells were separately isolated. 3 1×10 4 1.2×10 4 1.5×10 4 1.8×10 4 2.1×10 4 2.4×10 4 2.8×10 4 Cells were seeded into 9 wells of a 96-well plate (NEST), and the volume of each well was brought up to 100 μl with complete culture medium (DMEM / F12 + 10% fetal bovine serum + 1% penicillin-streptomycin solution). After 20 h, 100 μl of diluted CCK8 (cell counting kit-8) solution [10% CCK8 (APE×BIO) + 90% DMEM / F12 medium (Gibco)] was added. After 3.5 h, the absorbance at 450 nm was measured using a microplate reader (BioTek, Synergy H1). A standard curve was plotted with absorbance on the x-axis and cell count on the y-axis. Based on the growth curve, the cell doubling time was calculated (using a website); the average cell doubling time was approximately 85 h; the growth curve of DPC-X5 cells is shown below. Figure 3 As shown.

[0041] 4. DPC-X5 cells in logarithmic growth phase were obtained, enzymatically dissociated, and seeded onto sterile glass slides for growth. After 60 hours, the coverslips were washed with PBS (phosphate-buffered saline), fixed with 4% paraformaldehyde (Servicebio) for 15 minutes, air-dried, and then treated with 0.5% Triton X-100 (MCE) for 20 minutes before subsequent staining. The original tumor tissue and xenograft tumors were paraffin-embedded, cut into 4 μm thick sections, dried at 60°C for 5 hours, and then stained.

[0042] Immunohistochemical staining: After dewaxing and rehydration, slides were immersed in sodium citrate solution (10 mMol / L, pH=6.0), boiled (90 s) for antigen retrieval, incubated in 3% hydrogen peroxide solution at 37°C for 15 min, and then 100 µl of normal goat serum was added, followed by blocking at 37°C for 15 min. The slides were then incubated with anti-CK20, anti-CDX2, anti-MUC1, anti-Ki67, and anti-E-cadherin at 37°C for 12 h, respectively, followed by incubation with secondary antibody at room temperature for 50 min. DAB (diaminobenzidine) staining kit (Dako) was used for staining. After rinsing with running water for 5 min, hematoxylin was counterstained, dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and observed under an inverted microscope (Olympus, IX73+DP74). Results are as follows: Figure 4 As shown.

[0043] Figure 4 In this context, A indicates that CK20 is positively expressed in cells; Figure 4 In this context, B indicates that CDX-2 is positively expressed in cells; Figure 4 The "C" in the text indicates that MUC1 (mucin 1) is positively expressed in cells. Figure 4 The "D" in the text indicates that E-cadherin (epithelial cadherin) is positively expressed in cells. Figure 4 The "E" indicates that the positive expression rate of Ki67 (nuclear antigen for cell proliferation) in the cells is 50%.

[0044] 5. Take DPC-X5 cells in the logarithmic growth phase, add colchicine (Spectrum) to the culture medium to a concentration of 0.2 μg / ml, incubate for 90 min, then digest the cells into a cell suspension using 0.25% trypsin (Vivacell); centrifuge and discard the supernatant, resuspend the precipitate in 0.56% KCl solution, and incubate at 37℃ for 30 min. Add fixation solution 1 [a mixture of formic acid and glacial acetic acid (volume ratio of formic acid:glacial acetic acid = 3:1)], mix thoroughly, centrifuge and discard the supernatant, and resuspend again using fixation solution 1 to obtain a chromosome suspension. Place one drop of this suspension onto a glass slide and dry in an oven at 80℃ for 3 h. Digest the dried slide in trypsin for 1 min, then stain in Giemsa stain (BIOSIC) for 8 min. Remove the slide, rinse off the stain with running water, mount it, and observe under a 100x oil immersion microscope. Keratogenetic analysis was performed using ImageJ imaging software and the ChromosomeJ plugin. The results are as follows: Figure 5 As shown. In Figure 5 The top image shows the chromosome division phase in metaphase cells; the bottom image is an analysis diagram of the chromosome division phases after pairing and sorting using karyotype analysis software; chromosomes highlighted in the analysis diagram represent chromosomes with obvious structural abnormalities or those that do not match their corresponding sequence numbers.

[0045] DPC-X5 cells are 70% subtriploid and 30% subtetraploid, exhibiting complex chromosomal number and structural aberrations. Representative karyotypes include 60, XXX der(5), der(8), der(9), der(21). Figure 5 ).

[0046] The above experiments demonstrate that the present invention can be applied in cell models of ampullary carcinoma occurrence, development or metastasis, as well as in cell models of ampullary carcinoma differentiation mechanism, cell morphology and functional abnormalities, tumor invasion and metastasis mechanism, and to guide comprehensive clinical diagnosis and treatment.

[0047] 6. DPC-X5 cells in the logarithmic growth phase were collected, enzymatically dissociated, and prepared into a single-cell suspension. The cell density in the suspension was then adjusted to 1.2 × 10⁻⁶ cells / cells. 5 After thorough mixing, add 100 μl of the solution to each well of a 96-well plate (NEST). After 24 hours, dilute the antitumor drug to different concentrations using complete culture medium (DMEM / F12 + 10% FBS (fetal bovine serum) + 1% penicillin-streptomycin (BI)). Discard the culture medium from the 96-well plate. Add different concentrations of drug solution to the drug treatment groups, and complete culture medium to the control group. Both the drug treatment and control groups have four replicates, with 170 μl of drug solution or complete culture medium added to each well. After 72 hours of treatment, discard the drug solution and complete culture medium from the 96-well plate, and add 100 μl of diluted CCK8 solution [10% CCK8 (APE×BIO) + 90% DMEM / F12 (Gibco)] to each well. Simultaneously, add 100 μl of diluted CCK8 solution to each of the four blank wells (wells without cell suspension). After incubation at 37°C for 3.5 hours, the absorbance at 450 nm was measured using a microplate reader (BioTek, Synergy H1). Drug dose-response curves were plotted using GraphPad Prism 8.0.2 software (GraphPad Inc., San Diego, CA, USA), and the IC50 was calculated. This analysis was repeated three times. The results are as follows: Figure 6 As shown.

[0048] from Figure 6 It can be seen that this cell line is resistant to gemcitabine (IC50 > 600 μmol / L). Figure 6 A), resistance to oxaliplatin (IC50 = 102.60 μmol / L) Figure 6 (B in the text) is resistant to 5-FU (IC50 = 2718 μmol / L). Figure 6 C), resistance to paclitaxel (IC50 > 10 μmol / L) Figure 6 The result (D) indicates that it is a multidrug-resistant cell line. The above experiments demonstrate that this invention can be applied to study the pathogenesis of ampullary carcinoma and to screen drugs for its prevention and treatment.

[0049] 7. Take DPC-X5 cells in the logarithmic growth phase, enzymatically dissociate them to prepare a cell suspension, and adjust the cell density to 1×10⁻⁶. 7 / ml. 0.1ml of the mixed cell suspension was subcutaneously injected into the right posterior axilla of three NXG mice. The body weight and tumor growth of the NXG mice were monitored regularly. After 4 weeks, the mice were euthanized, and the xenograft tumors, liver, and lung tissues were dissected for observation. The xenograft tumors were fixed with 4% paraformaldehyde (Servicebio) for subsequent H&E staining and immunohistochemical staining. Results are as follows: Figure 7 As shown, Figure 7 AB, representing DPC-X5, when subcutaneously inoculated into NXG mice, can form xenografts with a tumor formation rate of 100%. Figure 7 In the figure, C indicates that no metastatic lesions were observed in the lungs and liver of mice after 4 weeks. These experiments demonstrate that this invention can be applied to establish an animal model of ampullary carcinoma.

[0050] In summary, this invention successfully established and systematically identified a novel human ampullary carcinoma cell line, DPC-X5. This cell line possesses a clearly defined tissue origin (STR identification), stable epithelial cell morphology and growth characteristics, unique molecular marker expression profiles and chromosomal karyotype, specific drug sensitivity profiles, and in vivo tumorigenicity. These combined characteristics make the DPC-X5 cell line a valuable experimental tool and model system for studying the biological behavior, molecular mechanisms, drug screening, and development of novel therapeutic strategies for ampullary carcinoma.

[0051] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A human ampullary cancer cell line, characterized in that... It was named the human ampullary cancer cell line (homo sapiens) DPC-X5 and was deposited at the China Center for Type Culture Collection on October 30, 2025, with accession number CCTCC NO: C202580.

2. The application of the human ampullary cancer cell line as described in claim 1 in establishing a cell model for the occurrence, development, or metastasis of ampullary cancer.

3. The application of the human ampullary cancer cell line as described in claim 1 in the study of the differentiation mechanism, cell morphology and functional abnormalities, tumor invasion and metastasis mechanism of ampullary cancer, and as a cell model to guide comprehensive clinical diagnosis and treatment.

4. The application of the human ampullary cancer cell line as described in claim 1 in studying the pathogenesis of ampullary cancer and screening drugs for the prevention and treatment of ampullary cancer.

5. The application of the human ampullary cancer cell line as described in claim 1 in establishing an animal model of ampullary cancer.

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