Human liver metastasis-derived colon cancer cell line crc-x1 and use thereof

Abstract

The human liver metastasis-derived colon cancer cell line CRC-X1 and its application belong to the field of microbial animal cell lines. The human colon cancer cell line is named Homo sapiens CRC-X1, and was preserved in the China Center for Type Culture Collection on November 22, 2025, with the preservation number CCTCC NO: C2025354. The human liver metastasis-derived colon cancer cell line CRC-X1 can be applied in the establishment of a cell model of colon cancer occurrence, development or metastasis; CRC-X1 can be applied in the cell model of studying the differentiation mechanism of colon cancer, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism, and guiding clinical comprehensive diagnosis and treatment. CRC-X1 can be applied in the study of the mechanism of colon cancer and the screening of drugs for preventing and treating colon cancer. CRC-X1 can be applied in the establishment of an animal model of colon cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a human liver metastasis-derived colon cancer cell line CRC-X1 and its applications. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors of the digestive tract worldwide. Distant metastasis is the primary challenge in the treatment of colorectal cancer, marking the progression of the tumor to advanced stage (stage IV). Liver metastasis is the most common type of metastasis, occurring in approximately 50% of colorectal cancer patients during their disease course. Based on the timing of occurrence, liver metastasis can be classified as synchronous or metachronous. The related mechanisms and clinical treatment strategies still require in-depth research. Existing studies have found that genes such as BRAF, KRAS, NRAS, PI3KCA, and TP53 are associated with liver metastasis, and the mutation type affects prognosis. More translational research is needed for colorectal cancer to advance practical clinical diagnosis and treatment and prolong the survival of colorectal cancer patients.

[0003] Human tumor cell lines, especially those with complete data and follow-up, are important tools for tumor biology research. Since the 1980s, scientists from the United States, Japan, and South Korea, among others, have established and identified numerous colon cancer cell lines, but few Chinese colon cancer cell lines have been reported. Cell lines derived from primary tumors play a significant role in exploring biology, tumorigenesis, detecting drug sensitivity, developing molecular therapeutic targets, and studying drug resistance mechanisms. A comprehensive tumor cell line library should reflect the diversity of tumor phenotypes and provide cell lines representing different tumor heterogeneities; considering the different etiologies of tumors and their etiological and ethnic-related genetic variations, it is important to use appropriate preclinical models that reflect these characteristics.

[0004] Newly established cell lines can maintain characteristics similar to the primary tumor to the greatest extent, and the results obtained from studies using these cell lines are closest to the actual situation in humans. Due to different genetic backgrounds, it is essential to establish disease models specific to the Chinese population in order to better study diseases in Chinese people. For these reasons, continuously establishing new cell lines and discarding outdated cell lines has become a crucial part of studying the pathogenesis and treatment of colorectal cancer. Summary of the Invention

[0005] The purpose of this invention is to address the insufficient number of colon cancer cell lines in existing technologies by providing a novel human liver metastasis-derived colon cancer cell line CRC-X1 and its applications.

[0006] This invention provides a human colon cancer cell line named human liver metastasis-derived colon cancer cell line (Homosapiens) CRC-X1, with accession number CCTCC NO: C2025354.

[0007] The human liver metastasis-derived colon cancer cell line CRC-X1 can serve as a cell model for studying the mechanisms of colon cancer occurrence, development, or metastasis. CRC-X1 can also be used to establish animal models of colon cancer.

[0008] This invention utilizes a surgical specimen obtained from a 73-year-old female patient. After digestion with a mixture of type II collagenase and neutral protease, primary culture was performed. A human liver metastasis-derived colon cancer cell line, homosapiens, named CRC-X1, was established using cell culture techniques. This human liver metastasis-derived colon cancer cell line (Homo sapiens) CRC-X1 was deposited on November 22, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2025354.

[0009] The human liver metastasis-derived colon cancer cell line CRC-X1 homo sapiens possesses the following biological characteristics:

[0010] 1. Cells adhere to the wall and grow without contact inhibition, which can lead to superimposed growth.

[0011] 2. The cell doubling time was 60 h.

[0012] 3. Immunohistochemical results showed that CRC-X1 is a moderately or poorly differentiated colon cancer cell line.

[0013] 4. Chromosome analysis showed that 90% of CRC-X1 cells were hypertriploid and 10% were hypertetraploid. The representative karyotypes were 71,XXXX der(5),inv(9),der(11),der(22).

[0014] 5. CRC-X1 cells are resistant to oxaliplatin but sensitive to 5-fluorouracil (5-FU).

[0015] 6. After inoculation into NCG mice, CRC-X1 can rapidly form subcutaneous xenografts with a tumor formation rate of 60%.

[0016] 7. The human liver metastasis-derived colon cancer cell line CRC-X1 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 colon cancer, and guiding comprehensive clinical diagnosis and treatment.

[0017] The CRC-X1 colon cancer cell line derived from human liver metastases can be used to establish cell models of colon cancer occurrence, development, or metastasis.

[0018] The human liver metastasis-derived colon cancer cell line CRC-X1 can be used as a cell model to study the differentiation mechanism, abnormal cell morphology and function, tumor invasion and metastasis mechanism of colon cancer, and to guide comprehensive clinical diagnosis and treatment.

[0019] The human liver metastasis-derived colon cancer cell line CRC-X1 can be used to study the pathogenesis of colon cancer and screen drugs for the prevention and treatment of colon cancer.

[0020] The colon cancer cell line CRC-X1 can be used to establish animal models of colon cancer.

[0021] This invention establishes a novel human liver metastasis-derived colon cancer cell line, CRC-X1. This cell line possesses various biological characteristics suitable for colon cancer research, including its occurrence, development, metastasis mechanisms, and applications in guiding comprehensive clinical diagnosis and treatment. The human colon cancer cell line provided by this invention will help advance colon cancer research and offer new ideas and methods for clinical treatment. Attached Figure Description

[0022] Figure 1 Pathological results of tumor tissue derived from CRC-X1 cells (scale bar = 100 μm).

[0023] Figure 2 Morphological observation of CRC-X1 cells under a microscope (scale bar = 50 μm).

[0024] Figure 3 The growth curve for CRC-X1 cells. Figure 3 In the graph, the horizontal axis represents cell culture time, and the vertical axis represents cell number.

[0025] Figure 4 Immunohistochemical results of tissue samples taken from CRC-X1. Figure 4 In the diagram, A indicates positive expression of CK20 in tumors; B indicates positive expression of CDX2 in tumors; C indicates negative expression of Her-2 in tumors; and D indicates a positive expression rate of Ki67 of 80% (scale bar = 50 μm).

[0026] Figure 5 The results are from chromosome analysis of CRC-X1 cells.

[0027] Figure 6 Results of drug sensitivity in CRC-X1 cells. Figure 6 In the above, A represents resistance to oxaliplatin, and B represents sensitivity to fluorouracil.

[0028] Figure 7 These are the results of an in vivo tumorigenicity experiment in CRC-X1 cell-immunodeficient mice. Figure 7In the study, A and B were CRC-X1 subcutaneously injected into NCG mice, which could form xenografts with a tumor formation rate of 60%. Detailed Implementation

[0029] 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.

[0030] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0031] Establishment and identification of the human liver metastasis-derived colon cancer cell line CRC-X1.

[0032] I. Establishment of the human liver metastasis-derived colon cancer cell line CRC-X1

[0033] Tumor tissue was collected from a clinically diagnosed colorectal cancer patient, digested with mixed enzymes, and then cultured in primary culture. This successfully established a continuously passaged colorectal cancer cell line, which has now reached over 60 passages with stable cell characteristics. The patient's pathology result was moderately to poorly differentiated colorectal cancer. Figure 1 As shown.

[0034] This invention utilizes a surgical specimen obtained from a 73-year-old female patient. After digestion with a mixture of type II collagenase and neutral protease, primary culture was performed. A human liver metastasis-derived colon cancer cell line (Homosapiens), named CRC-X1, was established using cell culture techniques. It was deposited on November 22, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2025354.

[0035] II. Detection of biological characteristics of the human liver metastasis-derived colon cancer cell line CRC-X1

[0036] 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. The morphological observation of CRC-X1 cells under the microscope is shown in the image below. Figure 2 As shown.

[0037] 2. CRC-X1 cells in the logarithmic growth phase were collected and dissociated by trypsin (VivaCell). These cells, along with the 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 tumor tissue. The STR typing results are shown in Table 1.

[0038] Table 1

[0039]

[0040] Note: "-" in the table indicates that the locus is not applicable to the matching probability calculation. Some STR loci have allelic differences, which are due to the heterogeneity of primary tumor tissue, clonal selection during in vitro culture, and genetic drift. This does not affect the determination of cell line origin identity.

[0041] As can be seen from Table 1, the STR typing results of CRC-X1 cells are highly consistent with those of the tumor tissue, indicating that the CRC-X1 cell line originated from the tumor tissue and maintained its genetic characteristics during in vitro culture.

[0042] 3. Single-cell suspensions were prepared using P50 CRC-X1 cells and seeded into 96-well plates in two groups for standard curve plotting. The cell number gradient in each group was 6 × 10⁻⁶. 3 8×10 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 The plate also includes 6 sets of experimental wells, with 6 wells in each set and 8×10⁸ wells per well. 3 Cells were selected, with 100 μl of cell culture in each well. After 6 h, 100 μl of CCK8 dilution [10% CCK8 (APE×BIO):90% RPMI 1640 (Servicebio)=1:9] was added to the standard curve wells and the first set of experimental wells, and the cells were incubated for 2 h. The absorbance at 450 nm was measured, and growth curves were plotted. Absorbance was measured for four consecutive days at the same time, cell counts were calculated, and growth curves were plotted. The doubling time was calculated using a website; the average cell doubling time was approximately 60 h. The growth curve of CRC-X1 cells is shown below. Figure 3 As shown.

[0043] 4. After embedding the original tumor tissue in paraffin, cut it into 4μm thick sections, dry it at 60℃ for 5 hours, and then perform subsequent staining.

[0044] Immunohistochemical staining: After dewaxing and rehydration, slides were immersed in sodium citrate solution (10 mMol / L, pH=6.0), boiled (90s) for antigen retrieval, incubated in 3% hydrogen peroxide solution at 37℃ for 15min, and then 100 µl of normal goat serum was added, followed by blocking at 37℃ for 15min. The slides were then incubated with anti-CK20, anti-CDX2, anti-Her-2, and anti-Ki67 at 37℃ for 12h, followed by incubation with secondary antibodies at room temperature for 50min. DAB (diaminobenzidine) staining kit (Dako) was used for staining. After rinsing with running water for 5min, 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.

[0045] Figure 4 The A in the text indicates that CK20 (keratin 20) is expressed in tumor tissues; Figure 4 In this context, B indicates positive expression of CDX2 (tailed homeobox transcription factor 2) in tumors; Figure 4 The C in the text indicates that Her-2 (human epidermal growth factor receptor-2) is negatively expressed in tumors; Figure 4 The "D" indicates that the positive expression rate of Ki67 (nuclear antigen for cell proliferation) is 80%.

[0046] 5. Take CRC-X1 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 (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. Perform karyotype analysis using ImageJ imaging software and the ChromosomeJ plugin. The results are as follows Figure 5 As shown.

[0047] 90% of CRC-X1 cells were hypertriploid and 10% were hypertetraploid. The representative karyotypes were 71,XXXX der(5),inv(9),der(11),der(22). Figure 5 ).

[0048] The above experiments demonstrate that the present invention can be applied in cell models of colorectal cancer occurrence, development or metastasis, as well as in cell models of colorectal cancer differentiation mechanisms, abnormal cell morphology and function, tumor invasion and metastasis mechanisms, and to guide comprehensive clinical diagnosis and treatment.

[0049] 6. CRC-X1 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 (RPMI-1640 + 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 group, and complete culture medium to the control group. Both the drug treatment group and the control group 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% RPMI-1640 (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.

[0050] from Figure 6 It can be seen that this cell line is resistant to oxaliplatin (IC50 > 400 μmol / L) Figure 6 All of the drugs in group A) are resistant to fluorouracil (IC50 = 2.128 μmol / L). Figure 6 (B) Sensitive. The above experiments demonstrate that this invention can be applied in studying the pathogenesis of colon cancer and screening drugs for the prevention and treatment of colon cancer.

[0051] 7. Take CRC-X1 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. 0.15ml of the mixed cell suspension was subcutaneously injected into the posterior right axilla of five NCG mice. The body weight and tumor growth of the NCG mice were monitored regularly. After 4 weeks, the mice were euthanized, and the xenograft tumors and 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 The AB in the formula represents CRC-X1, which, when subcutaneously inoculated into NCG mice, can form xenografts with a tumor formation rate of 60%. These experiments demonstrate that this invention can be applied to establish animal models of colon cancer.

[0052] In summary, this invention successfully established and systematically identified a novel human liver metastasis-derived colon cancer cell line, CRC-X1. 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 CRC-X1 cell line a valuable experimental tool and model system for studying the biological behavior and molecular mechanisms of colon cancer, drug screening, and developing novel therapeutic strategies.

[0053] 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 liver metastasis-derived colon cancer cell line, characterized by... It was named CRC-X1 Homo sapiens, a human liver metastasis-derived colon cancer cell line, and was deposited at the China Center for Type Culture Collection on November 22, 2025, with accession number CCTCC NO: C2025354.

2. The application of the human liver metastasis-derived colon cancer cell line as described in claim 1 in establishing cell models of colon cancer occurrence, development, or metastasis.

3. The application of the human liver metastasis-derived colon 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 colon cancer, and as a cell model to guide comprehensive clinical diagnosis and treatment.

4. The application of the human liver metastasis-derived colon cancer cell line as described in claim 1 in studying the pathogenesis of colon cancer and screening drugs for the prevention and treatment of colon cancer.

5. The application of the human liver metastasis-derived colon cancer cell line as described in claim 1 in establishing an animal model of colon cancer.

Images