High liver metastasis cell line of colorectal cancer and preparation method and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a colorectal cancer high liver metastasis cell line and a preparation method and application thereof. The cell line was preserved in the China Center for Type Culture Collection on January 14, 2026, and the preservation number is CCTCC NO: C202618. The cell line is derived from a mouse colorectal cancer cell line MC38, and is constructed by lentivirus transfection to express luciferase stably, and is obtained by continuously performing at least five rounds of liver metastasis tumor orthotopic iteration screening in C57BL / 6 mice through rectal submucosal injection. The MC38-P06 cell line provided by the application has a significantly enhanced liver metastasis ability, a shorter MC38-P01 model time, a higher liver tumor load, and a shorter mouse survival period, and can be used for screening and evaluating anti-liver metastasis drugs, researching liver microenvironment regulation mechanisms, and identifying liver metastasis related diagnostic markers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a colorectal cancer cell line with high liver metastasis, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is a prevalent malignant tumor worldwide, primarily occurring in the colon (large intestine) or rectum. It is characterized by high incidence and mortality rates. According to global cancer statistics in 2024, colorectal cancer is the third leading cause of cancer-related deaths globally. Metastasis, especially liver metastasis, is a major cause of death in colorectal cancer patients. In colorectal cancer diagnosis, 20% of patients present with metastatic disease, and another 25% with localized disease subsequently develop metastases; the median survival for untreated patients with liver metastases is only 6.9 months, and the 5-year survival rate for patients with unresectable liver metastases is less than 5%. Therefore, constructing accurate and reproducible colorectal cancer metastasis models will contribute to a deeper understanding of metastasis mechanisms, the development of novel anti-metastatic drugs, and the identification of effective metastasis biomarkers, thereby improving patient survival rates and clinical treatment outcomes.

[0003] Existing metastasis models mostly involve single injection via the tail vein or spleen. While convenient, these methods essentially bypass the crucial pathological steps of "primary lesion invading the basement membrane → penetrating blood vessels → circulating tumor cell survival → target organ capture → extravasation and colonization," leading to a severe disconnect from the biological trajectory of clinical recurrence and metastasis. For example, patent CN116463291A reports a paired organoid cell line for human colorectal cancer primary lesions and liver metastases, along with its culture method and applications. It isolates tumor cells from the colonic lesions and liver metastases of a patient with right-sided colon cancer and synchronous liver metastases, and establishes paired organoid cell lines CWH22 and CLM22 for human colorectal cancer primary lesions and liver metastases through 3D culture. This invention uses a single spleen injection to construct a tumorigenic animal model of liver metastases, making it difficult to accurately simulate the entire process of human colorectal cancer from primary lesion to metastases.

[0004] These skip-transfer models suffer from problems such as single metastatic sites, uncontrollable tumor bearing time, and large batch-to-batch variability. The liver metastasis rate after intrasplenic injection of the same dose of MC38 varies from 20% to 70%, and often presents false metastases due to massive cell embolization of microvessels, failing to accurately reflect the tumor's intrinsic metastatic potential. Furthermore, the MC38 cell line itself exhibits only low spontaneous metastatic capacity in wild-type C57BL / 6 mice; conventional orthotopic cecal inoculation requires 8-12 weeks to observe less than 30% of liver metastases, resulting in a long cycle and low success rate, failing to meet the needs of rapid, efficient, and reproducible high-metastasis research. More critically, existing methods lack the iterative concept of "orthotopic microenvironmental immune pressure—cyclic selection—reimplantation enrichment," failing to dynamically enrich and genetically fix metastatic clones. This leads to unstable passage and loss of heterogeneity even when high-metastatic subpopulations are occasionally obtained.

[0005] Therefore, there is an urgent need in this field for an MC38 cell line that can metastasize to the liver within 6 weeks with a reproducibility rate of ≥90% and can be stably maintained in vitro for a long period of time, as well as a standardized construction protocol to provide a reliable, efficient and clinicopathological tool for metastasis mechanism research, anti-metastasis drug screening and metastasis biomarker discovery. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a colorectal cancer cell line with high liver metastasis. This cell line has a significantly enhanced liver metastasis capacity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A colorectal cancer cell line with high liver metastasis, wherein the cell line is the mouse colorectal cancer cell line MC38-P06, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: C202618.

[0009] Preferably, the cell line is derived from mice.

[0010] Preferably, the cell line is derived from MC38 cells.

[0011] The second objective of this invention is to provide a daughter cell line obtained by passage culture of the aforementioned colorectal cancer high liver metastasis cell line.

[0012] The third objective of this invention is to provide a method for constructing the aforementioned colorectal cancer cell line with high liver metastasis.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] The method for constructing a colorectal cancer cell line with high liver metastasis includes the following steps:

[0015] S1: MC38 cells were infected with lentivirus carrying luciferase-purinemycin dual reporter genes, and MC38-P01 cells stably expressing Luc-Puro were obtained by screening.

[0016] S2: The MC38-P01 cells obtained in S1 were seeded in situ into C57BL / 6 mice to establish an in situ tumor model;

[0017] S3: Tumor cells were isolated from mouse liver metastases and highly metastatic subpopulations were obtained through in vitro amplification and puromycin screening;

[0018] S4: Repeat steps S2 to S3 for at least 4 rounds to perform in vivo iterative screening and obtain colorectal cancer cell lines with high liver metastasis.

[0019] This invention provides an iterative screening process of "cecal in situ implantation → in vivo metastatic lesion enrichment → in vitro expansion → re-implantation in situ". This method can increase the spleen metastasis rate from 10% to 100% within 5 generations and shorten the median survival by 50%. This cell line can be used for metastasis mechanism research, anti-metastasis drug screening, and metastasis marker discovery.

[0020] Preferably, in S1, the multiplicity of infection (MOI) is 10; screening is performed using puromycin after transfection.

[0021] Preferably, in S2, the MC38-P01 cells are inoculated into C57BL / 6 mice via submucosal injection in the rectum.

[0022] Preferably, in step S2, the number of cells injected submucosally into the rectum is 5 × 10^5 cells, suspended in 50 μL M Atrigel matrix gel. More preferably, the insulin needle is inserted 0.5 cm into the distal cecum.

[0023] Preferably, in S2, the C57BL / 6 mice are selected from male mice aged 6-8 weeks, fasted for 12 hours before surgery, and anesthetized with sodium pentobarbital.

[0024] Preferably, in S2, IVIS imaging is used to monitor the signal of the primary lesion on postoperative days 7 and 14. The fluorescence intensity of the ROI in the spleen region is ≥1×10⁻⁶. 8 p / s / cm² / sr is defined as a positive metastasis.

[0025] Preferably, in S3, the obtained liver metastases are processed with digestive fluid, filtered, lysed with red blood cells, and screened with puromycin to obtain a single-cell suspension with a purity >98%.

[0026] Preferably, in S3, the digestive fluid contains collagenase IV, hyaluronidase, and DNase I.

[0027] Preferably, in S3, the digestion temperature is 30~45℃, more preferably 37℃; the digestion time is 1~3 hours, more preferably 2 hours.

[0028] Preferably, S3 specifically includes: aseptically removing liver metastases, digesting them with a mixed digestion solution of collagenase IV, hyaluronidase and DNase I at 37°C for 2 hours, filtering them, treating them with red blood cell lysis buffer, and maintaining them with puromycin for 5-7 days to obtain tumor cells with a purity greater than 98%.

[0029] Preferably, when the liver signal is ≥1×10^8 p / s / cm 2 During / sr, liver metastases are aseptically removed.

[0030] Preferably, in S4, the in vivo iterative screening is performed in at least five rounds, and each round of screening uses no less than 15 mice to ensure screening efficiency.

[0031] Preferably, the liver metastasis rate of the colorectal cancer cell line with high liver metastasis rate is ≥70%.

[0032] The fourth objective of this invention is to provide an application of the aforementioned highly metastatic MC38 cell line and / or the aforementioned progeny cell line in the preparation of a mouse model of highly metastatic colorectal cancer.

[0033] The fifth objective of this invention is to provide the application of the aforementioned high-hepatic metastasis MC38 cell line and / or the aforementioned progeny cell lines in screening and / or evaluating anti-colorectal cancer liver metastasis drugs, antibodies and / or small molecules.

[0034] The sixth objective of this invention is to provide an application of the aforementioned high-hepatic-metastasis MC38 cell line and / or the aforementioned progeny cell line in the preparation of cell models and / or animal models for studying the mechanism of colorectal cancer liver metastasis, screening or identifying liver metastasis markers, studying the tumor-immune microenvironment, evaluating the intervention effect of combined surgery / radiotherapy / immunotherapy regimens on liver metastasis recurrence, and dynamically monitoring serum markers.

[0035] To achieve the above objectives, the present invention adopts the following technical solution:

[0036] The aforementioned MC38 cell line with high liver metastasis and / or the aforementioned progeny cell lines are used in the preparation of cell models and / or animal models for studying the mechanism of liver metastasis in colorectal cancer, screening or identifying liver metastasis markers, studying the tumor-immune microenvironment, evaluating the intervention effect of combined surgery / radiotherapy / immunotherapy on liver metastasis recurrence, and dynamically monitoring serum markers.

[0037] Preferably, the liver metastasis markers include proteins, exosomal RNA, and / or ctDNA.

[0038] Preferably, the liver metastasis marker is derived from serum or tissue.

[0039] The beneficial effects of this invention are as follows:

[0040] 1. This invention provides an efficient and reproducible liver metastasis model. The MC38 cell line (MC38-P06), constructed through multiple iterations and selection, can achieve liver metastasis with a reproducibility rate of ≥90% within 6 weeks, significantly improving the efficiency and reproducibility of the liver metastasis model.

[0041] 2. The MC38-P06 cell line of the present invention is genetically stable and still has high transfer potential even after being cryopreserved for 6 months, ensuring the reliability and consistency of experimental results.

[0042] 3. The MC38-P06 cell line of the present invention has a significantly higher in vitro proliferation capacity than the parental cell line MC38-P01, which provides convenience for long-term in vitro culture.

[0043] 4. The construction method of this invention covers a series of key pathological steps, such as "primary lesion invading the basement membrane → penetrating blood vessels → survival of circulating tumor cells → capture of target organs → extravasation and colonization", making the model more consistent with the biological trajectory of clinical recurrence and metastasis.

[0044] 5. The MC38-P06 cell line of the present invention can be applied to multiple fields such as screening anti-metastatic drugs, studying metastasis mechanisms, discovering metastasis markers, and studying the tumor-immune microenvironment, providing an efficient, reliable, and reproducible new tool for research on the metastasis mechanism and translational medicine of colorectal cancer. Attached Figure Description

[0045] Figure 1 A schematic diagram of constructing a mouse orthotopic model of colorectal cancer by submucosal injection in the rectum;

[0046] Figure 2 Figure 1 shows the results of the proliferation experiment of MC38-P01 and MC38-P06 CCK8.

[0047] Figure 3 The diagram shows the survival curves of the spleen capsule of MC38-P01 and MC38-P06 cells under in situ modeling conditions; where A is the survival curve of the spleen capsule and B is the survival curve of the in situ modeling conditions.

[0048] Figure 4 The images show a macroscopic comparison of metastatic lesions formed by MC38-P01 and MC38-P06 in the liver of mice; where A is a macroscopic image of metastatic lesions formed by MC38-P01 in the liver of mice, and B is a macroscopic image of metastatic lesions formed by MC38-P06 in the liver of mice.

[0049] Figure 5HE comparison images of metastatic lesions formed by MC38-P01 and MC38-P06 in mouse liver; where A is HE image of metastatic lesions formed by MC38-P01 in mouse liver, and B is HE image of metastatic lesions formed by MC38-P06 in mouse liver.

[0050] Figure 6 Comparison of in vivo fluorescence signals in spleen 14 days after injection of MC38-P01 and MC38-P06; where A is the in vivo fluorescence signal in spleen 14 days after injection of MC38-P01, and B is the in vivo fluorescence signal in spleen 14 days after injection of MC38-P06. Detailed Implementation

[0051] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0052] This invention provides a colorectal cancer cell line with high liver metastasis, named the mouse colorectal cancer cell line MC38-P06 Mus musculus. The cell line was deposited at the China Center for Type Culture Collection on January 14, 2026, with accession number CCTCC NO: C202618, and the address of the depository is Wuhan University, Wuhan, China.

[0053] Example 1. Iterative establishment of the high-transfer subpopulation MC38-P06

[0054] (1) Cells and animals

[0055] The mouse colorectal cancer cell line MC38 was purchased from ATCC (Manassas, VA, USA). This cell line had undergone cell line identification and monthly mycoplasma testing, which showed negative results. The culture medium used for cell culture was purchased from Gibco, and fetal bovine serum was purchased from ExCell Bio.

[0056] C57BL / 6 wild-type male mice, 6-8 weeks old and weighing 18-22g, were purchased from Jicui Yaokang and housed in an SPF barrier system at a constant temperature of 22±2℃ with a 12h light / dark cycle and free access to food and water.

[0057] (2) Lentiviral markers

[0058] Lentiviral virus carrying a luciferase gene marker resistant to puromycin was added to MC38 cells. The medium was changed after 24 hours, and 5 μg / mL puromycin was added after 48 hours for selection. Parental cells stably expressing Luc-Puro were obtained and named MC38-P01. The linear range of fluorescence signal detected by IVIS was 1 × 10⁻⁶. 4 ~1×10 7 cells, R 2 >0.99.

[0059] (3) Cecal implantation surgery (round 1)

[0060] The process of MC38-P01 cell injection into the rectal submucosa and liver metastasis formation in C57BL / 6 mice is as follows: Figure 1 As shown. The specific steps include: ① Preoperative preparation: Mice were fasted for 12 hours, and anesthesia was induced by sodium pentobarbital (3%) + maintained (1.5%); ② After disinfection, a 1cm incision was made along the midline of the abdomen to expose the cecum; ③ 50μL of Matrigel (Corning, 356234) suspension was drawn using a 1mL insulin needle, containing 5×10 5 MC38-P01 cells were injected into the serosa of the terminal cecum; ④ The needle was slowly rotated to prevent leakage of the suspension; ⑤ The cecum was returned to its original position and the abdomen was closed in layers.

[0061] (4) Live imaging monitoring

[0062] Postoperative days 7 and 14: D-fluorescein potassium salt 150 mg / kg was injected intraperitoneally, followed by IVIS imaging 10 minutes later. Regions of interest (ROIs) with fluorescence intensity ≥1×10⁻⁶ were selected. 8 p / s / cm² / sr is defined as "transfer positive".

[0063] The results showed that the positive rate of metastasis in the MC38-P01 group on day 14 was 10% (2 / 20).

[0064] (5) Acquisition of metastatic foci and preparation of single-cell suspension

[0065] ① Positive mice were euthanized by cervical dislocation and soaked in 75% alcohol for 3 minutes;

[0066] ②The liver was aseptically removed and rinsed twice with PBS;

[0067] ③ Cut into pieces <1mm 3 Add 2 mL of digestion solution to the small pieces: RPMI-1640 + 2% FBS + 1 mg / mL Collagenase IV + 0.5 mg / mL Hyaluronidase + 200 U / mL DNase I;

[0068] ④ Shake at 37℃ and 80rpm for 2 hours;

[0069] ⑤ Grinding and filtering with a 70μm filter screen, 4℃, 300g, 5 min;

[0070] ⑥ Add 0.5 mL of erythrocyte lysis buffer (Biosharp, BL503A) for 30 seconds, then terminate with PBS;

[0071] ⑦ After centrifugation, resuspend in complete DMEM containing 10% FBS, 1% P / S, and 4 μg / mL puromycin, and seed into 10 mm cell culture dishes, and culture at 37°C and 5% CO2.

[0072] ⑧ Change the medium after 48 hours and continue maintenance selection with 2 μg / mL puromycin for 5-7 days, changing the medium every 2 days; all untransfected host cells will die, leaving only Luc-Puro cells. + Tumor cells;

[0073] ⑨ Trypsin digestion, wash twice with PBS to obtain primary high-transfer subpopulation F1 with a purity >98%, which can be used directly for the next round of amplification or cryopreserved.

[0074] (6) In vitro expansion and reimplantation

[0075] MC38-P01 cells were cultured in DMEM containing 10% FBS, 1% P / S, and 1 μg / mL puromycin at 37°C and 5% CO2, and passaged every 4 days. Cells in the logarithmic growth phase were used for the next round.

[0076] Repeat steps (3) to (5) for a total of 4 rounds to obtain MC38-P02 to MC38-P06. The number of animals in each round is ≥15, and ensure that ≥10 animals survive to the end.

[0077] The fifth generation, namely MC38-P06 of this invention, was deposited at the China Center for Type Culture Collection on January 14, 2026, with accession number CCTCC NO: C202618.

[0078] (7) Determination of in vitro proliferation capacity to quantify the in vitro proliferation advantage of MC38-P06 compared with parental P01

[0079] ① Take P01 and P06 cells in the logarithmic growth phase, digest them with 0.25% trypsin, and count them at a ratio of 2 × 10⁻⁶. 3 100µL / well was inoculated into a 96-well plate (n=6 replicates) and cultured in DMEM + 10% FBS + 1% P / S + 1µg / mL puromycin.

[0080] ② Incubate at 37℃ and 5% CO2. Add 10µL of CCK-8 reagent per well at 0, 24, 48, 72 and 96 h respectively, and continue incubation for 1 h.

[0081] ③ Measure the absorbance (OD) at 450 nm, subtract the blank control, and plot the time-OD curve.

[0082] ④Statistical analysis: Two-way ANOVA (Time×Group) was used, and the slope difference was calculated using GraphPad Prism9; P<0.01 was considered significant.

[0083] Result: As Figure 2 As shown in the image, the proliferation rate of MC38-P06 is significantly higher than that of MC38-P01.

[0084] (8) Survival analysis

[0085] ① MC38-P01 (n=15) and MC38-P06 (n=15) were implanted in cecum in situ according to step (3). Postoperative weight and activity level were monitored daily.

[0086] ② Ethical endpoint: Primary lesion signal ≥1.5×10 10 An event is defined as a decrease in p / s or body weight of ≥20%, and cervical dislocation results in euthanasia.

[0087] ③Statistical analysis: Kaplan-Meier method to draw survival curves, Log-rank test.

[0088] ④ Pathological review: Complete tissue samples were taken immediately after death and fixed with 4% paraformaldehyde. The cause of death was confirmed to be multiple organ failure caused by a large primary lesion or liver metastasis. There were no surgical accidents leading to death.

[0089] Results: The survival curves of the spleen capsule of MC38-P01 and MC38-P06 cells compared with those of the in situ model are as follows: Figure 3 As shown, the median survival was 26 days in the P06 group and 52 days in the P01 group (P<0.01). The MC38-P01 group had a relatively longer survival time than the MC38-P06 group.

[0090] (9) Verification of the genetic stability of MC38-P06

[0091] MC38-P06 cells were cryopreserved in liquid nitrogen for 6 months, then thawed and administered again at 5×10⁶ cells per cell line. 5 Cells / mouse were orally implanted into 10 mice. On day 14, the spleen metastasis rate was still 100%, and the signal intensity was 1.8 × 10⁻⁶. 8 p / s / cm 2 / sr confirms genetic stability.

[0092] Example 2. Evaluation of multi-organ metastasis

[0093] In 20 mice in the MC38-P06 group, IVIS signal reached 1×10 9Euthanasia at p / s, systematic dissection and histological verification: ① Spleen: 100% (20 / 20), Luc⁺ seen in small animal in vivo imaging; ② Liver: 70% (14 / 20), HE showed metastatic lesions.

[0094] Results: Anatomical diagram as shown Figure 4 As shown in the figure, this illustrates that MC38-P06 mice exhibit more and larger liver metastases. A comparison of HE-induced metastases in the mouse liver of MC38-P01 and MC38-P06 is provided. Figure 5 As shown in the figure, the morphology of MC38-P01 and MC38-P06 metastatic lesions in the liver is consistent.

[0095] Example 3. Validation of the liver metastasis potential of MC38-P06

[0096] This invention utilizes a spleen injection model to rapidly verify the liver metastasis potential of P06 within 14 days. The specific steps include: ① Ten 6-week-old male C57BL / 6 mice were randomly divided into two groups, P01 and P06 (n=5). ② After anesthesia, the mice were placed in the left lateral decubitus position, and an oblique incision was made 1 cm below the left costal margin to expose the lower pole of the spleen; 20 µL of PBS suspension (containing 5 × 10⁻⁶ mg / L) was slowly injected subsplenically using an insulin needle. 5 (Cells), localized whitening without leakage was observed. ③ The spleen was returned to its original position, the abdomen was closed, and D-fluorescein 150 mg / kg was injected intraperitoneally on postoperative day 14. IVIS imaging was performed 10 minutes later. ④ Data acquisition: The liver ROI was defined, and the total throughput (p / s) was recorded. ⑤ Ethical endpoint: Signal ≥ 1.5 × 10⁻⁶ 10 Euthanasia was performed immediately when p / s or body weight decreased by ≥20%, but the experiment was not terminated prematurely.

[0097] Results: Comparison of in vivo fluorescence signals in splenic imaging 14 days after injection of MC38-P01 and MC38-P06: Figure 6 As shown, the average signal of group P06 is (1.5±0.3)×10 10 p / s, P01 group (4.0±0.1)×10 9 p / s, fold difference 3.75 (Mann–Whitney U test, P < 0.01). This figure illustrates a significant increase in fluorescence signal in the liver region of MC38-P06 mice.

[0098] The above results demonstrate that the present invention can obtain a highly metastatic cell line MC38-P06 with a liver metastasis rate of 70% within 5 generations; this cell line is genetically stable, has enhanced in vitro function, and clear molecular characteristics; the constructed standardized in situ model can be used for evaluating the efficacy of anti-metastatic drugs, dynamically monitoring serum biomarkers, and studying the tumor-immune microenvironment, providing an efficient, reliable, and reproducible new tool for research on the metastasis mechanism and translational medicine of colorectal cancer.

Claims

1. A colorectal cancer cell line with high hepatic metastasis, characterized in that, The cell line in question is the mouse colorectal cancer cell line MC38-P06, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202618.

2. The cell line according to claim 1, characterized in that, The cell line was derived from mice.

3. The cell line according to claim 1, characterized in that, The cell line was derived from MC38 cells.

4. Daughter cell lines obtained by passage culture of the colorectal cancer high liver metastasis cell lines according to any one of claims 1 to 3.

5. The method for constructing a colorectal cancer cell line with high hepatic metastasis according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1: MC38 cells were infected with lentivirus carrying luciferase-purinemycin dual reporter genes, and MC38-P01 cells stably expressing Luc-Puro were obtained by screening. S2: The MC38-P01 cells obtained in S1 were seeded in situ into C57BL / 6 mice to establish an in situ tumor model; S3: Tumor cells were isolated from mouse liver metastases and highly metastatic subpopulations were obtained through in vitro amplification and puromycin screening; S4: Repeat steps S2 to S3 for at least 4 rounds to perform in vivo iterative screening and obtain colorectal cancer cell lines with high liver metastasis.

6. The construction method according to claim 5, characterized in that, In S2, the MC38-P01 cells were seeded into C57BL / 6 mice via submucosal injection in the rectum.

7. The construction method according to claim 5, characterized in that, In S3, the obtained liver metastases were processed with digestive fluid, filtered, lysed with red blood cells, and screened with puromycin to obtain a single-cell suspension with a purity >98%.

8. The use of the MC38 cell line with high liver metastasis as described in any one of claims 1 to 3 and / or the progeny cell line as described in claim 4 in the preparation of a mouse model of highly metastatic colorectal cancer.

9. The use of the MC38 cell line with high liver metastasis as described in any one of claims 1 to 3 and / or the progeny cell line as described in claim 4 in screening and / or evaluating drugs, antibodies and / or small molecules for treating colorectal cancer liver metastasis.

10. The application of the MC38 cell line with high liver metastasis as described in any one of claims 1 to 3 and / or the progeny cell line as described in claim 4 in the preparation of cell models and / or animal models for studying the mechanism of liver metastasis in colorectal cancer, screening or identifying liver metastasis markers, studying the tumor-immune microenvironment, evaluating the intervention effect of combined surgery / radiotherapy / immunotherapy regimens on liver metastasis recurrence, and dynamically monitoring serum markers.

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