A method for constructing and validating a high-metastasis model of colorectal cancer based on metastatic selection pressure

Abstract

The present invention relates to a method for constructing and validating a high-metastasis model of colorectal cancer, and belongs to the field of biotechnology. Specifically, the present invention applies in vitro and in vivo selective evolutionary pressure to a colorectal cancer cell line (LoVo) to obtain a cell line (LoVo-HM) CCTCC NO: C2024232 with high metastasis ability; the screened LoVo-HM cell line and the LoVo cell line are verified using in vitro migration and invasion experiments and in vivo tumorigenesis experiments in animals, proving that the model was successfully established. The LoVo-HM cell line constructed by the present invention has strong migration, invasion and metastasis abilities, providing a new vector for the study of the mechanism of colorectal cancer liver metastasis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing and verifying a high-metastasis model of colorectal cancer. Background Art

[0002] The main metastatic site of CRC is the liver, with approximately 50%-60% of CRC patients developing liver metastasis, which is also one of the main causes of death in CRC patients. In recent years, although comprehensive systemic treatments such as surgery, targeted therapy, and immunotherapy have been able to prolong and improve patient survival and prognosis to a certain extent, the five-year survival rate of patients with CRC liver metastasis remains low, at only approximately 25%-40%. The occurrence and development of CRC liver metastasis is complex and is not only closely related to the aggressiveness of the tumor, but also involves multiple molecular mechanisms, including intercellular adhesion, matrix metalloproteinase activity, tumor microenvironment, immune escape, and other factors. Therefore, it is necessary to construct an efficient and stable CRC cell line with liver metastasis ability, which is of great significance for exploring the mechanisms of its pathogenesis and metastasis, as well as further exploring treatment strategies.

[0003] In the study of CRC liver metastasis models, CRC liver metastasis models are typically established through splenic injection or orthotopic colorectal cancer implantation. Because the liver is a highly heterogeneous organ, metastasis from tumors at other primary sites exerts multiple selective pressures on tumor cells. Only tumor cells that adapt to this environment can successfully metastasize and colonize the liver. Based on this theory, researchers have begun exploring ways to mimic the selective pressures of the liver microenvironment to generate CRC cell lines with enhanced metastatic potential. Currently, a common approach is to obtain highly metastatic cell lines through repeated in vitro and in vivo metastasis screening. For example, CRC cells are injected into the tail vein or portal vein of immunodeficient mice to induce the formation of liver metastases. Cancer cells from these metastases are then isolated, cultured in vitro, and injected back into mice. After multiple rounds of screening, CRC cell lines with strong liver metastasis potential, such as the HCT116 cell line, are ultimately obtained. However, this approach has limitations in simulating liver metastasis, including incomplete resemblance of the tumor microenvironment to human disease and low metastasis efficiency. This low metastasis efficiency is particularly prominent when using only animal models. In recent years, with more and more research on the mechanisms of CRC pathogenesis and metastasis, it has become particularly important to construct an efficient and reliable CRC liver metastasis cell line, which has also become a research hotspot in this field.

[0004] Although there have been some research results on the establishment of CRC liver metastasis models, there is still a lack of a systematic method to accurately simulate the liver metastasis selection pressure and effectively screen highly metastatic CRC cell lines. Therefore, establishing a method for constructing and validating a CRC high metastasis model based on liver metastasis selection pressure has important research and clinical value. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this study provides a method for constructing and validating a high-metastasis model for colorectal cancer based on metastatic selection pressure. To achieve the objectives of the present invention, the technical solutions proposed in the present invention are as follows:

[0006] The first aspect of the present invention provides a method for constructing a colorectal cancer high metastasis model, comprising the following steps:

[0007] (1) In vitro screening of cell subpopulations with strong migration ability in the colorectal cancer LoVo cell line;

[0008] (2) The LoVo cell subpopulation with strong migration ability was injected into the spleen of nude mice to construct a LoVo cell line with strong liver metastasis ability;

[0009] (3) A secondary in vitro screening of cell subpopulations with stronger liver metastasis ability among the cell lines obtained from the animal model was performed to obtain the LoVo-HM cell line.

[0010] Another aspect of the present invention is to provide a method for verifying the high transfer model constructed by the above construction method.

[0011] In a preferred embodiment of the present invention, the high metastatic ability of the LoVo-HM cell line refers to the fact that the liver metastasis ability and the peritoneal implantation ability of the LoVo-HM cell line are verified by comparison with the LoVo cell line.

[0012] The present invention has at least the following advantages and beneficial effects:

[0013] 1. The present invention uses multiple and repeated metastatic selection pressures to more realistically simulate the process of colorectal cancer cell metastasis in the human body, and can prepare a stable and reliable tumor cell line with high metastatic potential.

[0014] 2. Through multiple rounds of in vitro and in vivo experiments, the present invention screened out a tumor cell line with high adaptability to liver metastasis. The biological characteristics of this cell line are closer to the actual situation of tumor cell metastasis in the human body and it is a more representative biological model.

[0015] 3. The method designed in the present invention can reduce the experimental errors caused by the heterogeneity of colorectal cancer cells in the process of preparing the metastasis model and reduce the number of repeated model constructions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1a : Using the Transwell method, cell subpopulations with strong migration ability were screened in the colorectal cancer LoVo cell line, and this step was repeated three times;

[0018] Figure 1b : The LoVo cell subpopulation with strong migration ability was injected into the spleen of nude mice to construct a LoVo cell line with strong liver metastasis ability. This step was repeated three times.

[0019] Figure 1c : Using the Transwell method, a secondary in vitro screening of cell subpopulations with stronger metastatic ability among the cell lines obtained from the animal model was performed. This step was repeated three times to obtain the LoVo-HM cell line;

[0020] Figure 2a : The morphological differences between LoVo cells and LoVo-HM cells were captured by bright field microscopy;

[0021] Figure 2b : LoVo cells and LoVo-HM cells were cultured in vitro and their migration abilities were compared, showing that the migration ability of LoVo-HM cells was significantly enhanced;

[0022] Figure 2c : LoVo cells and LoVo-HM cells were cultured in vitro and their invasion abilities were compared, showing that the invasion ability of LoVo-HM cells was significantly enhanced;

[0023] Figure 3a : LoVo cells and LoVo-HM cells were injected into the spleens of two groups of nude mice, respectively, and the liver metastasis ability of the two cells was compared. The results showed that the weight and number of liver metastases were significantly higher after LoVo-HM cells metastasized to the liver.

[0024] Figure 3b : LoVo cells and LoVo-HM cells were injected into the spleens of two groups of nude mice, respectively, and the peritoneal implantation ability of the two cells was compared. It was shown that after LoVo-HM cells were implanted into the peritoneal cavity, the weight and number of implanted tumors were significantly higher. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0026] Example 1 Preparation and Construction of a Highly Metastatic Colorectal Cancer Model

[0027] 1.1 Preparation of reagents and consumables

[0028] (1) Consumables: 15mL sterile centrifuge tube, 10mL / 25cm 2 Culture flasks, 8 μm pore size Transwell chambers, 30G syringe needles, 70 μm cell strainers, 24-well culture plates, 5 mL rubber-tipped pipettes, 10-1000 μL pipettes, pipette tips, filters, sterile gloves, sterile pipettes, and aluminum foil.

[0029] (2) Reagents: 75% ethanol, sterile PBS solution, trypsin-EDTA solution, methanol;

[0030] (3) Culture medium: DMEM-high glucose medium containing 20% ​​FBS, serum-free DMEM-high glucose medium, 10% fetal bovine serum, 1% penicillin-streptomycin solution;

[0031] (4) Equipment: small animal operating microscope, 37°C constant temperature incubator, centrifuge.

[0032] 1.2 Pretreatment of LoVo cell lines

[0033] (1) LoVo cell recovery and culture: Take out the frozen LoVo cell line from liquid nitrogen, quickly thaw the cell suspension in a 37°C water bath for about 1-2 minutes, wipe the outer surface of the cryovial immediately after thawing with 75% ethanol, transfer the thawed cells to a 15 mL sterile centrifuge tube in a clean bench, add 5-10 mL of preheated DMEM-highglucose medium containing 20% ​​FBS and 1% penicillin-streptomycin and shake well, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add fresh DMEM-highglucose medium containing 20% ​​FBS to resuspend the cells, inoculate the cell resuspension into a preheated culture flask and add an appropriate amount of culture medium, place the culture flask in a constant temperature incubator at 37°C and 5% CO2, and observe the cell status every 1-2 days;

[0034] (2) Cell passaging: When the cells reach 80-90% mixed state, pour out the old culture medium in the culture flask, wash the cells once with 2-3 mL of preheated PBS solution, add 1 mL of trypsin-EDTA solution to cover the cell surface, incubate at room temperature for 1-2 minutes, add an appropriate amount of preheated DMEM-high glucose culture medium containing 20% ​​FBS to neutralize the trypsin, and then gently blow the cells with a pipette to form a single cell suspension and transfer to a sterile centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and re-add culture medium to resuspend the cells to ensure good cell viability.

[0035] 1.3 Screening of LoVo cell subpopulations with strong migration ability

[0036] (1) Cell preparation: LoVo cells were digested with trypsin and counted at 2×10 6 The cells were suspended in serum-free medium at a concentration of cells / mL;

[0037] (2) Cell seeding and culture: The chamber was inserted into a 24-well plate, and 600 μL of DMEM-high glucose medium containing 20% ​​FBS was added to the lower well of the 24-well plate as a chemoattractant. 100 μL of LoVo cells suspended in serum-free medium was added to the upper well of the Transwell chamber. The 24-well plate was placed in an incubator at 37°C and 5% CO2 and incubated for 24 h.

[0038] (3) Collect the lower layer of cells and prepare a suspension: remove the Transwell chamber on the upper layer of the 24-well plate, use a sterile pipette to gently transfer the culture medium in the lower layer of the 24-well plate to a 15 mL sterile centrifuge tube, add 1-2 mL sterile PBS, mix well, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, retain the precipitated cells, add an appropriate amount of serum-free DMEM-high glucose medium, gently blow the bottom of the centrifuge tube to completely resuspend the cell pellet, and adjust the cell concentration to 2 × 10 6 cells / mL and continue culturing until the cells expand to a certain number;

[0039] (4) Repeat the above steps (1)-(3) 3 times to screen out the LoVo cell subpopulation with strong migration ability and continue to culture ( Figure 1a ).

[0040] 1.4 Construction of a LoVo cell line with strong metastatic capacity in nude mice

[0041] (1) Cell preparation: The LoVo cell subpopulation with strong migration ability screened in the above steps was digested with trypsin, washed, and then suspended in physiological saline to adjust the concentration to 3×10 6 cells / mL;

[0042] (2) Nude mouse spleen injection: In a sterile environment, 100 μL of cell suspension was injected into the spleen of the nude mouse using a syringe with a 30G needle. The nude mouse was then placed in a sterile cage. The health status of the nude mouse was regularly observed and in vivo imaging was performed if necessary.

[0043] (3) Treatment of liver metastatic tumor cells: Nude mice were sacrificed after 4 weeks. Liver metastasis was observed using in vivo imaging technology and then dissected. Metastases in the liver were confirmed using sectioning and HE staining. The number and size of metastatic lesions were observed. Liver tissue with metastatic lesions was removed and placed in 5 mL of sterile PBS. The liver was cut into approximately 1-2 mm sections using sterile surgical tools. 3 Place the cut pieces into 2-3 mL of digestion solution containing digestive enzymes and incubate at 37°C for 30-60 minutes. Filter the digestion solution through a 70 μm cell sieve and wash the cell pellet with PBS. Centrifuge at 1000 rpm for 5 minutes and resuspend the cell pellet in DMEM-high glucose medium and continue culturing until the cells expand to a certain number.

[0044] (4) Repeat the above steps (1)-(3) three times to obtain the LoVo cell line with strong metastatic ability in the animal model, and continue to culture ( Figure 1b ).

[0045] 1.5 Secondary screening to obtain LoVo-HM cell line: Repeat steps 1.3(1)-(3) for 3 times with the LoVo cell line with strong metastatic ability in the animal model, and obtain LoVo-HM cell line through secondary in vitro screening ( Figure 1c ), the LoVo-HM cell line was sent to the preservation center for preservation. The preservation date was November 21, 2024, the preservation number was CCTCC NO: C2024232, and the preservation name was highly metastatic human colon cancer cell line LoVo-HM (Homosapiens).

[0046] Example 2: Verification of the high metastatic capacity of LoVo-HM cell line (CCTCC NO: C2024232)

[0047] 2.1 Consumables and Animal Preparation

[0048] (1) Consumables: 15mL sterile centrifuge tube, 10mL / 25cm 2 Culture flasks, 35 mm culture dishes, 8 μm pore size Transwell chambers, 30G needle syringes, 70 μm cell strainers, 24-well culture plates, 5 mL rubber-tipped pipettes, 10-1000 μL pipettes, pipette tips, filters, sterile gloves, sterile pipettes, aluminum foil, surgical tools (scalpels, forceps, sutures);

[0049] (2) Reagents: 75% ethanol, sterile PBS solution, trypsin-EDTA solution, methanol, crystal violet solution, anesthetic (isoflurane);

[0050] (3) Culture medium: DMEM-high glucose medium containing 20% ​​FBS;

[0051] (4) Equipment: small animal operating microscope, 37°C constant temperature incubator, centrifuge, inverted phase contrast microscope, cell counter, ruler;

[0052] (5) Experimental animals: BALB / c nude mice.

[0053] 2.2 Cell morphology observation

[0054] Culture LoVo and LoVo-HM cells in DMEM-high glucose medium supplemented with 20% FBS at 37°C and 5% CO2. Aspirate the remaining medium from the culture flask and wash the cells 1-2 times with PBS to remove any residual medium. Add an appropriate amount of trypsin-EDTA solution for digestion to prepare a single-cell suspension. Plate an appropriate amount of the single-cell suspension into a new culture dish and culture at an appropriate density for observation.

[0055] Turn on the inverted microscope and adjust the light source to an appropriate brightness. Remove the culture dish and gently remove any excess medium to avoid disturbing the optical path. Place the culture dish on the microscope stage, focus, and observe the cell morphology.

[0056] 2.3 Verification of the high metastatic capacity of the LoVo-HM cell line (CCTCC NO: C2024232)

[0057] (1) In vitro validation

[0058] a. Migration capability:

[0059] LoVo cells were trypsinized and counted at 2 × 10 6 Cells were added to the upper wells of the Transwell chamber at a concentration of 100 μL cells / mL and incubated in an incubator for 24 hours. The upper Transwell chamber of the 24-well plate was removed, and the lower cells were fixed and stained with crystal violet. The cells were photographed under a light microscope and the number of cells in three different fields of view was counted.

[0060] b. Invasiveness:

[0061] After the matrigel was evenly spread on the upper layer of the Transwell chamber, the LoVo cells were digested with trypsin and counted at a concentration of 2×10 6 Cells were added to the upper wells of the Transwell chamber at a concentration of 100 μL cells / mL and incubated in an incubator for 24 hours. The upper Transwell chamber of the 24-well plate was removed, and the lower cells were fixed and stained with crystal violet. The cells were photographed under a light microscope and the number of cells in three different fields of view was counted.

[0062] (2) In vivo animal validation

[0063] a. Liver metastasis ability:

[0064] The LoVo-HM and LoVo cell lines stably expressing luciferase were constructed by lentiviral infection. The two cell lines were cultured to the logarithmic growth phase and the cell concentration was adjusted to 3×10 6 cells

[0065] / mL, and cell counts were performed using a cell counter to ensure high cell viability (>90%). Approximately 100 μL of LoVo-HM and LoVo cell suspensions were injected into the spleens of nude mice, with five nude mice per group. Four weeks later, liver metastasis formation was observed using in vivo imaging. The nude mice were anesthetized, and the metastases in the liver were removed, maintaining the integrity of the liver tissue as much as possible. The size and weight of the liver after metastasis, as well as the number of metastatic lesions, were measured and statistically analyzed.

[0066] b. Abdominal cavity implantation capacity:

[0067] The LoVo-HM and LoVo cell lines stably expressing luciferase were constructed by lentiviral infection. The two cell lines were cultured to the logarithmic growth phase and the cell concentration was adjusted to 3×10 6 cells

[0068] / mL, and cell counts were performed using a cell counter to ensure high cell viability (>90%). Approximately 100 μL of LoVo-HM and LoVo cell suspensions were injected into the peritoneal cavity of nude mice, with 5 nude mice per group. Four weeks later, intraperitoneal tumor formation was observed using in vivo imaging. The nude mice were anesthetized, and liver metastases were removed, maintaining liver tissue integrity as much as possible. The size and weight of the liver after metastasis, as well as the number of metastatic lesions, were measured and statistically analyzed.

[0069] 2.4 Verification Results

[0070] like Figure 2a As shown, under the microscope, LoVo cells were observed to be more uniform in size, with smoother edges, more dispersed cells, larger intercellular spaces, relatively lower density, sparser cell numbers, good adhesion ability, and relatively limited expansion area; while the morphology of the LoVo-HM cell line was longer strips or irregular shapes, some cells had protrusion structures, relatively irregular edges, denser cells, higher density, strong adhesion, and diverse cell morphology.

[0071] like Figure 2b As shown in Figure 2, at the detection time point (24 hours), the LoVo-HM cells in the lower chamber were more densely packed.

[0072] The number of cells was greater, about 1.5 times that of LoVo cells. The statistical results showed that P<0.01, proving that the migration ability of LoVo-HM cells was significantly higher than that of LoVo cells.

[0073] like Figure 2c As shown in the figure, at the detection time point (24 hours), the number of LoVo-HM cells that passed through the matrix gel layer and successfully invaded to the other side of the chamber was much greater than that of LoVo cells, with a ratio of approximately 3-4:1. The statistical results showed that P<0.001, indicating that the invasive ability of LoVo-HM cells was significantly higher than that of LoVo cells.

[0074] like Figure 3a As shown in the results, in vivo imaging technology was used to observe a larger fluorescence range in the nude mice of the LoVo-HM group at the detection time point. After the liver was removed, the liver volume was observed to be enlarged with the naked eye. Statistical results showed that the weight of metastatic tumors increased significantly (P<0.05) and the number increased significantly (P<0.05).

[0075] like Figure 3b As shown in the results, in vivo imaging technology was used to observe a larger fluorescence range in the nude mice of the LoVo-HM group at the detection time point. After the abdominal implanted tumors were removed, the number of implanted tumors seen by the naked eye increased significantly. Statistical results showed that the weight of the implanted tumors increased significantly (P<0.05) and the number increased significantly (P<0.05).

[0076] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A LoVo-HM cell line, characterized in that The cell line was deposited on November 21, 2024, with a deposit number of CCTCC NO: C2024232 and a deposit name of highly metastatic human colon cancer cell line LoVo-HM.

2. Use of the LoVo-HM cell line as described in claim 1 in preparing a highly metastatic model of colorectal cancer metastasis to the liver.

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