Construction method and application of highly metastatic cell strain for directional colorectal cancer liver metastasis

Abstract

The invention relates to a construction method and application of a high metastasis cell strain for orienting colorectal cancer liver metastasis, and the construction method specifically comprises the following steps: constructing a stably transfected cell strain CT26-TL for stably expressing tdTomato and Luciferase by using a gene editing technology; a CT26-TL cell strain is used as an initial cell strain, a screening process is optimized to screen a cell strain with directional transfer, and the mouse colon cancer cell clone strain CT26-TL-CL9 with directional and stable liver high metastasis is finally obtained through three rounds of spleen injection screening, three rounds of cecum injection and spleen injection screening and six rounds of cecum injection screening. And the liver transfer rate can reach 100%. The CT26-TL-CL9 cell strain can simulate the whole process of intestinal cancer from a primary site to liver metastasis, and an animal model constructed by the CT26-TL-CL9 cell strain can be used for researching a molecular mechanism of colorectal cancer liver metastasis and developing drugs targeting colorectal cancer liver metastasis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. CN202411268634.5 filed on September 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing a highly metastatic cell line directed toward colorectal cancer liver metastasis and its application, specifically to applying the highly metastatic cell line to research on inhibiting colorectal cancer liver metastasis. Background Art

[0004] At present, the methods for constructing animal models of colorectal cancer liver metastasis include: (1) by injecting colorectal cancer cells CT26 into the cecal serosa of mice, an animal model of cecal-liver metastasis starting from the cecum is constructed, and its metastasis rate is only 40% (Zhang L, BuP. Generation of an orthotopic mouse model to study colorectal cancer metastasis [J]. STAR protocols, 2021, 2 (4)); (2) by subcutaneous injection of CT26 cell line to form subcutaneous tumors, after 2 weeks, the subcutaneous tumors are digested into single cell suspensions and cultured for two weeks, and then the cells are injected into the cecal serosa; after 4 weeks, the liver metastases are digested into single cell suspensions, and then injected into the cecal serosa. After a total of 3 rounds of screening, a highly metastatic cell line CT26-FL3 capable of spontaneous liver metastasis is obtained, and the metastasis rate reaches 80%-90% (Zhang Y, Davis C, Ryan J, et al. Development and characterization of a reliable mouse model of colorectal cancer metastasis to the liver[J].Clinical&experimental metastasis,2013,30:903-918.); (3) Chinese patent CN110760477A discloses a method for constructing a mouse liver highly metastatic colorectal cancer cell line, which involves labeling mouse colorectal cancer cells with antibiotics and bio-optical markers, and then inoculating the cells into the subcutaneous part of the mouse to form a tumor; after 4 weeks or when the tumor volume exceeds 2 cm 3 The mice were then killed, and the subcutaneous tumor tissue formed was digested into a single cell suspension, which was screened and cultured in vitro based on antibiotic resistance, and then cells that were positive for biomarkers were selected; the positive cells were injected into the subserosal layer of the cecum, and 4-6 weeks later or when the in situ tumor in the mouse grew to 1 cm 3The liver metastases of the mice were then removed, screened and cultured in vitro based on antibiotic resistance, and then cells that were positive for biomarkers were selected; the screening was repeated three times to obtain a colorectal cancer cell line with a liver metastasis rate of 80% after in situ injection into the cecum.

[0005] However, the above method has the following shortcomings: (1) When the CT26.WT cell line is injected into the serosa of the cecum of BABL / c mice, tumors can be formed in the cecum, but at the end of the experiment, the tumor cells injected into the cecum did not metastasize to the liver; (2) According to the method reported in the literature, CT26.WT is injected subcutaneously, and then the CT26.WT subcutaneous tumor is digested and cultured, and then inoculated into the serosa of the mouse cecum. After three rounds of screening, it is actually impossible to reproduce the 80% liver metastasis of CT26-FL3 cells; (3) After the CT26.WT cells are injected into the serosa of the mouse cecum, it is impossible to monitor the tumor growth and tissue distribution in real time in the mouse body; (4) The modeling cycle is generally long. Summary of the invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention utilizes gene editing technology to construct a stable cell line CT26-TL that stably expresses tdTomato and Luciferase; using the CT26-TL cell line as the starting cell line, the screening process is optimized for screening cell lines with directional metastasis, and finally the CT26-TL-CL9 cell line with directional and stable high liver metastasis is obtained, which can significantly shorten the modeling time to 3 weeks, and the liver metastasis rate reaches 100%.

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

[0008] The first aspect of the present invention is to provide a method for constructing a highly metastatic cell line directed toward colorectal cancer liver metastasis, comprising the steps of:

[0009] Step S1, using lentivirus as a vector to infect CT26.WT cells, and obtaining a stable cell line CT26-TL stably expressing tdTomato and Luciferase by flow cytometry sorting;

[0010] Step S2, constructing a liver metastasis model by injecting the CT26-TL cells obtained in step S1 into the spleen, and digesting and amplifying the metastatic lesions in vitro after the mice have liver metastasis; injecting the in vitro amplified CT26-TL cells into the spleen again, and performing 3 rounds of screening in total;

[0011] Step S3, injecting the CT26-TL cells obtained in step S2 into the cecum in situ, and injecting CT26.WT cells into the spleen at the same time, and after the mice have liver metastasis, digesting the metastatic lesions and amplifying and culturing them in vitro; sorting the CT26-TL cells with tdTomato fluorescent protein by flow cytometry, and injecting CT26.WT cells into the cecum in situ, and injecting CT26.WT cells into the spleen at the same time, for a total of 3 rounds of screening;

[0012] Step S4, injecting the CT26-TL cells obtained in step S3 into the cecum in situ to construct a cecum in situ liver metastasis model, and after liver metastasis occurs in mice, digesting the metastatic lesions and amplifying and culturing them in vitro; injecting the in vitro amplified CT26-TL cells into the cecum in situ again, performing a total of 6 rounds of screening, and finally obtaining a highly metastatic cell line with targeted colorectal cancer liver metastasis, which is named CT26-TL-CL9 cell line.

[0013] Furthermore, in step S1, the preparation step of the CT26-TL cell line includes:

[0014] Step S1-1, preparing virus packaging reagent: adding transfection reagent p-PEI, virus expression plasmid pCDH-tdTomato-Luciferase, virus packaging plasmid pMD2.G, and virus packaging plasmid psPAX2 in a predetermined ratio into serum-free culture medium;

[0015] Step S1-2, adding the virus packaging reagent (preferably 4 mL) to 293FT cells (preferably with a cell density of 80%), incubating for a predetermined time (preferably at 37° C., 5% CO2 for 6 h), and then adding fresh culture medium; after culturing for a predetermined time (preferably after culturing for 24 h), discarding the culture medium of the 293FT cells, adding serum-free culture medium (preferably 10 mL), and amplifying the virus (preferably at 32° C., 5% CO2 for 72 h);

[0016] Step S1-3, after the virus is amplified for a predetermined time (preferably after culturing for 72 h), the supernatant culture medium of 293FT cells is collected, filtered (preferably using a 0.45 μm filter), centrifuged (preferably at 4 ° C, 20000 rpm, centrifuged for 100 min), the supernatant is discarded, the culture medium (preferably 2 mL) is added to resuspend the virus, and then the infection enhancer (preferably HB, added at a ratio of 1:1000, the final concentration is 8 mg / mL) is added, and the virus is added to CT26.WT cells (preferably 5×10 5 The cells were inoculated in a 6 cm culture dish and cultured (preferably at 37° C., 5% CO 2 for 24 h);

[0017] Step S1-4: Perform flow cytometry sorting on the cells obtained in step S1-3 to obtain CT26-TL positive cell lines expressing tdTomato and Luciferase.

[0018] Furthermore, the step S1-1 specifically includes the steps of adding 30 μL of transfection reagent p-PEI, 3 μg of viral expression plasmid pCDH-tdTomato-Luciferase, 1.87 μg of viral packaging plasmid pMD2.G, and 3.43 μg of viral packaging plasmid psPAX2 to 4 mL of serum-free DMEM HG culture medium, mixing well, and standing at room temperature for 5 minutes.

[0019] Furthermore, the serum-free culture medium in step S1-1 is serum-free DMEM HG culture medium without 10% FBS; the serum-free culture medium in step S1-2 is serum-free DMEM HG culture medium without 10% FBS; the culture medium for the remaining 293FT cells is DMEM HG culture medium containing 10% FBS; and the culture medium for resuspension is RPMI-1640 culture medium containing 10% FBS.

[0020] It is understandable that the above-mentioned step of screening positive cells by flow cytometry can be performed by conventional steps.

[0021] Furthermore, in the spleen injection step of step S2, CT26-TL cells were resuspended in PBS solution to obtain a cell resuspension. In the first round of screening, the injected cell concentration was 10 7 / mL, in the second round of screening, the injection cell concentration was 3×10 6 / mL, in the third round of screening, the injection cell concentration was 1×10 6 / mL, and the injection volume in each round was 100 μL of cell resuspension.

[0022] Furthermore, in the cecal in situ injection step of step S3, the cells were diluted to 1×10 6 / 15μL, an equal amount of matrix gel was added to the cell suspension to obtain a cell suspension, and the injection volume for each round was 30μL of cell suspension; in the spleen injection step of step S3, CT26.WT cells were resuspended in PBS solution to obtain a concentration of 1×10 6 / mL of cell resuspension, the injection volume in each round was 100 μL of cell resuspension, and the cells obtained in the last round of screening were named CT26-TL-CL3 cell line.

[0023] Furthermore, in the cecal serosa injection step of step S4, CT26-TL-CL3 cells were diluted to 1×10 6 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension, and the injection volume for each round is 30μL of cell suspension.

[0024] Furthermore, in the above-mentioned construction method, the method for obtaining tumor cells by digestion from liver metastases includes the steps of: cutting the metastasis tissue into pieces, digesting the tissue, and forming the tissue into a single cell suspension; filtering the cell suspension, centrifuging, and discarding the supernatant; adding complete cell culture medium and resuspending the cell pellet; adding the resuspended cells to a cell culture dish and culturing overnight; the next day, discarding the culture medium, adding fresh complete cell culture medium, and waiting for the cells at the bottom of the culture dish to grow to a coverage of 60%-80%, the cells are passaged and amplified.

[0025] It is understandable that the reagents, culture conditions, etc. used in the above method of obtaining tumor cells from liver metastases by digestion can refer to conventional methods in the art.

[0026] Furthermore, the CT26-TL cell line is used for in vivo imaging or in vitro organ imaging after modeling.

[0027] Furthermore, the liver metastasis rate of the CT26-TL-CL9 cell line was 100%.

[0028] The second aspect of the present invention is to provide a highly metastatic cell line for directional colorectal cancer liver metastasis, which is a mouse colon cancer cell clone CT26-TL-CL9 Mus musculus, with a preservation number of CCTCC NO: C2024306, a preservation date of September 5, 2024, and a preservation unit of the China Center for Type Culture Collection (abbreviated as CCTCC). The preservation unit address is Wuhan University, Wuhan, China.

[0029] Furthermore, the CT26-TL-CL9 cell line is obtained by any construction method described in the first aspect.

[0030] Furthermore, the preservation and resuscitation method of the CT26-TL-CL9 cell line is as follows: CT26-TL-CL9 cells are frozen using a cell freezing solution containing 90% FBS and 10% DMSO, the cells are first placed in a -80°C refrigerator for cooling, and after 24 hours, the cells are transferred to liquid nitrogen for long-term storage; when the cells need to be used, the cells are taken out of the liquid nitrogen, quickly placed in a 37°C water bath to melt, centrifuged, the supernatant is discarded, the cells are resuspended in culture medium, inoculated, and cultured.

[0031] The third aspect of the present invention is to provide a highly metastatic cell line of targeted colorectal cancer liver metastasis obtained by any construction method described in the first aspect, or the use of a highly metastatic cell line of targeted colorectal cancer liver metastasis as described in any one of the second aspects in constructing an animal model of colorectal cancer liver metastasis.

[0032] Furthermore, the colorectal cancer liver metastasis animal model includes a colorectal cancer in situ-liver metastasis model.

[0033] The fourth aspect of the present invention is to provide a method for constructing a mouse model of colorectal cancer liver metastasis, which comprises injecting a highly metastatic cell line of targeted colorectal cancer liver metastasis obtained by any of the construction methods described in the first aspect or a highly metastatic cell line of targeted colorectal cancer liver metastasis obtained by any of the construction methods described in the second aspect into the cecal serosa of mice to construct a mouse model of colorectal cancer liver metastasis.

[0034] Furthermore, in the above construction method, the CT26-TL-CL9 cell line was diluted to 1×10 6 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension, and the cecal serosa injection volume is 30μL of cell suspension.

[0035] Furthermore, a colorectal cancer liver metastasis mouse model was successfully established 3 weeks after injection of the CT26-TL-CL9 cell line, with a liver metastasis rate of 100%.

[0036] The fifth aspect of the present invention is to provide an application of a colorectal cancer liver metastasis mouse model obtained by any of the construction methods described in the fourth aspect, which is selected from at least one of the following applications: use as an animal model in screening components that promote or inhibit colorectal cancer liver metastasis or in screening for colorectal cancer metastasis-related preventive or therapeutic drugs, and use as an animal model in studying the mechanism of colorectal cancer liver metastasis.

[0037] It is understandable that the above-mentioned components or drugs include antibiotics, traditional Chinese medicine ingredients, antagonistic bacteria or bacteriophages against liver metastasis pathogens, etc.

[0038] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0039] The present invention obtains a CT26-TL-CL9 cell line with a high metastasis rate (up to 100%) by optimizing the screening method of colorectal cancer cell lines with stable liver metastasis, which provides a feasible method for screening cell lines with directional metastasis. The cell line CT26-TL-CL9 with high liver metastasis obtained by the present invention can simulate the entire process of colorectal cancer metastasis from the primary site to the liver. The animal model of colorectal cancer liver metastasis constructed by using the CT26-TL-CL9 cell line can achieve a 100% liver metastasis rate in about three weeks. The constructed animal model can be used as an ideal model for studying the molecular mechanism of colorectal cancer liver metastasis and developing drugs targeting colorectal cancer liver metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0041] Figure 1 This is a schematic diagram of screening a stable and highly liver-directed metastatic cell line CT26-TL-CL9 in the present invention;

[0042] Figure 2 It is a schematic diagram of the results of obtaining CT26-TL positive cell lines expressing tdTomato and Luciferase by flow sorting in one embodiment of the present invention.

[0043] Figure 3 It is a schematic diagram of the signal imaging results of a colorectal cancer in situ-liver metastasis model constructed with CT26-TL-CL9 in one embodiment of the present invention; wherein, part A is in vivo imaging; part B is ex vivo liver tumor signal imaging; and part C is ex vivo spleen, kidney, heart, lung and brain signal imaging.

[0044] The above-mentioned colorectal cancer liver metastasis cell line CT26-TL-CL9 has been preserved. It is a mouse colon cancer cell clone CT26-TL-CL9 Mus musculus, with a preservation number of CCTCC NO: C2024306, a preservation date of September 5, 2024, and a preservation unit of the China Center for Type Culture Collection (CCTCC for short). The preservation unit address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials and reagents without the source in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0046] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0047] In certain embodiments of the present invention, Figure 1The screening schematic diagram shows a method for constructing a highly metastatic cell line for colorectal cancer liver metastasis, comprising the following steps: (1) using lentivirus as a vector to infect CT26.WT cells, and obtaining a stably transfected cell line CT26-TL that stably expresses tdTomato and Luciferase by flow cytometry sorting; (2) optimizing the screening process: 1) firstly injecting CT26-TL cells into the spleen to construct a liver metastasis model, and after the mice have liver metastasis, taking the metastatic lesions for digestion and in vitro amplification culture; then continuing to inject the in vitro amplified CT26-TL cells into the spleen for 3 rounds of screening; 2) subsequently, injecting CT26-TL cells into the cecum in situ, and injecting CT26.WT cells into the spleen at the same time. cells, and after the mice had liver metastasis, the metastatic lesions were digested and expanded in vitro; the CT26-TL cells with tdTomato fluorescent protein injected in situ into the cecum were used to flow sort the CT26-TL cells with tdTomato fluorescent protein; the sorted CT26-TL cells were further subjected to 3 rounds of screening; 3) finally, the CT26-TL cells were injected in situ into the cecum to construct a cecal in situ liver metastasis model, and after the mice had liver metastasis, the metastatic lesions were digested and expanded in vitro; then the in vitro expanded CT26-TL cells were injected in situ into the cecum again, and 6 rounds of screening were performed to obtain a stable and directional liver metastasis CT26-TL-CL9 cell line (the deposit number is CCTCC NO: C2024306).

[0048] In the following embodiment, the method for obtaining tumor cells by digestion from liver metastases includes the following steps: (1) at the end of the experiment, the mouse is euthanized, the abdominal cavity is opened, and the metastatic lesions in the liver are removed; (2) the metastatic lesions are placed in a sterile 1.5 mL EP tube, rinsed with sterile PBS three times, and the tissue is cut into small pieces with a diameter of about 1 mm; (3) (type II) collagenase (Yisheng, 40508ES60) with a final concentration of 1 mg / mL is added, and the tissue is incubated in a 37°C incubator for 1 hour for tissue digestion; (4) a 1 mL pipette tip is used to repeatedly blow and aspirate the tissue to form a cell suspension, and then the cell suspension is passed through a 70 μm filter to filter out inadequately digested tissue pieces; the cell digestion solution is transferred to a 15 mL centrifuge tube, 2 mL HBSS buffer (Gibco, 14025092) is added, and the tube is centrifuged at 1000 rpm for 5 minutes, and the supernatant is discarded; and 2 mL Repeat the washing twice with HBSS; (5) Add 2 mL of complete culture medium to resuspend the cell pellet; the complete culture medium contains RPMI-1640 (Gibco, 11875093), 10% fetal bovine serum (Cegrogen, A0500-3010), 1×GlutaMAXTM (Gibco, 35050061), and 100 U / ml penicillin-streptomycin (Gibco, 15140148); (6) Add the resuspended cells to a 6 cm cell culture medium. The cells were cultured overnight in a dish; (7) The next day, the culture medium was discarded, and the surface of the adherent cells was gently washed with PBS, repeated twice, and 2 mL of fresh complete culture medium was added; (8) When the cells at the bottom of the culture dish grew to 60%-80% coverage, trypsin was used for digestion, and then culture medium was added to terminate the process. The cell suspension was transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; (9) After the cells were resuspended, the cell suspension was divided into three equal parts and added to the cell culture dish containing culture medium for continued culture. The complete culture medium used for screening the different CT26-TL cell lines obtained above was the same as the complete culture medium required for CT26.WT culture, which contained RPMI-1640 (Gibco, 11875093), 10% fetal bovine serum (Cegrogen, A0500-3010), 1×GlutaMAXTM (Gibco, 35050061), and 100 U / ml penicillin-streptomycin (Yamei, CB010).

[0049] The following embodiments are only used to illustrate the technical solutions of the present invention.

[0050] Example 1 - Construction of CT26-TL cell line by lentiviral transfection

[0051] This example is to construct a stable cell line stably expressing tdTomato and Luciferase, and the specific steps include:

[0052] (1) Preparation of virus packaging reagent: Add 30 μL of transfection reagent p-PEI (Polysciences, 23966), 3 μg of virus expression plasmid pCDH-tdTomato-Luciferase, 1.87 μg of virus packaging plasmid pMD2.G (addgene, 12259) and 3.43 μg of virus packaging plasmid psPAX2 (addgene, 12260) to 4 mL of serum-free DMEM HG (Gibco, 11965092) culture medium (not containing 10% FBS), mix well, and let stand at room temperature for 5 min.

[0053] (2) Add 4 mL of virus packaging reagent to 293FT cells at a density of 80% and incubate at 37°C, 5% CO2 for 6 h.

[0054] (3) After 6 hours, freshly prepared DMEM HG medium containing 10% FBS (Cegrogen, A0500-3010) was slowly added.

[0055] (4) After 24 hours, the culture medium of 293FT cells was discarded, and 10 mL of serum-free DMEM HG medium (not containing 10% FBS) was added and cultured at 32° C., 5% CO 2 for 72 hours to amplify the virus.

[0056] (5) After 72 h, the supernatant culture medium of 293FT cells was collected and filtered through a 0.45 μm filter into a virus centrifuge tube. The culture medium was centrifuged at 4°C, 20,000 rpm, for 100 min and the supernatant was discarded.

[0057] (6) Add 2 mL of RPMI-1640 medium containing 10% FBS to resuspend the virus, and then add infection enhancer HB (8 mg / mL, Sigma, 107689) at a dilution of 1:1000. 5 The cells were inoculated into 6 cm culture dishes, and 24 h later, the virus was added into CT26.WT cells (ATCC CRL-2638) and cultured at 37° C., 5% CO 2 for 24 h.

[0058] (7) After 24 h, add 2 mL of freshly prepared RPMI-1640 medium containing 10% FBS and culture at 37°C, 5% CO2 for 24 h.

[0059] (8) Digestion was performed using trypsin (Gibco, 27250-018), and then terminated by adding RPMI-1640 medium containing 10% FBS. The cell suspension was transferred into a centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0060] (9) Add 10 mL of PBS to resuspend and centrifuge at 1000 rpm for 5 min.

[0061] (10) Add 1 mL of serum-free RPMI-1640 medium (not containing 10% FBS), resuspend the cells, and add the cells to a flow cytometry tube. At the same time, prepare a flow cytometry recovery tube and add 300 μL of culture medium to the flow cytometry recovery tube.

[0062] (11) When sorting on the machine, the control group is negative cells that do not express fluorescence. The cells are sorted using a sorting flow cytometer and the PE channel is selected to obtain CT26-TL positive cell lines expressing tdTomato and Luciferase (such as Figure 2 shown).

[0063] Example 2 - Screening of the highly metastatic cell line CT26-TL-CL9 with colorectal cancer-directed liver metastasis

[0064] This embodiment is a screening and optimization scheme for highly metastatic cell lines of colorectal cancer directed liver metastasis, which specifically includes the following steps:

[0065] (1) Digest the CT26-TL cells obtained in Example 1, filter the cells through a 70 μm filter to obtain single cells, centrifuge and resuspend them in an appropriate volume of PBS to adjust the cell concentration to 10 7 / mL.

[0066] (2) The mice that had been previously depilated were anesthetized by induction and maintenance of anesthesia with 1-3% isoflurane, and the epidermis and peritoneum were cut open by a scalpel for about 0.5 cm at the lower edge of the left rib cage of the mouse.

[0067] (3) Gently pick out the mouse spleen with a sterile cotton swab and place it on a sterile gauze moistened with saline to prepare for cell injection. Thoroughly mix the cell suspension and draw up 100 μL of the cell suspension with a 29G insulin syringe. Insert the needle into the spleen about 1.5 cm at a straight angle and slowly inject the cell suspension.

[0068] (4) Gently press with a cotton swab until the blister produced by the injection disappears, then remove the insulin needle. Continue pressing the needle hole for 2-3 minutes.

[0069] (5) Use No. 4 suture to ligate the blood vessels at both ends of the spleen and remove the spleen.

[0070] (6) Suture the wound of the mouse, and then place the mouse on a 37°C heating pad to keep warm and wait for it to recover. Place the recovered mouse in a sterile cage, mark the date of surgery, and return it to a ventilated cage for breeding.

[0071] (7) The experimental endpoint was 4 weeks after modeling. The mice were euthanized, the liver metastases were digested, and the obtained cells were expanded and cultured. The cells were digested and the cell concentration was adjusted to 3×10 6 / mL CT26-TL.

[0072] (8) The second round of spleen injection modeling was performed using the same procedure.

[0073] (9) At the end of the experiment, liver metastases were collected and the obtained cells were expanded and cultured. The cells were digested and the cell concentration was adjusted to 1×10 6 / mL CT26-TL.

[0074] (10) Perform the third round of spleen injection using the same procedure.

[0075] (11) At the end of the experiment, liver metastases were collected and the obtained cells were expanded and cultured.

[0076] (12) The digested cells were resuspended in RPMI-1640 medium containing 10% FBS (Cegrogen, A0500-3010), centrifuged again, and the supernatant was discarded.

[0077] (13) Resuspend the cells in a small amount of serum-free RPMI-1640 medium (not containing 10% FBS), take 10 μL of the cell suspension and dilute it appropriately, and count the live cells.

[0078] (14) The cells were diluted to an appropriate concentration using serum-free RPMI-1640 medium (without 10% FBS) for subsequent construction of a mouse model of cecal orthotopic colorectal cancer liver metastasis. The cell concentration was adjusted to 1×10 6 / 15μL CT26-TL.

[0079] (15) Add an equal amount of Matrigel (Yisheng, 40183ES10) to the cell suspension diluted to an appropriate concentration, mix well, and place on ice. Pipette 30 μL of the cell suspension into a 30G insulin syringe and place on ice for later use.

[0080] (16) Anesthesia was induced and maintained in mice with pre-depilated abdomen using 1-3% isoflurane. The epidermis and peritoneum were cut open about 1 cm along the linea alba about 1.5 cm above the mouse anus.

[0081] (17) The cecum was gently picked out using a cotton swab moistened with saline and placed on a sterile gauze moistened with saline in preparation for cell injection.

[0082] (18) Gently press one side of the cecum with a cotton swab moistened with saline, and place the prepared insulin needle tip with the cell suspension facing upwards. Carefully pierce the cecum subserosa and inject the cell suspension into the subserosa. After successful injection, a transparent bubble-like structure will bulge on the surface of the serosa. Allow the needle tip to remain in the subserosa for about 20 seconds, remove the needle, and immediately glue the needle hole with a small amount of medical glue.

[0083] (19) At the same time, the spleen of the mouse was gently picked out with a sterile cotton swab and placed on a sterile gauze moistened with saline to prepare for cell injection. The cell suspension was thoroughly mixed and 100 μL of the cell suspension (1×10 6 / mLCT26.WT). Insert the needle into the spleen at a straight angle for about 1.5 cm and slowly inject the cell suspension.

[0084] (20) Gently press with a cotton swab until the blister produced by the injection disappears and then remove the insulin needle. Continue to press the needle hole for 2-3 minutes.

[0085] (21) Use No. 4 suture to ligate the blood vessels at both ends of the spleen and remove the spleen.

[0086] (22) The wound of the mouse was sutured and the mouse was placed on a 37°C heating pad to keep warm. After the mouse recovered, it was placed in a sterile cage, marked with the date of surgery, and returned to a ventilated cage for breeding.

[0087] (23) The experimental endpoint was 3 weeks after modeling. The mice were euthanized, the liver metastases were digested and cultured, and the cells expressing tdTomato were obtained by flow cytometry and named CT26-TL-CL1. The cell concentration was adjusted to 1×10 6 / 15μL CT26-TL-CL1, and the second and third rounds of screening were performed using the same operation method to obtain the CT26-TL-CL3 cell line.

[0088] (24) The cell concentration was adjusted to 1×10 6 / 15μL CT26-TL-CL3, then add an equal amount of matrix gel, mix well and place on ice. Pipette 30μL of cell suspension into a 30G insulin syringe and place on ice for later use.

[0089] (25) Gently press one side of the cecum with a cotton swab moistened with saline, and place the prepared insulin needle tip with the cell suspension facing upwards. Carefully pierce the cecum subserosa and inject the cell suspension into the subserosa. After successful injection, a transparent bubble-like structure will bulge on the surface of the serosa. Allow the needle tip to remain in the subserosa for about 20 seconds, remove the needle, and immediately glue the needle hole with a small amount of medical glue.

[0090] (26) The wound of the mouse was sutured and the mouse was placed on a 37°C heating pad to keep warm. After the mouse recovered, it was placed in a sterile cage, marked with the date of surgery, and returned to a ventilated cage for breeding.

[0091] (27) The experimental endpoint was 3 weeks after modeling, when the mice were euthanized and the liver metastases were harvested for digestion and culture to obtain CT26-TL-CL4. The same operation method was used for rounds 2 to 6 of screening to obtain the CT26-TL-CL9 cell line.

[0092] The above CT26-TL-CL9 cell line has been preserved, and its preservation number is CCTCC NO: C2024306. CT26-TL-CL9 cells were frozen using a cell freezing solution containing 90% FBS and 10% DMSO. The cells were first placed in a -80°C refrigerator for cooling; after 24 hours, the cells were transferred to liquid nitrogen for long-term storage. When the cells need to be used, the cells were taken out of the liquid nitrogen, quickly placed in a 37°C water bath to melt, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in RPMI-1640 medium containing 10% FBS, and the cells were inoculated in a cell culture dish and placed in a 37°C cell culture incubator for culture.

[0093] Example 3 - Construction of a mouse model of colorectal cancer liver metastasis using cell line CT26-TL-CL9

[0094] In this example, the cell line CT26-TL-CL9 obtained in Example 2 was used to construct a mouse model of colorectal cancer liver metastasis, which specifically includes the following steps:

[0095] (1) Cecal modeling was performed in the same manner as in Example 2: After digestion, the cells were resuspended in a small amount of serum-free RPMI-1640 medium without 10% FBS, and the concentration of CT26-TL-CL9 cells was adjusted to 1×10 6 / 15μL, then add an equal amount of matrix gel, mix well and place on ice; draw 30μL of cell suspension into a 30G insulin syringe and inject into the subserosa of the cecum of mice.

[0096] (3) At the end of the experiment, 3 weeks later, in vivo imaging detected 100% of the mice with in situ cecal tumor formation ( Figure 3 Part A), ex vivo liver imaging detected 100% spontaneous liver metastasis in mice with the cecum orthotopic model ( Figure 3 Part B), while there was no metastatic signal in the spleen, kidney, heart, lung and brain ( Figure 3 of the FDA).

[0097] The above experimental results indicate that the CT26-TL-CL9 cell line is a colorectal cancer cell line with 100% liver metastasis, and the model can be successfully constructed in 3 weeks, greatly shortening the modeling time.

[0098] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for constructing a highly metastatic cell line directed toward liver metastasis of colorectal cancer, characterized in that: Includes steps: Step S1, using lentivirus as a vector to infect CT26.WT cells, and obtaining a stable cell line CT26-TL stably expressing tdTomato and Luciferase by flow cytometry sorting; Step S2, constructing a liver metastasis model by injecting the CT26-TL cells obtained in step S1 into the spleen, and digesting and amplifying the metastatic lesions in vitro after the mice have liver metastasis; injecting the in vitro amplified CT26-TL cells into the spleen again, and performing 3 rounds of screening in total; Step S3, injecting the CT26-TL cells obtained in step S2 into the cecum in situ, and injecting CT26.WT cells into the spleen at the same time, and after the mice have liver metastasis, digesting the metastatic lesions and amplifying and culturing them in vitro; sorting the CT26-TL cells with tdTomato fluorescent protein by flow cytometry, and injecting CT26.WT cells into the cecum in situ, and injecting CT26.WT cells into the spleen at the same time, for a total of 3 rounds of screening; Step S4, injecting the CT26-TL cells obtained in step S3 into the cecum in situ to construct a cecum in situ liver metastasis model, and after liver metastasis occurs in mice, digesting the metastatic lesions and amplifying and culturing them in vitro; injecting the in vitro amplified CT26-TL cells into the cecum in situ again, performing a total of 6 rounds of screening, and finally obtaining a highly metastatic cell line with targeted colorectal cancer liver metastasis, which is named CT26-TL-CL9 cell line.

2. The construction method according to claim 1, characterized in that: In step S1, the preparation steps of the CT26-TL cell line include: Step S1-1, preparing virus packaging reagent: adding transfection reagent p-PEI, virus expression plasmid pCDH-tdTomato-Luciferase, virus packaging plasmid pMD2.G, and virus packaging plasmid psPAX2 in a predetermined ratio into serum-free culture medium; Step S1-2, adding a virus packaging reagent to 293FT cells, incubating for a predetermined time, and then adding fresh culture medium; after culturing for a predetermined time, discarding the culture medium of 293FT cells, adding serum-free culture medium, and amplifying the virus; Step S1-3, after the virus is amplified for a predetermined time, the supernatant culture medium of the 293FT cells is collected, filtered, centrifuged, the supernatant is discarded, the culture medium is added to resuspend the virus, an infection enhancer is added, and the virus is added to CT26.WT cells for culture; Step S1-4: Perform flow cytometry sorting on the cells obtained in step S1-3 to obtain CT26-TL positive cell lines expressing tdTomato and Luciferase.

3. The construction method according to claim 1, characterized in that: In the spleen injection step of step S2, CT26-TL cells were resuspended in PBS solution to obtain a cell resuspension. In the first round of screening, the injection cell concentration was 10 7 / mL, in the second round of screening, the injection cell concentration was 3×10 6 / mL, in the third round of screening, the injection cell concentration was 1×10 6 / mL, and the injection volume in each round was 100 μL of cell resuspension; In the cecal in situ injection step of step S3, cells were diluted to 1×10 6 / 15μL, an equal amount of matrix gel was added to the cell suspension to obtain a cell suspension, and the injection volume for each round was 30μL of cell suspension; in the spleen injection step of step S3, CT26.WT cells were resuspended in PBS solution to obtain a concentration of 1×10 6 / mL of cell resuspension, and the injection volume in each round was 100μL of cell resuspension. The cells obtained in the last round of screening were named CT26-TL-CL3 cell line; In the cecal serosa injection step of step S4, CT26-TL-CL3 cells were diluted to 1×10 6 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension, and the injection volume for each round is 30μL of cell suspension.

4. The construction method according to claim 1, characterized in that: The method for obtaining tumor cells by digestion in liver metastasis includes the following steps: cutting up metastasis tissue, digesting the tissue, and forming the tissue into a single cell suspension; filtering the cell suspension, centrifuging, and discarding the supernatant; adding complete cell culture medium, and resuspending the cell pellet; adding the resuspended cells to a cell culture dish and culturing overnight; discarding the culture medium the next day, adding fresh complete cell culture medium, and when the cells at the bottom of the culture dish grow to a coverage of 60%-80%, the cells are passaged and amplified.

5. The construction method according to claim 1, characterized in that: The CT26-TL cell line is used for in vivo imaging or in vitro organ imaging after modeling, and the liver metastasis rate of the CT26-TL-CL9 cell line is 100%.

6. A highly metastatic cell line directed towards liver metastasis of colorectal cancer, characterized in that: The cell line is a mouse colon cancer cell clone CT26-TL-CL9 Mus musculus, and the deposit number is CCTCC NO: C2024306.

7. Use of a highly metastatic cell line for targeted colorectal cancer liver metastasis obtained by the construction method described in any one of claims 1 to 5, or a highly metastatic cell line for targeted colorectal cancer liver metastasis as described in claim 6 in constructing an animal model of colorectal cancer liver metastasis.

8. A method for constructing a mouse model of colorectal cancer liver metastasis, characterized in that: A mouse model of colorectal cancer liver metastasis is constructed by injecting the highly metastatic cell line directed to colorectal cancer liver metastasis obtained by the construction method described in any one of claims 1 to 5 or the highly metastatic cell line directed to colorectal cancer liver metastasis described in claim 6 into the cecal serosa of mice.

9. The construction method according to claim 8, characterized in that: CT26-TL-CL9 cells were diluted to 1×10 6 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension, and the cecal serosa injection volume is 30μL of cell suspension.

10. An application of a mouse model of colorectal cancer liver metastasis obtained by the construction method according to claim 8 or 9, characterized in that: The application is selected from at least one of the following applications: use as an animal model in screening components that promote or inhibit colorectal cancer liver metastasis or in screening colorectal cancer metastasis-related preventive or therapeutic drugs, and use as an animal model in studying the mechanism of colorectal cancer liver metastasis.

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