CN116463291A - Human colorectal cancer primary focus and liver transfer focus pairing organoid cell line and culture method and application thereof - Google Patents

Abstract

The invention discloses a paired organoid cell line of human colorectal cancer primary focus and liver metastases, a culture method and application thereof, and belongs to the technical field of digestive system tumor cell lines. It includes separating tumor cells from the colon lesion and liver metastases tumor tissue of a patient with right colon cancer combined with simultaneous liver metastases and through 3D culture to obtain the human right colon cancer primary tumor organoid cell line CWH22 with the preservation number CCTCC NO.C202218 and the paired human right colon cancer liver metastases organoid cell line CLM22 with the preservation number CCTCC No. This pair of organoid cell lines can be stably subcultured in vitro, has good tumorigenicity in nude mice, and can be used for research on the pathogenesis, drug resistance mechanism, and new drug screening of colorectal cancer and liver metastasis.

Description

Human colorectal cancer primary tumor and liver metastases paired organoid cell line and its culture Method and Application

technical field

The present invention relates to a process of culturing and establishing a pair of right-sided colon cancer organoids and corresponding liver metastases organoids, and a related animal tumor formation model constructed thereof, belonging to the field of digestive system tumor cell line technology models, and specifically relates to a paired organoid cell line of a primary human colon cancer and a liver metastases, and a culture method and application thereof.

Background technique

Colorectal cancer is one of the most common malignant tumors in humans. At present, the incidence of colorectal cancer in the Chinese population has been on the rise. It accounts for the fifth and fourth incidence of all cancers in men and women, and the fifth death rate among all cancers. The 5-year survival rate of colorectal cancer varies greatly among different stages: 90% for stage I and only 15% for stage IV. The liver is the most common metastatic site of stage IV colorectal cancer. 20-25% of colorectal cancer patients have liver metastases when they are first diagnosed, and 50% of patients develop liver metastases during treatment after primary tumor resection. Although radical surgical resection is the standard treatment for liver metastases from colorectal cancer, less than 20% of patients with liver metastases can undergo good metastasectomy, and all of them inevitably continue to relapse in the liver. Therefore, exploring the potential mechanism, molecular markers and effective treatment strategies of colorectal cancer liver metastasis is extremely important for early blocking or delaying the occurrence of liver metastasis, effectively controlling tumor recurrence and progression, and prolonging the overall survival of patients.

In the research and exploration of tumors, the reliability of research models is the key to effectively transform basic research into clinical application. Tumor organoids have proven to be useful experimental models for studying disease progression, affected signaling pathways, tumor microenvironment, and relevant drug treatments, among others. Colorectal cancer organoids are extracted from patients’ colorectal primary or metastatic tumor tissues and cultured in 3D organoids in matrigel to produce cultures with a three-dimensional spatial structure that are closer to the physiological level in vivo, which can simulate the characteristics of corresponding tumors in vitro to the greatest extent, and realize more translational medical applications, such as predicting the possibility of metastasis in a specific patient, the sensitivity to a specific treatment method, and screening effective drugs for salvage therapy.

The mechanism of liver metastases in colorectal cancer is still unclear, and there is no clear predictor to predict the sensitivity to standard treatment regimens. Patients who are resistant to standard multi-line therapy and have no other options for further treatment are the dilemma of current treatment. Patient-derived organoids play an important role in the study of colorectal cancer metastasis and drug resistance mechanisms, but there are currently no publicly available organoid cell lines. The pair of colon cancer primary tumors and corresponding liver metastases organoid cell lines established by us will play an important role in improving the pertinence and effectiveness of the treatment plan, further screening effective therapeutic drugs, reducing the recurrence rate, and prolonging the survival of patients.

Contents of the invention

In order to achieve the above technical purpose, the present invention discloses a paired organoid cell line of human colorectal cancer primary focus and liver metastases and its culture method and application. The cell line can not only be stably subcultured in vitro, but also has strong proliferative activity and the ability to form nude mouse subcutaneous, cecal in situ and liver metastatic tumors. To provide new in vivo and in vitro research support for further in-depth research on the etiology, pathogenesis, drug resistance mechanism, and new drug screening of colorectal cancer and liver metastasis.

In order to better realize the technical purpose of the present invention, the present invention discloses a paired organoid cell line of the primary human colorectal cancer and liver metastases, including the organoid cell line CWH22 of the primary human right colon cancer with the preservation number CCTCCNO.

Further, the cell line was established by in vitro 3D organoid culture of a patient with right colon cancer complicated with simultaneous liver metastases by simultaneously resecting the primary tumor and the tumor tissue of the corresponding liver metastases.

The second object of the present invention is to disclose a method for cultivating a paired organoid cell line between the primary human colorectal cancer and liver metastases, which includes the following steps:

1) Material collection: 0.5 ^cm3 of tumor tissue from the patient’s primary tumor and corresponding liver metastases were collected during the operation, placed in a sterile sampling tube containing Advanced DMEM/F12, placed in a 4°C ice box, and quickly sent to the laboratory;

2) Digestion: Wash and discard the mucus, necrotic tissue and residual blood cells on the surface of the sample obtained in step 1) in a sterile ultra-clean bench, then rinse once with iodine (for clinical mucous membrane disinfection) and three times with PBS, cut the tissue repeatedly with scissors in a plate, add 2 mL of 50% digestive enzyme TrypLE (diluted in 2 mL of DMEM) for digestion at room temperature, and fully mix the tissue suspension containing digestive enzyme every 5 minutes. About 20 minutes, add an equal volume of Ca ^{2 +} HBSS buffer to neutralize and mix again;

3) Filtration: filter the suspension obtained in step 2) with a filter of not less than 200 mesh diameter, and centrifuge, resuspend the precipitate with 5 mL of culture medium, wash and centrifuge repeatedly to discard necrotic cell fragments, and centrifuge at a speed of 500-1500 r/min;

4) Seed cells: Take the above-mentioned washed cells for live cell counting, resuspend the cells with Matrigel at low temperature to 8000 cells/30 μL, seed in a preheated 24-well plate, 30 μL/well, place in a 37°C incubator for 30 minutes, and add 650 μL of medium to each well after the vaulted Matrigel solidifies;

5) Conventional subculture: the medium was changed every 3 to 5 days, and the subculture operation was performed on the 10th to 14th day. Digest with TrypLE digestive enzyme 250 μL/well, pipette and mix every 5 minutes, digest into a single cell suspension in 10 to 15 minutes, resuspend with HBSS neutralization and centrifugation, and count, continue to pass at 8000 cells per well, and then repeat steps 4) and 5).

The third objective of the present invention is to disclose a paired organoid cell line of the above-mentioned human colorectal cancer primary tumor and liver metastases, which is used to construct an animal model of cecal orthotopic tumor formation and/or an animal model of liver metastases tumor formation, and to study the pathogenesis and drug resistance mechanism of rectal cancer.

The fourth object of the present invention is to disclose a method for establishing a tumor-forming animal model of liver metastases, which includes the following steps:

1) Organoid culture: After the organoid cells were stably overexpressed luciferase with lentivirus infection, the organoids were cultured routinely, and the medium was changed respectively on the 4th, 7th, 9th, and 11th day after passage, and the CWH22 and CLM22 organoids in the logarithmic growth phase (10-12 days of each passage) were taken for nude mouse spleen injection to transfer tumors;

2) Digestion of organoids: the process of digesting and harvesting cells is the same as the conventional subculture operation, try to digest into a single-cell suspension state, and count them, resuspend in DMEM to 1.25*10 ⁷ /mL and put them in a 4°C incubator for later use;

3) Spleen injection: after anesthetizing the nude mice with pentobarbital, fix the nude mice and perform abdominal skin disinfection, make a longitudinal incision of about 1 cm along the left lower costal margin of the abdomen at the front of the axillary line 0.5 cm to the abdominal cavity, gently pick out the spleen and inject about 80 μL of mixed cell suspension from the distal end of the spleen. After 10 minutes, the splenic blood vessels were ligated, the spleen was cut off and sutured, and the spleen was closely observed to ensure smooth recovery.

4) Tumor formation monitoring: regular in vivo imaging was used to observe and record the tumor formation of liver metastases in nude mice.

The fifth object of the present invention is to disclose a method for constructing an animal model of cecum in situ tumor formation, which includes the following steps:

1) Organoid culture: routinely culture luciferase-overexpressing organoids CWH22 and CLM22, and change the medium on the 4th, 7th, 9th, and 11th days after passage, and inject the organoids in the logarithmic growth phase into the cecum wall of nude mice; wherein, each passage grows on the 10th to 12th day;

2) Organoid collection: Collect organoids on the 10th to 12th day, digest with TrypLE digestive enzyme for 5 minutes, neutralize and collect organoid precipitates, about 3*10 ⁶ cells, resuspend in 60 μL Matrigel after pre-cooling, and place in a 4°C incubator for later use;

3) In situ injection of the cecum: after anesthetizing the nude mice with pentobarbital, fix the nude mice and perform abdominal skin disinfection, make a longitudinal incision about 1.5 cm along the midline of the lower abdomen to the abdominal cavity and find the cecum, move the end of the cecum to the center of the field of vision, and draw 20 μL of the organoid suspension with a pre-cooled insulin injection needle for submucosal injection of the cecum. After the injection is completed and no leakage is observed, the abdominal muscles and skin are sutured, and close observation is made to ensure a smooth recovery;

4) Tumor formation monitoring: Regular in vivo imaging was used to observe and record the in situ tumor formation in the cecum of nude mice.

The sixth object of the present invention is to disclose the application of the above-mentioned organoid cell line paired with primary human colon cancer and liver metastases in the preparation of drugs for screening and treating colorectal cancer and/or liver metastases.

Further, the therapeutic drug is administered to an in vitro cell model, and the drug that inhibits cell proliferation or causes cell apoptosis after administration is a therapeutically sensitive drug, or the test compound is administered to an in vitro cell model, and the candidate compound is a compound that inhibits cell proliferation or causes cell apoptosis after administration.

The seventh object of the present invention is to disclose a method for screening tumor drugs for the treatment of colorectal cancer and/or liver metastases, which includes digesting and counting the above-mentioned CWH22 and CLM22 organoids in the logarithmic growth phase, resuspending them in Matrigel and planting them in a 96-well plate, 2*10 ⁵ cells/mL, 10 μL/well, after the Matrigel is solidified, adding 100 μL/well of medium, and culturing for 5 to 6 days. The culture medium continued to be cultured, and the medium was changed once every 3 days. After 6 days of drug action, the 3D cell activity was detected to calculate the IC50 of the corresponding drug.

Further, the drug gradient comprises at least one of the following: 5-fluorouracil, oxaliplatin, irinotecan (SN-38), etoposide (VP-16), sotorasib (sotorasib, AMG-510), regorafenib, dasatinib or bortezomib (PS-341).

Beneficial effect:

1. The human right colon cancer primary focus organoid CWH22 and the corresponding liver metastases organoid CLM22 cultured in the present invention are identified as new cell lines by STR, and come from the same patient, providing new experimental materials close to clinical tumor biology for the study of human colorectal cancer and liver metastasis.

2. The human colorectal cancer primary tumor organoid CWH22 and the corresponding liver metastases organoid CLM22 screened by the present invention have good 3D organoid morphology, stable properties, sustainable and stable passage, and good tolerance to virus infection. Studies have found that they have very strong cecum in situ tumor formation and liver metastasis tumor formation ability by spleen injection, and can successfully prepare animal models of colorectal cancer liver metastasis. Experiments have found that injecting 1 million cells into the cecum in situ or in the spleen can form obvious tumors in about 25 days. It can be used to study the metastasis mechanism of colorectal cancer and the drug sensitivity of comprehensive treatment in nude mice.

3. The human colorectal cancer primary focus organoid cell line CWH22 and the corresponding metastatic focus organoid cell line CLM22 cultivated in the present invention, all exome (WES) sequencing revealed that both had KRAS exon 2 missense mutation (NM_033360.3:p.Gly12Cys/c.34G>T), APC gene frameshift mutation (NM_000038.5:p.Tyr935fs/ c.2805_2806delCA), SMAD4 gene missense mutation (: c.1610A>G), KMT2C missense mutation (NM_170606.2:p.Gln3158His/c.9474G>T) and CDKN2B gene frameshift mutation (NM_004064.4:p.Ser110fs/c.323_329dupATGT CAG) etc. In addition, the common related driver genes NRAS/HRAS/BRAF/PIK3CA, TP53, etc. in colorectal cancer were all wild type. This group of paired organoid cell lines is a typical colorectal cancer organoid cell line with KRAS, APC and SMAD4 mutations at the same time, so it can be used as an effective tool cell for the study of the occurrence and development of colorectal cancer with KRAS/APC/SMAD4 triple mutations and TP53 wild-type subgroup and the mechanism of drug resistance.

Description of drawings

Figure 1 shows the preoperative examinations of the patients from which the CWH22 and CLM22 organoid cells obtained in the present invention are derived. Among them, Fig. 1A shows the preoperative colonoscopy examination, and Fig. 1B shows the preoperative liver CT and MR plain scan of the patient.

Figure 2 shows the results of HE and immunohistochemical staining corresponding to the resected primary tumor and metastatic tumor tissue of the patient in Figure 1. CK20(+), CDX-2(+), Her-2(4B5)(-), Her-2(positive control)(+), P53(scattered+), Ki-67(LI about 60%), MLH1(+), PMS2(+), MSH2(+), MSH6(+), Villin(+). Moderately differentiated colorectal adenocarcinoma was diagnosed based on morphology and immunohistochemistry.

Figure 3 shows the bright field morphology and dynamic growth records of organoids CWH22 and CLM22 obtained from the primary tumor and metastatic tumor tissue cultured in Figure 2. The scale bars are 500 μm and 200 μm, respectively.

Figure 4 is a comparison of the relevant staining conditions of the tumor tissue obtained in Figure 1 and the cultured organoids in Figure 3. Figure 4A shows the macroscopic morphology of the primary tumor and the metastatic tumor tissue taken in Figure 1, HE staining of the tissue section, and the expression of CK20 and CDX2. Figure 4B shows the bright-field morphology of cultured organoids and corresponding HE staining of organoid slices, and the expression of CK20 and CDX2.

Figure 5 shows the whole exon sequencing of CWH22 and CLM22, and the mutation consistency and mutation characteristics analysis of corresponding tumor tissues.

Fig. 6 is a study on liver metastasis and tumor formation of CWH22 and CLM22 cells in nude mice. Figure 6A shows the intrasplenic injection of 1 million CWH22 and CLM22 cells into two groups of nude mice (5 mice/group), live imaging signals on day 45 and observation of metastatic tumor formation in the liver; Figure 6B shows the corresponding HE staining and the corresponding expression of CK20 and CDX2.

Figure 7 shows the macroscopic findings and HE staining of CWH22 organoid cells after cecum injection to form tumors in situ.

Figure 8 is the dose-response curves of 5-fluorouracil, oxaliplatin, irinotecan (SN-38), etoposide (VP-16), sotorasib (AMG-510), regorafenib, dasatinib and bortezomib (PS-341) under the corresponding concentration gradient of cells CWH22 and CLM22 in Figure 3 And the corresponding IC50 test results.

Figure 9 is a comparison of imaging re-examinations of clinical patients before and after 8 courses of mFOLFOX+Bevacizumab treatment.

Detailed ways

Below in conjunction with specific example, further set forth the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

For the experimental methods that do not indicate specific conditions in the following examples, conventional methods in the art can be used, for example, refer to "Molecular Cloning Experiment Guide" (Third Edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions suggested by the supplier.

The present invention relates to the preservation of biological materials, wherein the preservation unit is the China Typical Culture Collection Center, the address is Wuhan University, Wuhan, China, postcode: 430072, and the preservation time is October 21, 2022.

Various instruments, raw materials and reagents not specifically described in the following examples are commercially available products well known in the art and can be obtained through commercial channels. The specific materials used and their sources listed in the following examples are exemplary only and are not intended to limit the present invention. Materials identical or similar to the type, model, quality, property or function of the following tissues, cells, reagents and instruments can be used to implement the present invention.

Example 1

A method for establishing a primary human right colon cancer organoid cell line CWH22 and a corresponding liver metastasis organoid cell line CLM22 is disclosed, which includes the following steps:

1.1 The source of the specimen:

The tumor tissue sample came from a 65-year-old patient with a right-semi-colon mass and liver metastases. He was admitted to the hospital in January 2021 because of "a physical examination found that the liver mass occupied more than half a month." Plain CT and MR scans showed "a huge blocky mass in the left lobe-caudal lobe of the liver, considering neoplastic lesions." Colonoscopy showed "new growths in the ascending colon, Ca?". Later, "right hemicolectomy + left hemihepatectomy" was performed in our hospital at the same time. Postoperative pathological examination showed: moderately differentiated adenocarcinoma of the right colon, invading the subserosa of the intestinal wall; (1) Proximal resection margin of the anastomotic ring: no cancer; (2) Distal resection margin of the anastomotic ring: no cancer; (3) Radial resection margin/mesangial vessel stump: no cancer, focally adjacent to the resection margin; (4) vessel invasion: visible; (5) nerve invasion: no; (6) mesenteric lymph nodes: 13 no cancer; (7) extralymphatic tumor implantation ( ENTD): Visible, N=1. (8) Carcinoma was seen in segment IV, segment V, segment VIII of the liver, and beside the common hepatic artery; (9) Cancer was not found in one lymph node of the gallbladder, appendix, and hepatoduodenal ligament. Immunohistochemistry: MLH1(+), PMS2(+), MSH2(+), MSH6(+), CDX-2(+), Her-2(4B5)(-), Her-2(positive control)(+), P53(scattered+), Ki-67(LI about 60%). Genetic testing revealed: KRAS gene exon 2 mutation; MSS, BRAF amplification; TMB 4.61Muts/Mb, ranking higher than 80.98% of patients.

The patient's preoperative colonoscopy, CT and MR imaging findings are shown in Figure 1. The staining of the patient's tumor tissue sections is shown in Figure 2.

1.2 Specimen processing:

1) Material collection: 0.5 cm ³ of the patient's primary tumor and metastatic tumor tissue were collected during the operation, placed in a sterile sampling tube containing AdvancedDMEM/F12, placed in a 4°C ice box, and quickly sent to the laboratory;

2) Digestion: Wash and discard the mucus, necrotic tissue and residual blood cells on the surface of the sample obtained in step 1) in a sterile ultra-clean bench, then rinse once with iodine (for clinical mucous membrane disinfection) and three times with PBS, cut the tissue repeatedly with scissors in a plate, add 2 mL of 50% digestive enzyme TrypLE (diluted in 2 mL of DMEM) to digest at room temperature, fully mix the tissue suspension containing digestive enzyme every 5 minutes, about 20 minutes, add an equal volume of Ca ^{2 +} HBSS buffer to neutralize and mix again;

3) Filtration: filter the suspension obtained in step 2) with a filter of not less than 200 mesh diameter, and centrifuge, resuspend the precipitate with 5 mL of culture medium, wash and centrifuge repeatedly to discard necrotic cell fragments, and centrifuge at a speed of 500-1500 r/min;

4) Seed cells: Take the above-mentioned washed cells for live cell counting, resuspend the cells with Matrigel at low temperature to 8000 cells/30 μL, seed in a preheated 24-well plate, 30 μL/well, place in a 37°C incubator for 30 minutes, and add 650 μL of medium to each well after the vaulted Matrigel solidifies;

5) Conventional subculture: the medium was changed every 3 to 5 days, and the subculture operation was performed on the 10th to 14th day. Digest with TrypLE digestive enzyme 250 μL/well, pipette and mix every 5 minutes, digest into a single cell suspension in 10 to 15 minutes, resuspend with HBSS neutralization and centrifugation, and count, continue to pass at 8000 cells per well, and then repeat steps 4) and 5). That is, organoid cell lines with good 3D organoid morphology, stable properties, sustainable passage, and good tolerance to virus infection can be obtained.

Example 2

The biological characteristics of the organoid cell line CWH22 of the primary focus of human right colon cancer and the corresponding organoid cell line CLM22 of liver metastases are disclosed.

2.1 Morphological observation of CWH22 and CLM22 organoid cell lines

The subcultured CWH22 and CLM22 organoids were taken, and the morphological changes of organoid growth were observed and recorded under the Japanese Olympus IMT-2 inverted microscope. The results are shown in Figure 3. The organoids were passaged in the form of single cells, and a few small luminal epithelial epithelial differentiated structures could be seen on the 4th day, and obviously entered the logarithmic growth phase after the 7th day.

2.2 Similarity of CWH22 and CLM22 organoid structures to primary tumors

The retrieved primary colorectal cancer and liver metastases tissues were fixed and embedded and stained with HE, CK20, and CDX2. After 14 days of normal culture, the organoids were fixed and embedded with Thermo tissue embedding gel, and then prepared into wax block sections for HE, CK20, and CDX2 staining. The results are shown in Figure 4. Organoids and corresponding tumor tissues have consistent epithelial-like arrangement differentiation and positive expression of CK20 and CDX2.

2.5 Gene detection of CWH22 and CLM22 organoid cell lines

The fourth generation of normal cultured CWH22 and CLM22 organoids were collected, and DNA was extracted from the patient’s tumor tissue and normal mucosal tissue that were resected and quick-frozen during the operation. Whole-exome sequencing was performed after passing the quality inspection. Figure 5A shows the high-frequency mutation genes (APC, KRAS, SMAD4, CDKN1B, KMT2C, etc.) And the positive mutations in the corresponding organoids CWH22 and CLM22 are summarized. Figure 5B shows the results of the mutation consistency analysis between the cultured organoids and the primary tumor, suggesting that the organoids better retain the mutation characteristics of the primary tumor, and the established pair of organoids derived from this patient are APC/KRAS/SMAD4 triple mutations, TP53 wild-type colon cancer organoid cell line and the organoid cell line corresponding to the source of liver metastases.

2.6 STR Detection of CWH22 and CLM22 Organoid Cell Lines

Short tandem repeat ((short tandem repeat, STR)), also known as microsatellite DNA, refers to a type of DNA sequence formed by tandem repetition of several base pairs as the core unit on the chromosome, generally 2 to 6 base pairs. The number of repetitions is more than 10 to 60, and the gene fragment is less than 400 base pairs. There will be individual differences in the number of repetitions of each core unit, thus forming alleles with different fragment lengths. Therefore, the number of repetitions of a group of STR sequences is almost unique among different individuals, and it is an individual's genetic identity feature, and it is also the main method of cell biology to identify cell identity and origin.

The collected 4th passage CWH22 and CLM22 organoid cells and normal mucosal tissues of patients excised and frozen during operation were sent to CCTCC, China Center for Type Culture Collection, for STR detection. STR loci include: D19S433, D5S818, D21S11, D18S51, D6S1043, AMEL, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S 391, D2S1338, FGA.

The analysis results are shown in Table 1. It can be seen that the CWH22 and CLM22 organoid cell lines cultivated in the present invention are real, have the same spectrum type as the parental tissue, and are derived from the same patient. In the German Microorganism Culture Collection DSMZ database (Deutsche Sammlung vonMikroorganismen und Zellkulturen) and the American Type Culture Collection (ATCC) database, the STR sequence search was performed, but no identical STR detection results were found. That is, no cell line that completely matches the STR locus of the cell was found, indicating that this is a new cell line; no multi-allelic sites were found, indicating that the cell line was a single cell without other cell contamination.

Table 1 CWH22, CLM22 and the list of STR detection results of normal mucosal tissues of patients

2.7 Detection of tumorigenicity of liver metastasis by CWH22 and CLM22 organoid cells injected into spleen

Organoids expressing luciferase (overexpressed luciferase virus (PLVX-Luc-IRES-Neo) was purchased from Shanghai Dianjun Biotechnology Co., Ltd.): pre-spread 1:2 diluted Matrigel*10 wells in a 96-well plate, cover the bottom with 30 μL/well, and place it in a 37°C incubator for 30 minutes until it gels; synchronously digested cells into a single-cell suspension, and resuspended in medium to 3*10⁴/mL, 100 μL/well was planted in the above 10 wells; after 48 hours, 9 of the wells were taken (the other well was the blank control) and replaced with 100 μL/well of the above-mentioned virus-containing culture medium (with polybrene 5 μg/mL); after 12 hours of action, the conventional organoid medium was replaced, and after 24 hours of continuous culture, G418 (500 μg/mL) was screened for about 5-7 days. 1*10⁵Cells were added to 20 μL in vivo imaging substrate, and the luciferase reporter gene luminescence value > 50,000 was positive expression.

The luciferase-overexpressing CWH22 and CLM22 organoids were conventionally subcultured, and the organoid cells in the logarithmic growth phase were digested into a single cell suspension and counted, resuspended in DMEM to 1.25*10 ⁷ /mL, injected 80 μL/nude mouse through the spleen, and 10 minutes later, the splenic blood vessels were ligated, the spleen was cut off, and sutured. Regular in vivo imaging was used to observe and record the liver metastases in nude mice. Figure 6A shows the liver metastases of the primary tumor and metastatic organoids 45 days after spleen injection, and Figure 6B shows the HE staining and the expression of CK20 and CDX2 in the liver tumors of the two groups of cells.

2.8 In situ tumor formation of CWH22 organoid cells injected into cecum

CWH22 and CLM22 organoids were conventionally subcultured, and the organoids in the logarithmic growth phase were digested with TrypLE enzyme for 5 minutes to release the organoid structure. About 3*10 ⁶ cells were centrifuged and washed, pre-cooled, resuspended in 60 μL Matrigel and placed at 4°C; 20 μL of the organoid suspension was drawn with a pre-cooled insulin injection needle for submucosal injection in the cecum. After the mice were euthanized on the 45th day, the cecum was removed to observe the tumor formation, and HE staining was performed for identification. Figure 7 shows the macroscopic findings and HE staining of in situ tumor formation in the cecum on day 45, suggesting that the arrangement of moderately differentiated adenoma cells can be seen in the submucosa.

Example 3

Disclosed is the application of a pair of organoid cell lines CWH22 for the primary focus of human right colon cancer and the corresponding organoid cell line CLM22 for liver metastases having the above biological characteristics in judging drug sensitivity for the treatment of colorectal cancer.

3.1 Detection of IC50 of related chemotherapeutic drugs

The CWH22 and CLM22 organoids in the logarithmic growth phase were digested and counted, resuspended in Matrigel and planted in a 96-well plate, 2* ¹⁰⁵ cells/mL, 10 μL/well. After the Matrigel solidified, 100 μL/well of medium was added. After 5-6 days of culture (the diameter of the organoid was about 100 μm), the gradient drug medium was replaced to continue the culture. The medium was changed once every 3 days. See Figure 8.

The IC50 of CWH22 and CLM22 are as follows: 5-fluorouracil is 3.3 μm and 3.78 μm; oxaliplatin is 15.47 μm and 18.38 μm; SN-38 is 7.54 nm and 10.67 nm; VP-16 is 5.84 μm and 6.98 μm; regorafenib is 3.32 μm and 3.68 μm; 510 are 15.02μm and 10.95μm respectively; PS-341 are 8.06nm and 7.71nm respectively.

Compared with the IC50 of other colorectal cancer cell lines such as Lovo, SW620, T84 and SW480 in GDSC (https://www.cancerrxgene.org/), the drug sensitivity of CWH22 and CLM22 to 5-fluorouracil and oxaliplatin was clarified. As shown in Figure 9, the postoperative re-examination of the clinical patient showed scattered lesions in the liver. After receiving 8 courses of 5-fluorouracil + oxaliplatin + bevacizumab (mFOLFOX + Bevacizumab) treatment, the lesions were stable and regressed significantly, suggesting that the tumor was initially sensitive to 5-fluorouracil and oxaliplatin treatment.

To sum up, the organoid CWH22 of primary human colorectal cancer and the corresponding organoid CLM22 of liver metastases screened by the present invention have better 3D organoid morphology, stable properties, sustainable and stable passage, and good tolerance to virus infection and other operations. Studies have found that it has a very strong ability to form tumors in cecum in situ and liver metastasis by spleen injection, and can successfully create an animal model of colorectal cancer liver metastasis. To provide new in vivo and in vitro research support for further in-depth research on the etiology, pathogenesis, drug resistance mechanism, and new drug screening of colorectal cancer and liver metastasis.

The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention shall be covered within the scope of protection of the present invention.

Claims (10)

1. A paired organoid cell line of primary human colorectal cancer and liver metastases, characterized in that it includes the organoid cell line CWH22 of the primary human right colon cancer with the preservation number CCTCC No. 2. The organoid cell line paired with the primary human colorectal cancer and liver metastases according to claim 1, wherein the cell line is obtained by in vitro 3D organoid culture of the primary tumor and the corresponding liver metastases. 3. A method for cultivating a paired organoid cell line of a human colorectal cancer primary focus and liver metastases, characterized in that it comprises the following steps: 1) Material collection: 0.5 ^cm3 of the tumor tissue from the primary tumor and the corresponding liver metastases were collected during the operation, placed in a sterile sampling tube containing AdvancedDMEM/F12, placed in a 4°C ice box, and sent to the laboratory quickly; 2) Digestion: Wash and discard the mucus, necrotic tissue and residual blood cells on the surface of the sample obtained in step 1) in a sterile ultra-clean bench, then rinse once with iodine and PBS three times, cut the tissue repeatedly with scissors in a plate, add 2 mL of 50% digestive enzyme TrypLE to digest at room temperature, and mix the tissue suspension containing digestive enzyme every 5 minutes. About 20 minutes, add an equal volume of HBSS buffer containing Ca ²⁺ to neutralize and mix again ; 3) Filtration: filter the suspension obtained in step 2) with a filter of not less than 200 mesh diameter, and centrifuge, resuspend the precipitate with 5 mL of culture medium, wash and centrifuge repeatedly to discard necrotic cell fragments, and centrifuge at a speed of 500-1500 r/min; 4) Seed cells: Take the above-mentioned washed cells for live cell counting, resuspend the cells with Matrigel at low temperature to 8000 cells/30 μL, seed in a preheated 24-well plate, 30 μL/well, place in a 37°C incubator for 30 minutes, and add 650 μL of medium to each well after the vaulted Matrigel solidifies; 5) Conventional subculture: the medium was changed every 3 to 5 days, and the subculture operation was performed on the 10th to 14th day. Digest with TrypLE digestive enzyme 250 μL/well, pipette and mix every 5 minutes, digest into a single cell suspension in 10 to 15 minutes, resuspend with HBSS neutralization and centrifugation, and count, continue to pass at 8000 cells per well, and then repeat steps 4) and 5). 4. A paired organoid cell line of human colorectal cancer primary focus and liver metastases according to claim 1 or 2 or the cell line obtained by cultivating the method according to claim 3 is used to construct an animal model of cecum in situ tumor formation and/or an animal model of liver metastases tumor formation and to study the mechanism of colorectal cancer occurrence and drug resistance. 5. A method for establishing a tumor-forming animal model of liver metastases, characterized in that it comprises the steps of: 1) Organoid culture: After the organoid cells were stably overexpressed luciferase with lentivirus infection, the organoids were cultured routinely, and the medium was changed on the 4th, 7th, 9th, and 11th day after subculture, and the CWH22 and CLM22 organoids in the logarithmic growth phase were taken for nude mouse spleen injection and tumor transfer operation; 2) Digestion of organoids: the process of digesting cells is the same as that of conventional subculture operations, digested into a single cell suspension state, and counted, resuspended in DMEM to 1.25*10 ⁷ /mL and placed in a 4°C incubator for standby; 3) Spleen injection: After anesthetizing the nude mice with pentobarbital, fix the nude mice and perform abdominal skin disinfection, make a longitudinal incision about 1 cm along the left lower costal margin of the abdomen at the front of the axillary line 0.5 cm to the abdominal cavity, pick out the spleen and inject about 80 μL/nude mouse with a mixed cell suspension from the distal end of the spleen, ligate the splenic blood vessels 10 minutes later, cut off the spleen and suture it, and observe closely to ensure smooth recovery. 4) Tumor formation monitoring: regular in vivo imaging was used to observe and record the tumor formation of liver metastases in nude mice. 6. A method for constructing an animal model of cecum in situ tumor formation, characterized in that it comprises the steps of: 1) Organoid culture: routinely culture luciferase-overexpressing organoids CWH22 and CLM22, and change the medium on the 4th, 7th, 9th, and 11th days after subculture, and inject the organoids in the logarithmic growth phase into the cecum wall of nude mice; 2) Organoid collection: collect organoids on the 10th to 12th day, digest with TrypLE digestive enzyme for 5 minutes, neutralize and collect organoid precipitates, about 3*10 ⁶ cells, resuspend in 60 μL Matrigel after pre-cooling, and place in a 4°C incubator for later use; 3) Caecum in situ injection: after pentobarbital anesthetized the nude mice, fix the nude mice and perform abdominal skin disinfection, make a longitudinal incision about 1.5 cm along the midline of the lower abdomen to the abdominal cavity and find the cecum, move the end of the cecum to the center of the field of vision, draw 20 μL of the organoid suspension with a pre-cooled insulin injection needle for submucosal injection of the cecum, and observe that there is no leakage after the injection, suture the abdominal muscles and skin, and closely observe to ensure smooth recovery; 4) Tumor formation monitoring: Regular in vivo imaging was used to observe and record the in situ tumor formation in the cecum of nude mice. 7. The use of a paired organoid cell line of human colorectal cancer primary focus and liver metastases according to claim 1 or 2 or the cell line cultured by the method according to claim 3 in the preparation of tumor drugs for screening colorectal cancer and/or liver metastases. 8. The application according to claim 7, characterized in that, the therapeutic drug is applied to an in vitro cell model, and the drug that inhibits cell proliferation or causes cell apoptosis is a therapeutic sensitive drug after application, or the test compound is applied to an in vitro cell model, and the candidate compound is a candidate compound that inhibits cell proliferation or causes cell apoptosis after application. 9. A method for screening tumor drugs for the treatment of colorectal cancer and/or liver metastases, characterized in that it comprises taking the CWH22 and CLM22 organoids in the logarithmic growth phase cultivated in claim 3, digesting and counting them, resuspending them in matrigel and planting them in a 96-well plate, 2* ¹⁰⁵ cells/mL, 10 μL/well, after the matrigel is solidified, adding 100 μL/well of the medium, and replacing it with a gradient drug medium to continue culturing for 5 to 6 days, and changing the medium once every 3 days , after 6 days of drug action, through 3D cell activity detection, calculate the corresponding drug IC50. 10. The method according to claim 9, wherein the drug gradient medium comprises at least one of the following drugs: 5-fluorouracil, oxaliplatin, irinotecan, etoposide, sotoracib, regorafenib, dasatinib or bortezomib.