Esophageal cancer cell strain with high invasion and drug resistance characteristics as well as preparation method and application of esophageal cancer cell strain

Abstract

The invention provides an esophageal cancer cell strain with high invasion and drug resistance characteristics as well as a preparation method and application thereof, and relates to the technical field of microorganisms, the esophageal cancer cell strain is resistant to paclitaxel and resistant to multiple drugs such as fluorouracil and cis-platinum to different degrees, and the tumor forming capacity which is not possessed by original TE-1 is obtained. Meanwhile, the migration and invasion capabilities are remarkably enhanced, liver metastasis occurs in a nude mouse subcutaneous model, and a unique tool is provided for research of a drug resistance related metastasis mechanism. The esophageal cancer cell strain is constructed by adopting a combined strategy of in-vitro induction and in-vivo selection, the mutation rate of tumor cells can be remarkably improved, the construction period of drug-resistant cells is shortened, and the esophageal cancer cell strain has relatively good operability and repeatability. Due to the unique characteristics of the esophageal cancer cell strain, the esophageal cancer cell strain has a relatively good application prospect in screening anti-tumor drugs, researching a tumor drug resistance mechanism or constructing an animal model.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to an esophageal cancer cell line with high invasion and drug resistance characteristics, and a preparation method and application thereof. BACKGROUND

[0002] Esophageal cancer is still a high mortality rate of malignant tumor in the world, and chemotherapy is the cornerstone of drug treatment of esophageal cancer. However, tumor drug resistance is an important reason for drug treatment failure and tumor recurrence. Therefore, elucidating the mechanism of tumor drug resistance is particularly crucial for improving the level of drug treatment of esophageal cancer.

[0003] In order to study the mechanism of antitumor drug resistance, researchers usually need to construct a drug-resistant cell line first. At present, the method for constructing drug-resistant cells mainly adds drugs to tumor cells cultured in vitro for a long time, and gradually makes tumor cells resistant to drugs by giving tumor cells drug pressure for a long time. However, this process is difficult to simulate the process of drug resistance of chemotherapeutic drugs in actual clinical treatment, and the reasons include: (1) solid tumors are three-dimensional, and tumor cells are often in a hypoxic state, while the in vitro culture system cannot simulate the hypoxic state of the tumor; (2) there are no blood vessels, stroma, fibroblasts, immune cells and other tumor tissue components in the in vitro culture system, which makes it difficult to simulate the process of chemotherapeutic drugs from the peripheral blood circulation system through the blood vessel wall, tumor non-cell components and into tumor cells; (3) immune cells in the in vitro culture system cannot reproduce the influence of the interaction between tumor cells and immune cells on tumor cell drug resistance. In addition, the construction of drug-resistant cells in vitro usually takes a long time, and the cells are easily contaminated during the construction process, which also increases the difficulty of studying the mechanism of antitumor drug resistance. SUMMARY

[0004] The first object of the present application is to provide an esophageal cancer cell line with high invasion and drug resistance characteristics, which presents different degrees of drug resistance to multiple drugs, and the migration and invasion ability is significantly enhanced.

[0005] The second object of the present application is to provide a preparation method of the above-mentioned esophageal cancer cell line, which can significantly improve the mutation rate of tumor cells, shorten the construction period of drug-resistant cells, and has better operability and repeatability.

[0006] The third object of the present application is to provide an application of the above-mentioned esophageal cancer cell line, which has a better application prospect in screening antitumor drugs, studying the mechanism of tumor drug resistance or constructing animal models due to its high invasion and drug resistance characteristics.

[0007] The embodiments of the present application are implemented as follows: An esophageal cancer cell strain Homo sapiens with high invasion and drug resistance characteristics, which is preserved in the China General Microbiological Culture Collection Center, has a preservation number GDMCC No. 67553, and is preserved on December 29, 2025, and has an address of No. 59, Building 5, 100, Guangzhou Martyrs' Courtyard Road.

[0008] A preparation method of the esophageal cancer cell strain, comprising: S1. Culturing esophageal cancer cells in vitro, and inducing mutation by a chemical inducer; S2. Screening the cell strain after the induced mutation in vitro by a drug; S3. Inoculating the cells screened in vitro into a nude mouse subcutaneously, and screening in vivo.

[0009] An application of the esophageal cancer cell strain in screening an anti-tumor drug, researching a tumor drug resistance mechanism, or constructing an animal model.

[0010] The esophageal cancer cell strain provided by the embodiment of the present application has the advantages that: The present application provides an esophageal cancer cell strain Homo sapiens with high invasion and drug resistance characteristics, a preparation method and an application thereof. The esophageal cancer cell strain is resistant to paclitaxel, and also presents different degrees of resistance to multiple drugs such as fluorouracil and cisplatin, and has a tumorigenic ability that the original TE-1 does not have. At the same time, the migration and invasion ability of the esophageal cancer cell strain is significantly enhanced, and liver metastasis occurs in a nude mouse subcutaneous model, which provides a unique tool for the research of drug resistance related metastasis mechanism. The esophageal cancer cell strain is constructed by a combined strategy of 'in vitro induction + in vivo selection', which can significantly improve the mutation rate of tumor cells, shorten the construction cycle of drug-resistant cells, and has better operability and reproducibility. The unique characteristics of the esophageal cancer cell strain make it have a better application prospect in screening an anti-tumor drug, researching a tumor drug resistance mechanism, or constructing an animal model. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0012] Figure 1 A preparation flowchart of the esophageal cancer cell strain Homo sapiens with high invasion and drug resistance characteristics provided in the embodiment 1 of the present application; Figure 2 A photo of the esophageal cancer cell strain Homo sapiens with high invasion and drug resistance characteristics provided in the embodiment 1 of the present application and the parent cell under a phase contrast microscope; Figure 3 Survival rate of the parental cell and the drug-resistant cell at the same time point and different concentrations of paclitaxel provided in the test example 1 of the present application; Figure 4 Survival rate of the parental cell and the drug-resistant cell at the same paclitaxel concentration and different time points provided in the test example 1 of the present application; Figure 5 Survival rate of the parental cell and the drug-resistant cell at the same time point and different concentrations of cisplatin provided in the test example 2 of the present application; Figure 6 Survival rate of the parental cell and the drug-resistant cell at the same time point and different concentrations of fluorouracil provided in the test example 2 of the present application; Figure 7 Difference in the colony formation ability of the parental cell and the drug-resistant cell provided in the test example 3 of the present application; Figure 8 Difference in the three-dimensional spheroid formation ability of the parental cell and the drug-resistant cell provided in the test example 4 of the present application; Figure 9 Difference in the migration ability of the parental cell and the drug-resistant cell provided in the test example 5 of the present application; Figure 10 Difference in the invasion ability of the parental cell and the drug-resistant cell provided in the test example 6 of the present application; Figure 11 Difference in the cell cycle distribution of the parental cell and the drug-resistant cell under the action of the same concentration of paclitaxel provided in the test example 7 of the present application; Figure 12 Difference in the apoptosis rate of the parental cell and the drug-resistant cell under the action of the same concentration of paclitaxel provided in the test example 8 of the present application; Figure 13 Difference in the tumorigenicity of the parental cell and the drug-resistant cell in the subcutaneous tumor model of the nude mice provided in the test example 9 of the present application; Figure 14 Subcutaneous tumor formation of the drug-resistant cell in the subcutaneous tumor model of the nude mice provided in the test example 10 of the present application; Figure 15 Volcano plot of the different genes in the transcript sequencing of the parental cell and the drug-resistant cell provided in the test example 11 of the present application. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments are not specified by the manufacturers, they are all the conventional products that can be purchased in the market.

[0014] The following will specifically illustrate an esophageal cancer cell strain with high invasion and drug resistance characteristics, and a preparation method and application thereof.

[0015] The esophageal cancer cell strain is preserved in the China General Microbiological Culture Collection Center, has a preservation number of GDMCC No. 67553, a preservation date of December 29, 2025, and a preservation address of No. 59, Building 5, Guangzhou, China.

[0016] The esophageal cancer cell strain has a drug resistance index of no less than 20 to paclitaxel, and has cross drug resistance to cisplatin and fluorouracil, with a drug resistance multiple of no less than 2.

[0017] Meanwhile, the esophageal cancer cell strain has a tumorigenic ability that the original TE-1 does not have, and can be used for modeling. The migration and invasion ability of the esophageal cancer cell strain is significantly enhanced, and liver metastasis occurs in a nude mouse subcutaneous model, thereby providing a unique tool for research on drug resistance related metastasis mechanisms.

[0018] The embodiment of the present application further provides a preparation method of the esophageal cancer cell strain. S1. Culturing esophageal cancer cells in vitro, and inducing mutation by a chemical inducer.

[0019] The original esophageal cancer cells are TE-1 cells, which are a human esophageal squamous cell carcinoma cell line widely used in esophageal cancer research, have a wide source, and are an important in vitro model for exploring biological behavior, drug screening and molecular mechanisms of esophageal cancer.

[0020] The addition of the chemical inducer can improve the mutation rate of tumor cells and significantly shorten the time for constructing drug-resistant cells. Alternatively, the chemical inducer is ethyl methanesulfonate, and the concentration of the chemical inducer is 200-300 μg / ml. Under the induction of ethyl methanesulfonate, the mutation efficiency of the cells is higher.

[0021] S2. Screening the cell strain after the mutation induction in vitro by a drug.

[0022] The in vitro drug pressure screening adopts a high-concentration impact method. Specifically, the mutated cells are subjected to drug impact treatment by using paclitaxel with a concentration of 100-200 μM. The drug impact treatment is performed for 2-5 times, the single drug impact time is 2-20 h, and the impact time of the multiple drug impacts is sequentially increased.

[0023] After each drug shock, the medium containing paclitaxel needs to be removed, the cells are washed with PBS and fresh medium is added for continued culture until the cells regain the ability of autonomous proliferation. Alternatively, when the confluence rate of the cells reaches 80%, it can be considered that the cells have regained the ability of autonomous proliferation, and a part of the cells can be selected for cryopreservation, and the remaining cells are transferred to a new culture medium for continued culture for the next round of drug shock. After all rounds of drug shock are completed, the cells that have regained the ability of autonomous proliferation are sequentially cultured to obtain the primary drug-resistant cells TE-1 / PTX.

[0024] The primary drug-resistant cells TE-1 / PTX that survive the in-vitro drug pressure screening are inoculated at a low density, and single-cell clones are separated and cultured to screen subclone cell strains with strong proliferation ability for subsequent in-vivo passage screening.

[0025] S3. The cells screened in-vitro are inoculated subcutaneously into nude mice for in-vivo passage screening.

[0026] Because there are no blood vessels, stroma, fibroblasts, immune cells and other tumor tissue components in the in-vitro culture system, it is difficult to simulate the process in which the chemotherapeutic drug passes through the blood vessel wall and tumor non-cell components from the peripheral blood circulation system to enter the tumor cells. In the in-vitro culture system, immune cells also cannot reproduce the influence of the interaction between tumor cells and immune cells on the drug resistance of tumor cells.

[0027] To restore the characteristics of tumor tissue, such as hypoxia, containing blood vessels, fibroblasts, stroma and other complex components, after in-vitro culture, the cells screened in-vitro are further inoculated into nude mice for in-vivo screening, so as to strengthen drug resistance.

[0028] Further, in the step S3, the cells screened in-vitro are inoculated subcutaneously into nude mice, and when the tumor volume reaches 100mm 3 , paclitaxel treatment of 3-7 mg / kg / 3d is started; and when the tumor volume reaches 1500mm 3 , the tumor is peeled off and passaged.

[0029] The peeled tumor in each passage is washed with PBS and placed in physiological saline containing penicillin and streptomycin for cleaning, cut into small pieces, and the tumor tissue with good activity is selected for passage. After 2-5 times of repeated passage, a stable drug-resistant tumor tissue can be obtained.

[0030] After the drug-resistant tumor tissue is digested with collagenase IV and dispase, a single-cell suspension can be obtained by filtration, and then in-vitro culture can be performed to obtain the esophageal cancer cell strain TE-1 / PTX6 with high invasion and drug resistance.

[0031] The embodiment of the present application also provides an application of the above esophageal cancer cell strain in screening of anti-tumor drugs, research of tumor drug resistance mechanism or construction of an animal model.

[0032] The features and properties of the present application are further described in detail below in conjunction with the examples.

[0033] Example 1

[0034] This example provides an esophageal cancer cell line with high invasiveness and drug resistance, and the preparation process is as shown in Figure 1 , and specifically as follows: S1. TE-1 cells were inoculated in a 100 mm culture dish, and when the cell confluence rate reached 50%, ethyl methanesulfonate (250 μg / ml) was added to the culture medium for chemical mutation induction at 37°C for 24 hours.

[0035] S2. High-dose paclitaxel (150 μM) was added to the culture medium for shock treatment, and after 4 hours of incubation, the culture medium containing paclitaxel was removed, the cells were washed with PBS for 3 times, and then fresh culture medium was added for continuous culture until the cells recovered the ability of autonomous proliferation. When the cell confluence rate reached 80%, the cells were digested, and 70% of the cells were frozen, and the remaining 30% of the cells were inoculated in a new culture dish. When the cell confluence rate reached 80%, high-dose paclitaxel (150 μM) was added to the culture medium for shock treatment, and after 8 hours of incubation, the culture medium containing paclitaxel was removed, the cells were washed with PBS for 3 times, and then fresh culture medium was added for continuous culture until the cells recovered the ability of autonomous proliferation. When the cell confluence rate reached 80%, the cells were digested, and 70% of the cells were frozen, and the remaining 30% of the cells were inoculated in a new culture dish. When the cell confluence rate reached 80%, high-dose paclitaxel (150 μM) was added to the culture medium for shock treatment, and after 16 hours of incubation, the culture medium containing paclitaxel was removed, the cells were washed with PBS for 3 times, and then fresh culture medium was added for continuous culture until the cells recovered the ability of autonomous proliferation. Thus, the primary drug-resistant cells TE-1 / PTX were obtained. 70% of the cells were frozen, and the remaining 30% of the cells were used for further experiments.

[0036] S3. TE-1 / PTX cells were inoculated in a culture dish at an extremely low density (3000 cells per 100 mm culture dish), and after 14 days of culture, subclones were selected under a microscope using a 10 μl pipette and inoculated in a 96-well plate, respectively. Then, the culture was gradually expanded to a 12-well, 24-well, 6-well plate and a 100 mm culture dish to obtain stable subcell clones and freeze them.

[0037] S4. The drug-resistant cells after cloning were inoculated subcutaneously in nude mice, and when the tumor volume reached 100 mm 3 , paclitaxel treatment (5 mg / kg / 3d) was started; when the tumor volume reached 1500 mm 3Mice were euthanized in a sterile laminar flow hood, tumor tissue was dissected, washed twice with PBS, and then placed in a solution containing penicillin (100 U / ml) and streptomycin (100 μg / ml). The tumor tissue was cut into small pieces using ophthalmic scissors, stained with trypan blue, and selected from those with better viability. These pieces were then placed in 100 mm culture dishes containing fresh culture medium and further minced into 1-3 mm pieces using ophthalmic scissors and forceps. 3 Tissue blocks were prepared. Three to five tissue blocks were placed in a sterile inoculation needle, and the needle core was then inserted from the rear. The nude mouse was subsequently anesthetized with isoflurane, and the skin was disinfected with iodine. The tissue was then inoculated subcutaneously into the right forelimb of the nude mouse using the inoculation needle. The inoculation continued until the tumor volume reached 100 mm. 3 Paclitaxel treatment was initiated when the tumor volume reached 1500 mm. 3 Then, the tumor tissue was dissected again, cut into small pieces, and inoculated subcutaneously into nude mice, with the tumor volume reaching 100 mm. 3 Repeat the administration process three times in vivo to obtain stable drug-resistant tissue.

[0038] S5. After euthanizing the mice, dissect the drug-resistant tumor tissue, cut it into small pieces, wash away blood and impurities with PBS, transfer the pea-sized tissue pieces to a 1.5ml conical tube, add 1ml of digestion solution (collagenase IV, dispersin), and use ophthalmic scissors to further mince the tissue in the digestion solution to a volume of approximately 1-2mm. 3 The tissue suspension was then transferred to a 10ml test tube, and 3ml of digestion solution was added. Digestion was carried out at 37℃ for 20 minutes, shaking 3-5 times during digestion. After digestion, the cell suspension was filtered through a 70μm sieve. Subsequently, the cells were centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. Cells were reselected with fresh culture medium, transferred to culture dishes for further culture, and then cryopreserved. Thus, we obtained stable esophageal cancer paclitaxel-resistant TE-1 / PTX6 cells.

[0039] Figure 2 This image shows phase-contrast micrographs of esophageal cancer paclitaxel-resistant cells TE-1 / PTX6 and parental TE-1 cells. The paclitaxel-resistant cells TE-1 / PTX6 (…) are visible. Figure 2 (Top right and bottom right images) Compared to parental cell TE-1 ( Figure 2 The top left and bottom left images show obvious changes.

[0040] Experimental Example 1 This experiment used the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and parental cells TE-1 to test their changes in sensitivity to paclitaxel. The test methods and results are as follows: (1) TE-1 and TE-1 / PTX6 were seeded into 96-well plates, 1500 cells per well. After cell adhesion the next day, the culture medium in the wells was discarded, and 200 μl of paclitaxel containing gradient concentrations was added to each well. After culturing for 72 hours, 10 μl of CCK-8 staining was added, and the cells were incubated at 37°C for 20 minutes. The absorbance at A450 nm was measured, the cell viability was calculated, and the IC50 of paclitaxel was calculated using SPSS software. 50 .

[0041] The formula for calculating cell viability is: Cell viability (%) = (A DayX - A 空白 ) / (A Day0 -A 空白 100%, In the formula, A DayX To obtain the absorbance value measured after X days of cultivation, A Day0 To obtain the absorbance value measured on day 0 of cultivation, A 空白 The instrument is blank.

[0042] The calculation results are as follows Figure 3 As shown, the IC50 of drug-resistant TE-1 / PTX6 cells against PTX (paclitaxel) can be observed. 50 The value was 35.21 nM, and the IC50 value of the parental TE-1 cells against PTX was 1.74 nM, with a resistance index (RI) as high as 20.24. The resistant cells TE-1 / PTX6 showed significantly reduced sensitivity to paclitaxel.

[0043] (2) TE-1 and TE-1 / PTX6 were seeded in 96-well plates at 800 cells per well. After cell adhesion the next day, the culture medium in the wells was discarded, and 200 μl of RPMI 1640 medium containing paclitaxel (concentration: 5 nM) was added to each well for 5 days. 10 μl of staining agent CCK-8 was added to the wells daily. After incubation at 37°C for 20 minutes, the absorbance at A450 nm was measured, and the cell viability was calculated using the same formula as above. The results are as follows. Figure 4 As shown.

[0044] from Figure 4 It can be seen that under the action of 5 nM paclitaxel, the survival rate of TE-1 cells continued to decrease, dropping to 27% after 5 days; while the drug-resistant cells TE-1 / PTX6 continued to proliferate, and the cell survival rate reached 284% after 5 days, indicating that TE-1 / PTX6 can proliferate normally in the culture medium containing 5 nM paclitaxel.

[0045] Experimental Example 2 The test example adopts the drug-resistant cell TE-1 / PTX6 prepared in Example 1 and the parent cell TE-1 to test the sensitivity change of 5-FU and Cisplatin, and the test method and results are as follows: TE-1 and TE-1 / PTX6 are inoculated in a 96-well plate at 2000 cells per well, and the culture medium in the well is discarded the next day, and different concentrations of chemotherapeutic drugs (5-FU / Cisplatin) are added. The CCK-8 method is used to detect the sensitivity of the parent cell TE-1 and the drug-resistant strain TE-1 / PTX6 cells to 5-FU and Cisplatin at 72 hours, and the IC 50 values are calculated. The results are shown in Figure 5 and Figure 6 .

[0046] As can be seen from Figure 5 , the sensitivity of the drug-resistant cell TE-1 / PTX6 to Cisplatin has decreased to a certain extent, and the resistance has increased by 2.47 times. As can be seen from Figure 6 , the drug-resistant cell TE-1 / PTX6 also has improved resistance to 5-FU, and the resistance has increased by 3.44 times. It shows that the drug-resistant cell TE-1 / PTX6 induced by paclitaxel exhibits resistance to other chemotherapeutic drugs, and has cross-resistance.

[0047] Test Example 3 The test example adopts the drug-resistant cell TE-1 / PTX6 prepared in Example 1 and the parent cell TE-1 to test the cloning ability, and the test method and results are as follows: The logarithmic phase parent cell TE-1 and the drug-resistant strain TE-1 / PTX6 cells are inoculated in a 6-well plate at a density of 1000 cells per well. The next day, after the cells adhere, 2ml of fresh culture medium is added in the control group; in the experimental group, 2ml of fresh culture medium containing 5nM paclitaxel is added, and the cells are treated for 12 hours and 24 hours, and then the culture medium is discarded. After washing with PBS for 2 times, 2ml of fresh culture medium is added. After continuing to culture for 7 days, the culture medium is discarded, and after washing with PBS for 2 times, methanol is added to fix the cells for 10 minutes. Excess methanol is washed with PBS, and crystal violet staining agent is added for staining for 20 minutes. Excess staining solution is washed with ultrapure water, and after the plate is dried, it is photographed, and the results are counted by Image J, as shown in Figure 7 .

[0048] Figure 7The left panel shows the microscopic view of the culture medium, and the right panel shows the results of clone counting. It can be seen that the number of clones formed by the drug-resistant strain TE-1 / PTX6 cells is more and the diameter is larger, indicating that the drug-resistant cell clone formation ability is enhanced. For TE-1 cells, after paclitaxel treatment for 12 and 24 hours, the number of clones is significantly reduced; for TE-1 / PTX6 cells, paclitaxel treatment has no obvious effect on the number of clones, indicating that TE-1 / PTX6 cells are not sensitive to paclitaxel.

[0049] Test Example 4 In this test example, the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and the parent cells TE-1 were subjected to three-dimensional sphere formation test, and the test method and results are as follows: The well-grown cells were washed with PBS for 2 times, digested and centrifuged, and the serum-containing culture medium was removed and washed with PBS for 2 times.

[0050] The cells were resuspended with stem cell culture medium (DMEM / F12+1×B27+20ng / ml EGF+20ng / ml bFGF), mixed and counted into single cell suspension, and the cell concentration was adjusted to 1000 cells / ml. 300ul of cell suspension was added to each well of the ultra-low adsorption 96-well plate, i.e. 300 cells per well, and cultured at 37℃ and 5% CO2.

[0051] Every 2-3 days, half of the liquid was changed, and the cell spheres were observed under a microscope. On the 5th day and the 10th day, the morphology, size and number of cell spheres were recorded by taking pictures, and the sphere formation efficiency (SFE) was calculated. SFE=(number of cell spheres with a diameter greater than 75um in each well / total number of originally inoculated cells in each well)×100%. The results are shown in Figure 8

[0052] Figure 8 The left panel shows the microscopic view of the culture medium, and the right panel shows the results of sphere formation number counting. Compared with TE-1, the number of clones formed by the drug-resistant strain TE-1 / PTX6 cells is more and the diameter is larger, indicating that the drug-resistant cell clone formation ability is enhanced.

[0053] Test Example 5 In this test example, the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and the parent cells TE-1 were subjected to cell migration experiment, and the test method and results are as follows: ​Select the well-grown cells in the logarithmic growth phase, digest the cells with 0.25% trypsin for 3-5 min, transfer to a 10 mL centrifuge tube after stopping the digestion, centrifuge at 1000 rpm for 5 min, wash 1-2 times with PBS, suspend the cells with serum-free medium, and adjust the cell density to 3x10 5

[0054] Select a Transwell plate with a filter pore size of 8.0 μm, add 600 μL of medium containing 20% FBS to the lower chamber of the 24-well plate, then place the Transwell chamber in the 24-well plate with tweezers, add 100 μL of cell suspension to the upper chamber, repeat 3 wells per group, and incubate in the incubator for 12-48 h.

[0055] After incubation, carefully remove the chamber with tweezers, aspirate the medium in the upper chamber, wash 2-3 times with PBS, and gently wipe off the cells in the upper chamber with a cotton swab. Add 600 μL of 4% paraformaldehyde to a new well, and fix the chamber in the lower chamber at room temperature for 15-30 min.

[0056] After fixation, remove the chamber, aspirate the fixative in the upper chamber, and move it to a well pre-loaded with about 800 μl of 0.1%-0.2% crystal violet, and stain at room temperature for 15-30 min. Wash 2-3 times with PBS to remove unbound crystal violet, and gently wipe the upper side of the chamber with a cotton swab to remove the dye that is not specifically bound to the upper surface of the chamber for subsequent microscopic counting.

[0057] Place the stained chamber under a microscope (400x), randomly select 5-10 fields of view, and count the cells that have migrated to the lower chamber. Repeat 3 times, and use image analysis software ImageJ to quantitatively analyze the cell images, as shown in Figure 9 .

[0058] Figure 9 The left panel of Figure 1 shows a microscopic image of the medium, and the right panel shows the statistical results of the number of migrated cells. It can be seen that, compared with the parent cell TE-1, the drug-resistant cell TE-1 / PTX6 has stronger migration ability; for the parent cell TE-1, paclitaxel inhibits 75% of cell migration, and for the drug-resistant cell TE-1 / PTX6, paclitaxel only inhibits 36% of cell migration.

[0059] Test Example 6 In this test example, the drug-resistant cell TE-1 / PTX6 prepared in Example 1 and the parent cell TE-1 were used to perform a cell invasion experiment, and the test method and results are as follows: ​The Matrigel gel was removed from the -20 °C refrigerator and thawed in the 4 °C refrigerator. The Matrigel gel was diluted with serum-free cell culture medium at a ratio of 1:8 on ice. 50-60 μl of the mixed gel-containing medium was evenly added to the bottom membrane of the upper chamber of the Transwell, and placed in a 37 °C incubator to allow the Matrigel to polymerize into a gel film. After 1-3 h, the excess liquid in the upper chamber was removed, 100 μl of serum-free culture medium was added to each well, and the culture was placed in a 37 °C incubator for 30 min for membrane hydration.

[0060] The cells in the logarithmic growth phase were selected, and the cells were digested with 0.25% trypsin for 3-5 min. After stopping the digestion, the cells were transferred to a 10 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, resuspended with PBS for 1-2 times, and suspended with serum-free medium to adjust the cell density to 1×106 / mL.

[0061] 600 μL of medium containing 20% FBS was added to the lower chamber of the 24-well plate, and then the Transwell chamber was placed in the 24-well plate with tweezers. 100 μL of cell suspension was added to the upper chamber, and each group was repeated in 3 wells. The culture was placed in an incubator for 48-96 h.

[0062] After the culture ended, the chamber was carefully removed with tweezers, the medium in the upper chamber was aspirated, and the cells in the Matrigel and upper chamber were gently wiped with a cotton swab. 600 μL of 4% paraformaldehyde was added to a new well, and the chamber was placed in the lower chamber for fixation at room temperature for 30 min.

[0063] After the fixation ended, the chamber was removed, the fixation liquid in the upper chamber was aspirated, and the chamber was moved to a well where about 800 μl of 0.1%-0.2% crystal violet had been added. The chamber was stained at room temperature for 15-30 min. The chamber was washed with PBS for 2-3 times to remove the crystal violet that was not combined with the cells, and the upper side of the chamber was gently wiped with a cotton swab to remove the dye that was not specifically combined with the upper surface of the chamber for subsequent microscopic counting.

[0064] The stained chamber was placed under a microscope (400 times), and 5-10 fields of view were randomly selected for counting the cells that migrated to the lower chamber. The counting was repeated 3 times, and the cell images were quantitatively analyzed using image analysis software ImageJ. The results are shown in Figure 10 .

[0065] Figure 10 The left panel of FIG. 1 shows a microscopic image of the medium, and the right panel shows the statistical results of the number of invasive cells. It can be seen that the drug-resistant cell TE-1 / PTX6 has stronger invasion ability than the parent cell TE-1; for the parent cell TE-1, paclitaxel inhibits 80% of cell invasion, and for the drug-resistant cell TE-1 / PTX6, paclitaxel has no obvious effect on cell invasion.

[0066] Experimental Example 7 This experiment used the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and parental cells TE-1 for cell cycle detection. The test methods and results are as follows: Cell collection: Select cells in good growth condition, discard the cell culture medium, and wash the bottom of the dish twice with PBS. Add 1 mL of trypsin to each dish for digestion. After digestion is stopped, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in PBS and wash 1-2 times.

[0067] Cell fixation: Resuspend the cell pellet in 1 ml of 75% ethanol pre-cooled at -20℃, mix gently, and fix at 4℃ for 72 h.

[0068] Staining: Centrifuge at 1000 rpm for 5 minutes, gently discard the supernatant, resuspend the pellet in 1 ml of pre-chilled PBS, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 0.5 ml of the prepared PI staining working solution (0.5 ml staining buffer with 25 μl PI staining solution and 10 μl RNase A) to each cell sample, gently mix and resuspend the cells, and incubate at 37°C in the dark for 30 minutes.

[0069] Detection on flow cytometer: Red fluorescence was detected using a 488 nm excitation wavelength.

[0070] Test results are as follows Figure 11 As shown. Figure 11 The left figure shows the flow cytometry histogram of "cell cycle analysis," and the right figure is a quantitative statistical bar chart of cell cycle distribution. As can be seen from the figures, treatment with 5 nM paclitaxel did not significantly affect the cell cycle in parental and resistant cells. However, treatment with 10 nM paclitaxel significantly increased the proportion of G2 / M phase cells in parental cells, while the proportion of G2 / M phase cells in resistant cells did not change significantly.

[0071] Experimental Example 8 In this experiment, the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and parental cells TE-1 were used to conduct apoptosis experiments. The test methods and results are as follows: Cells were seeded at a rate of 2 × 10⁵ cells per well in six-well plates. After adhesion, the medium was replaced with drug-containing medium and treated for 24 h. Cells were washed twice with PBS, digested with 0.25% trypsin without EDTA, and centrifuged at 1000 rpm for 5 min at room temperature to collect the cells. Cells were resuspended in pre-chilled PBS at 4 °C, washed once by centrifugation at 1000 rpm for 5 min, and the supernatant was discarded.

[0072] Annexin V-FITC / PI apoptosis detection kit was used to detect cell apoptosis. 100 μL of 1×Binding Buffer was added to suspend the cells, and 5 μL of Annexin V-FITC was added and mixed, then incubated at room temperature for 15 minutes in the dark.

[0073] 5 μL of PI was added 5 minutes before the machine, and 400 μL of 1×Binding Buffer was added, filtered into the flow tube with a filter membrane.

[0074] The flow cytometer was used to detect within 1 h. Annexin V-FITC was green fluorescence (excitation wavelength 488 nm, emission wavelength 525 nm), and PI was red fluorescence (excitation wavelength 535 nm, emission wavelength 615 nm). GraphPad Prism 10.1.2 software was used for plotting.

[0075] The test results are shown in Figure 12 . The left graph of Figure 12 shows the flow cytometry scatter plot, and the right graph is the apoptosis percentage statistical box plot. As can be seen from the figure, after 24 hours of paclitaxel treatment, the early apoptosis rate of TE-1 cells was about 18%, while the early apoptosis rate of drug-resistant cells was only 1.2%.

[0076] Test Example 9 This test example uses the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and the parent cells TE-1 to perform in vivo tumor formation experiment, and the test method and results are as follows: 5-6 week old BALB / c Nude mice were selected, with a body weight of about 15-18 g. Prepare TE1 and TE-1 / PTX6 esophageal cancer cells in the logarithmic growth phase, with a cell density of about 80-90%. Discard the old culture medium, wash the cells twice with PBS to remove serum, digest the cells with trypsin, collect them into a centrifuge tube, centrifuge at 1000 rpm for 5 min, resuspend the cells with PBS and count the cells, adjust the cell concentration to 2×10 7 / mL, and place on ice.

[0077] Nude mice were inoculated: the left hand held the nude mice, and after disinfecting the right upper limb of each mouse subcutaneously, 200 μL of cell suspension was injected, i.e. 1×10 7 cells were inoculated into each mouse, and TE1 and TE-1 / PTX6 esophageal cancer cells were each inoculated into 5 mice. After inoculation, the nude mice were returned to their cages for further feeding, and the mice were observed daily for tumor formation and growth status.

[0078] Post-tumor treatment: tumor formation can be observed after about 1 week. The body weight and tumor volume of the mice were recorded every 2-3 days. Tumor volume = (long diameter × short diameter 2) / 2.

[0079] Mouse dissection and sample collection: When the maximum tumor diameter of the mouse reaches 20 mm or the maximum volume reaches 2000 mm². 3 The experiment was terminated at the designated time. Mice were euthanized by cervical dislocation, and subcutaneous tumors were isolated. The tumors were washed with pre-cooled PBS, blotted dry with filter paper, and their weight was recorded and photographed. The tumor tissue and internal organ tissues were fixed and embedded in 4% paraformaldehyde.

[0080] Test results are as follows Figure 13 As shown. Figure 13 Figure A shows a photograph of the tumor. Seventeen days after inoculation, TE-1 cells formed only four subcutaneous xenografts (top of Figure A), while TE-1 / PTX6 cells formed five subcutaneous xenografts (bottom of Figure A). Furthermore, it can be clearly seen that the tumors generated by the drug-resistant TE-1 / PTX6 cells are significantly larger in volume than those of the parental TE-1 cells. Figure 13 Figure A shows a slice of the tumor, where the drug-resistant TE-1 / PTX6 cells are more densely packed. Figure 13 Figures C, D, and E show the curves of tumor volume, mass, and mouse body weight over time, respectively. It can be seen that the average volume of the TE-1 xenograft is less than 150 mm². 3 The average tumor volume of TE-1 / PTX6 reached 650 mm. 3 The average weight of the TE-1 subcutaneous xenograft was less than 0.1g, while the average weight of the TE-1 / PTX6 subcutaneous xenograft was 0.26g; meanwhile, there was no significant difference in body weight between the two groups of mice.

[0081] Experimental Example 10 This experiment used the drug-resistant cells TE-1 / PTX6 prepared in Example 1 and parental cells TE-1 to perform a liver metastasis experiment of drug-resistant cells. The test methods and results are as follows: Ten 5-6 week old BALB / c Nude nude mice, weighing approximately 15-18g, were selected. TE-1 and TE-1 / PTX6 esophageal cancer cells in the logarithmic growth phase with a cell density of approximately 80%-90% were prepared. The old culture medium was discarded, and the cells were washed twice with PBS to remove serum. Cells were digested with trypsin, collected in centrifuge tubes, centrifuged at 1000 rpm for 5 min, resuspended in PBS, and counted. The cell concentration was adjusted to 2 × 10⁶ cells / mL. 7 / mL, placed on ice.

[0082] Nude mouse inoculation: Hold and restrain the nude mice with your left hand. After disinfecting the right upper limb of each mouse, inject 200 μL of cell suspension subcutaneously, i.e., 1 × 10⁶ cells per mouse. 7Ten mice were inoculated with TE-1 and TE-1 / PTX6 esophageal cancer cells respectively. After inoculation, the nude mice were put back into their cages for further feeding, and the mice were observed daily for tumor formation and growth.

[0083] Tumor treatment: After about one week, tumor formation was observed. After 30 days of feeding, the mice were sacrificed by cervical dislocation, the liver was removed, fixed with 4% paraformaldehyde, and embedded for HE staining and immunohistochemical detection.

[0084] The test results are shown in Figure 14 . As can be seen from the figure, after 30 days of inoculation, the HE staining results of the liver of the mice inoculated with TE-1 / PTX6 cells showed that a large number of tumor cells appeared in the liver (the upper panel in Figure 14 ); the immunohistochemical detection results showed that the Ki67 staining results of the tumor cells in the liver were positive, indicating that these cells proliferated rapidly (the middle panel in Figure 14 ); these tumor cells showed strong positive staining for human squamous cell carcinoma antigen (SCCA), suggesting that the tumor cells in the liver were subcutaneous tumor metastasis sites of esophageal cancer (the lower panel in Figure 14 ).

[0085] Test Example 11 In this test example, the drug-resistant cells TE-1 / PTX6 prepared in Example 1 were subjected to transcript sequencing together with the parent cells TE-1, and the test method and results are as follows: TE-1 and TE-1 / PTX6 were inoculated in 100 mm culture dishes. When the cells grew to 80% confluence, they were washed twice with PBS, 1.5 ml of TRIzol was added, the cells were scraped off with a cell scraper, and collected into a 2 ml cryogenic tube for transcript sequencing.

[0086] The test results are shown in Figure 15 . As can be seen from the figure, compared with TE-1, 1343 genes were up-regulated in TE-1 / PTX6, and 1983 genes were down-regulated. There were significant differences between the two in terms of genes.

[0087] In summary, the application provides an esophageal cancer cell line with high invasion and drug resistance characteristics, and a preparation method and application thereof. The esophageal cancer cell line is resistant to paclitaxel, and also presents different degrees of resistance to multiple drugs such as fluorouracil and cisplatin, and obtains the tumorigenicity that the original TE-1 does not have. At the same time, the migration and invasion ability of the esophageal cancer cell line is significantly enhanced, and liver metastasis occurs in a nude mouse subcutaneous model, providing a unique tool for the study of drug resistance related metastasis mechanism. The esophageal cancer cell line is constructed by using the combined strategy of "in vitro induction + in vivo selection", which can significantly improve the mutation rate of tumor cells, shorten the construction cycle of drug-resistant cells, and has better operability and reproducibility. The unique characteristics of the esophageal cancer cell line make it have better application prospect in screening antitumor drugs, studying tumor drug resistance mechanism or constructing animal models.

[0088] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An esophageal cancer cell line having high invasiveness and drug resistance, characterized in that, It is preserved in the China General Microbiological Culture Collection Center, with the preservation number GDMCC No. 67553, the preservation date being December 29, 2025, and the preservation address being No. 59, Building 5, Guangzhou Martyrs' Middle Road, Guangzhou.

2. The esophageal cancer cell line according to claim 1, characterized in that, The drug resistance index of the esophageal cancer cell line to paclitaxel is not less than 20.

3. The esophageal cancer cell line according to claim 2, wherein, The esophageal cancer cell line has cross-resistance to cisplatin and fluorouracil, and the drug resistance multiple is not less than 2.

4. A method for preparing the esophageal cancer cell line according to any one of claims 1 to 3, characterized by, It comprises: S1. Culturing esophageal cancer cells in vitro and inducing mutations by chemical inducers; S2. Screening the cell line after inducing mutations in vitro by drug pressure; S3. Inoculating the cells screened in vitro into nude mice subcutaneously for in vivo passage screening.

5. The preparation method according to claim 4, characterized in that, In S1, the chemical inducer is ethyl methanesulfonate, and the concentration is 200-300 μg / ml.

6. The production method according to claim 5, wherein In S2, the cells after inducing mutations are treated by drug shock using paclitaxel with a concentration of 100-200 μM.

7. The production method according to claim 6, wherein The drug shock treatment is performed 2-5 times, and the single drug shock time is 2-20 h, and the shock time of multiple drug shocks is increased successively.

8. The preparation method according to claim 7, characterized in that, In S2, it further comprises: The surviving cells after in vitro drug pressure screening are inoculated at a low density, single cell clones are separated and cultured, and subclone cell lines with strong proliferation ability are screened for subsequent in vivo passage screening.

9. The production method according to claim 8, characterized by, In step S3, the in vitro selected cells are inoculated subcutaneously in nude mice and, when the tumor volume reaches 100 mm 3 , treatment with paclitaxel at 3-7 mg / kg / 3d is started; when the tumor volume reaches 1500 mm 3 , the tumor is excised and passaged.

10. The esophageal cancer cell line according to any one of claims 1-3 in the screening of antitumor drugs, the study of tumor drug resistance mechanism or the construction of animal models.

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