Colorectal cancer radiotherapy resistant strain based on in-vivo iteration as well as construction method and application of colorectal cancer radiotherapy resistant strain

Abstract

The invention relates to the technical field of biology, in particular to a colorectal cancer radiotherapy resistant strain based on in-vivo iteration as well as a construction method and application of the colorectal cancer radiotherapy resistant strain. The influence of an immune microenvironment on tumor cells is fully considered, radiotherapy iteration is carried out on the tumor cells in a mouse body, MC38 tumor cells resistant to radiotherapy in the immune microenvironment are screened, and the colorectal cancer radiotherapy resistant strain is obtained and named as MC38-R5, the cell strain is high and stable in radiotherapy tolerance, the preservation number is CGMCC No.46314, and the cell strain is named as MC38-R5. The cell strain has a good application prospect in a plurality of aspects such as a cell model for researching a mouse colorectal cancer radiotherapy resistance mechanism and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a colorectal cancer radiotherapy-resistant strain based on in vivo iteration, a construction method thereof, and an application thereof. Background Art

[0002] The incidence and mortality of colorectal cancer (CRC) rank among the top in China. Compared with Western countries, the characteristics of CRC in China are as follows: the proportion of rectal cancer in colorectal cancer is about 60%, and 60-70% of them are low rectal cancers. Most patients are in the middle and advanced stages at the first visit. According to the 2022 NCCN guidelines for colorectal cancer, radiotherapy should be given to patients with middle and advanced rectal cancer. Radiotherapy can effectively reduce the local recurrence rate and improve the prognosis. It is reported that complete remission, partial remission, and minor remission each account for 1 / 3 after radiotherapy, and about 30% of patients show radiotherapy resistance.

[0003] The generation of tumor radiotherapy resistance is a complex process involving multiple genes, multiple factors, and multiple mechanisms. Multiple factors such as DNA damage repair, cell cycle arrest, autophagic regulation, the existence of tumor stem cells, changes in the tumor microenvironment, and tumor metabolism are the main ways for tumor cells to resist radiation damage. Research shows that there is a close relationship between the tumor immune microenvironment and radiotherapy resistance. Radiotherapy can help tumors survive by creating a hypoxic environment, limit the elimination of immune effector cells against tumors, and promote the activation of immunosuppressive cells, thus forming an immunosuppressive microenvironment, resulting in anti-radiotherapy. Radiation-activated macrophages may cause radiation resistance of cancer cells by inducing the high expression of tumor necrosis factor-α (TNF-α) and promoting angiogenesis, thereby promoting the recurrence of tumors after radiotherapy. Since DNA damage-inducible kinase ABL1 binds to the promoter of the colony-stimulating factor (CSF1) gene in the nucleus and enhances its transcription, blocking the migration of macrophages with a CSF1 inhibitor can radiosensitize tumors. In addition, studies on cervical cancer and breast cancer have confirmed that M2-type tumor-associated macrophages can cause radiotherapy resistance of tumor cells. In addition, it has been reported that radiotherapy can promote the enhancement of glycolytic metabolism in pancreatic cancer cells, promote the secretion of the metabolic molecule lactate, and lactate activates the function of myeloid-derived suppressor immune cells MDSCs, promoting an immunosuppressive microenvironment, and then leading to the progression and recurrence of pancreatic cancer. After radiotherapy, the regulatory CD4 + FOXP3 + T cells (Treg cells) also increase simultaneously. Treg cells exhibit immunosuppressive effects by producing CTLA4 signals and TGFβ. However, there is currently a lack of models for studying radiotherapy resistance and the tumor immune microenvironment.

[0004] At present, many tumor cells have successfully constructed acquired radiotherapy-resistant cell lines. The main construction methods are the "low-dose long-term induction method", the "high-dose short-term screening method", and the "gradient irradiation method" for cell line establishment. The "low-dose long-term induction method" generally irradiates tumor cells repeatedly about 30 times at a dose of 2 Gy to induce a radiotherapy-resistant cell subset in the tumor cells. This process takes a long time and requires repeated processing of the cells, increasing the probability of cell contamination. The "high-dose short-term screening method" is to increase the proportion of radiotherapy-resistant cell subsets in tumor cells through single or multiple sub-lethal dose irradiations, and finally screen out the radiotherapy-resistant cell subsets. This method requires first screening out the dose that causes sub-lethal damage to the cells through a series of dose points. During the screening process, the cells are prone to deformation and death, and the radiobiological parameters of the established cell line cannot represent the radioresistance of clinical recurrent cancer.

[0005] Constructing a new radiotherapy-resistant model for colorectal cancer, searching for treatment targets and mechanisms for radiotherapy resistance in colorectal cancer, is of great significance for improving the prediction and treatment of radiotherapy resistance in rectal cancer, thereby reducing the recurrence rate of rectal cancer patients and improving the prognosis. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a colorectal cancer radiotherapy-resistant strain MC38-R5 based on in vivo iteration.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a colorectal cancer radiotherapy-resistant strain, named colorectal cancer radiotherapy-resistant strain MC38-R5, which is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 46314.

[0009] Existing methods for radiotherapy-resistant cell lines only consider the impact of radiotherapy rays on the repair of tumor cell DNA damage, while ignoring the influence of the immune microenvironment on tumor cells. Radiotherapy can kill tumor cells and cause the release of pro-inflammatory molecules, which promotes the infiltration of immune cells such as T cells, dendritic cells (DCs), and natural killer cells (NK cells) into tumors. Radiotherapy can induce micronuclei in tumor cells, activate cytoplasmic nucleic acid sensors, especially the cyclic GMP-AMP synthase-interferon-inducible gene pathway, and reshape the tumor immune microenvironment. After radiotherapy, the pro-inflammatory pathway NF-κB is upregulated, accompanied by an increase in inflammatory factors such as IL-1β, IL-6, and IL-8, which may enhance the invasiveness and migratory ability of tumors. During the process of radiotherapy for tumor cells, the immune microenvironment, as an indispensable part of radiotherapy treatment, cannot be ignored. The present invention fully considers the influence of the immune microenvironment on tumor cells, performs radiotherapy iteration on tumor cells in mice, and screens for MC38 tumor cells that are resistant to radiotherapy under the immune microenvironment.

[0010] In the present invention, the mouse colorectal cancer cell line MC38 was inoculated into mice, and the colorectal cancer radiotherapy-resistant strain MC38-R5 was obtained after multiple radiotherapy treatments. This cell line has strong and stable radiotherapy tolerance. The present invention uses a mouse subcutaneous tumor model to detect the sensitivity of cells to radiotherapy in mice. The results show that the resistance of MC38-R5 cells in vivo and in vitro is higher than that of the parental MC38 cell line. Therefore, this cell line has good application prospects in many aspects such as being used as a cell model for studying the radiotherapy resistance mechanism of mouse colorectal cancer.

[0011] Preferably, the radiotherapy-resistant strain is the progeny cells obtained by in vivo iterative screening of the colorectal cancer cell line MC38.

[0012] In a second aspect, the present invention provides a method for constructing the above-mentioned colorectal cancer radiotherapy-resistant strain, comprising the following steps:

[0013] (1) Take the colorectal cancer cell line MC38 in good growth state for mouse subcutaneous tumor inoculation, and treat it with 8 Gy radiotherapy 3 times;

[0014] (2) Take the tumor tissue with less shrinkage after radiotherapy for dissociation and flow sorting;

[0015] (3) Take the MC38 cells obtained by flow sorting, repeat steps (1)-(2), and iteratively obtain the above-mentioned colorectal cancer radiotherapy-resistant strain.

[0016] The present invention selects a fractionation scheme of 8 Gy × 3 times as the stereotactic radiotherapy scheme; this scheme can fully activate the radiotherapy-induced immune response, activate the cGAS-STING signaling pathway, and activate the anti-tumor immune response.

[0017] Preferably, in the step (2), EPCAM cells are sorted by flow cytometry, which are MC38 cells. +

[0018] Preferably, in the step (3), the number of repetitions is 4 times.

[0019] In a third aspect, the present invention provides the application of the colorectal cancer radiotherapy-resistant strain in constructing a cell model for studying the mechanism of colorectal cancer radiotherapy resistance.

[0020] In a fourth aspect, the present invention provides the application of the colorectal cancer radiotherapy-resistant strain in constructing a cell model for screening radiosensitizing drugs for colorectal cancer radiotherapy.

[0021] In a fifth aspect, the present invention provides the application of the colorectal cancer radiotherapy-resistant strain in constructing a cell model for screening radiotherapy-resistant genes or therapeutic targets for colorectal cancer.

[0022] The beneficial effects of the present invention are as follows:

[0023] When constructing the radiotherapy-resistant cell model, the present invention takes into account the influence of the tumor immune microenvironment, performs radiotherapy iteration on tumor cells in mice, screens MC38 tumor cells resistant to radiotherapy under the immune microenvironment, and obtains a colorectal cancer radiotherapy-resistant strain named MC38-R5. This cell strain has strong and stable radiotherapy tolerance, and the preservation number is CGMCC No. 46314.

[0024] The present invention iterates multiple times in mice to fully ensure sufficient radiotherapy resistance ability in mice; the obtained cell strain has good application prospects in multiple aspects such as a cell model for studying the mechanism of colorectal cancer radiotherapy resistance in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram for constructing a colorectal cancer radiotherapy-resistant strain based on in vivo iteration.

[0026] Figure 2 It is a result diagram of detecting the radiotherapy resistance of MC38-R5 by CCK8.

[0027] Figure 3 It is a result diagram of detecting the radiotherapy resistance of MC38-R5 by plate cloning experiment.

[0028] Figure 4 It is a result diagram of detecting the radiotherapy resistance of MC38-R5 by mouse subcutaneous tumor model. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] ​Unless otherwise specified, the reagents used in the present invention are all commercially available products.

[0031] The colorectal cancer cell line MC38 used in the present invention was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This cell line has undergone cell line identification and monthly regular mycoplasma detection with negative results. The culture medium for culturing the cells was purchased from Gibco, and the fetal bovine serum was purchased from ExCell Bio.

[0032] Example 1: Construction of the in vivo iterative radiotherapy-resistant colorectal cancer cell line MC38

[0033] 1. Cell culture

[0034] The colorectal cancer cell line MC38 was cultured in DMEM medium containing 10% fetal bovine serum under the culture conditions of 37 °C and 5% CO2 (in an incubator). Cells with good growth status were selected for subsequent experiments.

[0035] 2. Subcutaneous tumor inoculation and radiotherapy in mice

[0036] The experimental animals were C57BL / 6 mice, 4 weeks old, female, with a body weight of 17 - 20 g.

[0037] On day 0, the cultured MC38 cells (5×10 5 cells / mouse) were injected subcutaneously into the back of the mice. When the tumor size exceeded 100 mm 3 , 8 Gy * 3 times of radiotherapy was performed, with a one-day interval between each radiotherapy. The tumor size was measured every two days (tumor volume calculation formula: length × width 2 × 0.5). When the tumor necrosis or the tumor volume reached 2000 mm 3 , the mice were sacrificed by cervical dislocation and the death endpoint was recorded. The tumors were dissected, weighed, and photographed, and the tumor tissues with relatively small shrinkage after radiotherapy were dissociated and sorted by flow cytometry for subsequent experiments.

[0038] 3. Dissociation and flow cytometry sorting of subcutaneous tumors in mice

[0039] (1) Select the tumor tissue with the worst therapeutic effect after radiotherapy and place it in a 10 cm dish, and wash away the blood and hair with 1×PBS;

[0040] (2) Put it into a 1.5 mL EP tube, separate the tissue into 1 mm tissue blocks with ophthalmic scissors, add digestive solution (RPMI1640 + 5% FBS + 200 U / mL DNase I + 1 mg / mL Collagenase IV + 0.5 mg / mL Hyaluronidase), mix well, and directly pour it into a 15 mL tube. Add digestive solution to 5 mL for each sample;

[0041] (3) Place it on a shaker and incubate at 80 rpm and 37 °C for 30 min; Prepare the filter membrane and pre-cool the centrifuge at 4 °C intermittently;

[0042] (4) Pour the digested tissue sample into a 70 μm filter screen, place a culture dish below, grind the tissue with the end of a 5 mL syringe until there are no obvious tissue particles, aspirate 1 mL of the filtrate to wash the filter screen, and collect the filtrate below into a 15 mL centrifuge tube; Centrifuge at 1000×g for 10 min at 4 °C and discard the supernatant;

[0043] (5) Lyse red blood cells: Add 0.5 mL of red blood cell lysate, incubate at room temperature for 30 s, add 3 mL of 1×PBS to resuspend, centrifuge again at 300×g for 5 min, and discard the supernatant;

[0044] (6) Staining: Add 100 μL of APC-EPCAM flow antibody diluted 1:200 (antibody for staining intestinal epithelial marker EPCAM), incubate on ice in the dark for 30 min, and add 150 μL of PBS to wash once;

[0045] (7) Perform flow sorting on the machine to sort out EPCAM + cells, which are MC38 cells and continue to be used for culture.

[0046] 4. Culture and re-inoculation of sorted cells

[0047] Culture the sorted EPCAM + cells again. After culturing a certain number, inject them subcutaneously into mice, and repeat steps 2 and 3.

[0048] The schematic diagram of the construction process of the present invention is as Figure 1 shown. The cells iterated to the fifth generation are called MC38-R5, and the original parental cells are called MC38-F0.

[0049] The MC38-R5 cell line obtained in the present invention has been deposited; Depositary institution: China General Microbiological Culture Collection Center, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Deposit date: December 30, 2024, Deposit number: CGMCC No. 46314.

[0050] Example 2: Identification of radiotherapy-resistant colorectal cancer strain MC38 with in vivo iteration

[0051] 1. Determination of cell radiotherapy sensitivity by CCK8 method

[0052] (1) Use a 96-well plate to plate MC38-R5 and MC38-F0 cells, control the cell density to 1000 cells / well, and add 200 μL of culture medium to each well.

[0053] (2) After mixing evenly, the samples were placed in a 5% CO2, 37 °C cell culture incubator for culture. After the cells adhered to the wall, the cells were irradiated with 6MV-X rays generated by a Varian 2300C / D linear accelerator. The irradiation field was 30 cm × 30 cm, the source-skin distance was 100 cm, the irradiation depth was 1.3 cm, the gantry angle was 180 °C, the dose was 4 Gy, and an acrylic tissue compensator was used to compensate for the dose; after the irradiation was completed, CCK8 solution was added. 10 μL of CCK8 solution and 90 μL of DMEM empty culture (a total of 100 μL) were added to each well, and the culture was continued for 2 h.

[0054] (3) The absorbance value (OD value) of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0055] (4) Thereafter, CCK8 solution was added at the same time every day to measure the absorbance value for 5 consecutive days. Finally, the data were summarized and analyzed, and a cell growth curve was plotted with time as the abscissa and OD 450 as the ordinate.

[0056] The experimental results are as Figure 2 shown. The results showed that there was no significant difference in the proliferation rate between MC38-F5 and MC38-F0 without radiotherapy. The proliferation rate of MC38-F5 cells was relatively faster than that of MC38-F0 cells after radiotherapy, indicating a certain degree of radioresistance.

[0057] 2. Determination of cell radiosensitivity by colony formation assay

[0058] (1) Cells in the logarithmic growth phase were digested and centrifuged, then resuspended with complete medium, and then cell counting was performed. MC38-R5 and MC38-F0 cells were seeded into 5 six-well plates at a certain number respectively;

[0059] (2) Different numbers of cells were seeded in each six-well plate according to different radiotherapy doses: 400 cells / well for 0 Gy, 800 cells / well for 2 Gy, 1000 cells / well for 4 Gy, 5000 cells / well for 6 Gy, and 8000 cells / well for 8 Gy; 3 replicates were set for each group, and the culture medium was supplemented to 3 mL, and the cells were placed in a 5% CO2, 37 °C cell culture incubator for continued culture;

[0060] (3) The next day, irradiation was performed according to the radiotherapy dose required for cell seeding in the six-well plates. After irradiation, the cells were changed with fresh medium and placed in the cell culture incubator for continued culture for 14 days or until visible colonies appeared;

[0061] (4) The culture medium was aspirated, washed once with 1×PBS, the PBS was discarded, 4% paraformaldehyde was added for fixation for 30 min, and then washed once with 1×PBS;

[0062] (5) Add crystal violet staining solution, stain for 20 min, rinse with tap water, and air dry;

[0063] (6) Use a camera to take separate photos of the entire six-well plate and each well, and finally count the number of visible clones with the naked eye.

[0064] The experimental results are as Figure 3 shown. The results show that the number of clones of MC38-R5 is significantly higher than that of MC38-F0 cells after radiotherapy.

[0065] According to the results of the colony formation assay, a multi-target single-hit model was constructed to fit the dose-survival curve, and relevant radiobiological parameters were obtained. Among them, the survival fraction SF2 at 2 Gy is an important indicator representing cell radiosensitivity. The larger the SF2, the stronger the radioresistance. D0 represents the mean lethal dose and represents the radiosensitivity of the cell population; the larger the D0, the stronger the resistance of the cells to radiation. Dq is the quasi-threshold dose: the dose at which the straight part of the survival curve extends upward and intersects the horizontal axis where the survival rate is equal to 1. Dq represents the size of the cell's ability to repair sublethal damage. A smaller Dq value indicates a weaker repair ability.

[0066] The results are as follows: The SF2 value of MC38-R5 cells is 0.915, and the SF2 value of the parental cell MC38-F0 is 0.663, which is 1.38 times that of the parental cell.

[0067] The D0 value of MC38-R5 cells is 0.968, and the D0 value of the parental cell MC38-F0 is 0.686, which is 1.41 times that of the parental cell.

[0068] The Dq value of MC38-R5 cells is 0.921, and the Dq value of the parental cell MC38-F0 is 3.891, which is 4.22 times that of the parental cell.

[0069] All of the above indicators that are positively correlated with radioresistance suggest that MC38-R5 has significant radioresistance.

[0070] 3. Determination of cell radiosensitivity using a mouse subcutaneous tumor model

[0071] (1) Inoculate tumor cells: Randomly divide 24 4-week-old SPF-grade C57BL / 6 mice into 4 groups, with 6 mice in each group. Two groups are inoculated with each of the MC38-R5 and MC38-F0 cells, and are divided into an irradiation group and a non-irradiation group. Wipe and disinfect the gluteal and dorsal positions of C57BL / 6 mice with a 75% alcohol cotton ball. With the bevel of the needle facing up, enter the subcutaneous at an angle of 10-15 °C to the skin. Gently pick the skin and then insert the needle further until 2 / 3 of the needle is inserted. Slowly inject 0.1 mL of cell suspension. It is appropriate that there is a small raised papule on the skin epidermis and there is no leakage. After inoculating each group of tumors, make labels such as name and date;

[0072] (2) Irradiation: On the 7th day after injection, the subcutaneous tumors of C57BL / 6 mice were observed, and obvious spheroid formation was seen. Thereafter, the tumor volume (measuring the major axis and minor axis) was observed and recorded every 2 days. When the transplanted tumor grew to 100 mm 3 , the C57BL / 6 mice in the irradiation group were anesthetized, and the subcutaneous tumors of the C57BL / 6 mice in the irradiation group were irradiated with a dose of 8 Gy, 3 times, with an interval of two days between each irradiation. Then, the volume and weight of all transplanted tumors were continuously observed and recorded.

[0073] (3) Tumor excision: When the transplanted tumors were about 4 weeks old, the trends among groups were relatively stable and the differences were obvious. The skin of C57BL / 6 mice in each group was peeled off after cervical dislocation, and the subcutaneous transplanted tumors were completely dissected; the major axis and minor axis of the transplanted tumors were measured with vernier calipers, and the weight of the transplanted tumors was measured with a weighing instrument and completely recorded; the transplanted tumors were arranged in descending order from largest to smallest, with the control group on top and the experimental group below, and photographed and saved.

[0074] The experimental results are as Figure 4 shown. The results indicate that without radiotherapy, there is no significant difference in the growth of MC38-R5 and MC38-F0 cells in vivo. However, under radiotherapy, the tumor of MC38-F0 cells becomes significantly smaller after radiotherapy, while the change of MC38-R5 cells is not significant, suggesting that MC38-F5 has significant radioresistance.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A colorectal cancer radiotherapy-resistant strain, characterized in that, The colorectal cancer radiotherapy-resistant strain is named as colorectal cancer radiotherapy-resistant strain MC38-R5, and is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 46314.

2. The colorectal cancer radiotherapy-resistant strain according to claim 1, characterized in that, The radiotherapy-resistant strain is the progeny cells obtained by in vivo iterative screening of the colorectal cancer cell line MC38.

3. A method for constructing a colorectal cancer radiotherapy-resistant strain as described in any one of claims 1-2, characterized in that, It includes the following steps: (1) Take the colorectal cancer cell line MC38 with good growth state for subcutaneous tumor inoculation in mice, and treat it with 8 Gy radiotherapy for 3 times; (2) Take the tumor tissue with less shrinkage after radiotherapy for dissociation and flow sorting; (3) Take the MC38 cells obtained by flow sorting, repeat steps (1)-(2), and iteratively obtain the colorectal cancer radiotherapy-resistant strain.

4. The method for constructing a radioresistant colorectal cancer cell line according to claim 3, characterized in that, In the step (2), EPCAM + cells are sorted by flow cytometry, which are MC38 cells.

5. The method for constructing a radioresistant strain of colorectal cancer according to claim 3, characterized in that In step (3), the number of repetitions is 4 times.

6. Use of the colorectal cancer radiotherapy-resistant strain according to any one of claims 1-2 in constructing a cell model for studying the mechanism of colorectal cancer radiotherapy resistance.

7. Use of the colorectal cancer radiotherapy-resistant strain according to any one of claims 1-2 in constructing a cell model for screening radiosensitizing drugs for colorectal cancer radiotherapy.

8. Use of the colorectal cancer radiotherapy-resistant strain according to any one of claims 1-2 in constructing a cell model for screening colorectal cancer radiotherapy resistance genes or therapeutic targets.

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