In-vivo mouse colorectal cancer radiotherapy resistant cell line and construction method thereof

Abstract

The invention discloses an in-vivo mouse colorectal cancer radiotherapy resistant cell line and a construction method thereof. The cell line is a colorectal cancer radiotherapy resistant cell line derived from mouse MC38 cells. An MC38-RR cell line with radiotherapy resistance is induced by adopting a method of repeatedly radiating a tumor inoculated to the subcutaneous part of a mouse, the preservation name of the MC38-RR cell line is MC38-RR, the preservation unit is China General Microbiological Culture Collection Center, the preservation date is February 26, 2025, and the preservation number is CGMCC No. 46328. Experiments find that the medium-high dose radiation resistance of the constructed MC38-RR cell line is significantly higher than that of a parent MC38 cell line.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an in vivo mouse radiotherapy-resistant cell line for colorectal cancer and its construction method. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, ranking third in incidence and second in mortality. Radiotherapy, as a crucial treatment for colorectal cancer, plays a vital role in locally controlling tumor growth and improving patient survival. However, in clinical practice, not all patients are sensitive to radiotherapy; tumor cell resistance to radiotherapy is a key factor affecting treatment efficacy. Radiotherapy resistance refers to the ability of tumor cells to survive and continue to proliferate after receiving a certain dose of radiotherapy, leading to treatment failure and cancer recurrence. Currently, increasing research suggests that the mechanism of radiotherapy resistance may be related to the tumor microenvironment. Radiotherapy can induce various immune cell-related immune stimulation and inhibition, as well as induce tumor hypoxia, extracellular matrix remodeling, acidic microenvironment, various inflammatory responses, and angiogenesis, all of which may mediate radiotherapy resistance. Nevertheless, the mechanisms by which the microenvironment leads to radiotherapy resistance in colorectal cancer are not yet fully elucidated, particularly lacking in vivo mouse cell models that can simulate clinical radiotherapy resistance. Therefore, constructing an in vivo mouse colorectal cancer cell line that reflects radioresistance characteristics is of great significance for in-depth research on the molecular mechanisms of radioresistance, evaluation of radiotherapy efficacy, and development of new radiosensitization strategies. This application aims to provide a method for constructing an in vivo mouse colorectal cancer radioresistance cell line, which will provide a powerful experimental tool for studying radioresistance mechanisms, screening radiosensitizing drugs, and evaluating radiotherapy efficacy.

[0003] Patent application CN106367391A discloses a method for constructing a radiotherapy-resistant cell line of human rectal cancer cells SW480; patent applications CN118599778A and CN113583963A disclose a radiotherapy-resistant cell line of mouse sarcoma and a radiotherapy-resistant cell line of mouse oral squamous cell carcinoma, respectively; however, both human and mouse radiotherapy-resistant cell lines adopt an in vitro radiotherapy mode, which is easy to operate, observe and analyze, but cannot fully represent the actual situation in the organism because it lacks the complex interaction and regulatory mechanisms such as the microenvironment of the organism.

[0004] Currently, there are no clear reports describing how to construct radioresistant cell lines for colorectal cancer in vivo, which poses a challenge to in-depth exploration of the molecular mechanisms of radioresistance and improving radiotherapy efficacy. The purpose of this invention is to propose a method for constructing radioresistant cell lines for colorectal cancer, aiming to provide new research avenues and guidance for in-depth research into the molecular mechanisms of radioresistance in this cancer, identification of potential therapeutic targets, and ultimately, enhancement of radiosensitivity. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide an in vivo mouse radiotherapy-resistant cell line for colorectal cancer. This application utilizes in vivo radiotherapy in mice to construct the radiotherapy-resistant cell line, resulting in more biologically significant outcomes.

[0006] This application adopts the following technical solution:

[0007] A mouse radiotherapy-resistant cell line for colorectal cancer, with the accession name MC38-RR, deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 26, 2025, accession number CGMCC No. 46328.

[0008] Furthermore, it originates from MC38 cells, which are cells that can still survive after multiple generations of radiotherapy in mice.

[0009] The present invention also provides a method for preparing the above-mentioned in vivo mouse radiotherapy-resistant colorectal cancer cell line, comprising the following steps:

[0010] 1) Inject MC38 cells subcutaneously into C57BL / 6 mice to induce tumor growth. Wait until the tumors grow to 100-150 mm. 3 When the tumors are of similar size, select 5 mice and irradiate the corresponding tumor locations with X-rays.

[0011] 2) Continue feeding the mice after radiotherapy until the tumor size is ≥500mm. 3 Afterwards, the mice were euthanized and the tumors were removed, then cut into 5-10mm sections. 3 Small pieces were implanted subcutaneously into new mice and repeatedly irradiated with X-rays;

[0012] 3) Repeat step 2) 5 times (collecting surviving tumor tissue and preparing a tumor cell suspension, then passaged stably in vivo for 5 generations) to obtain primary tumor cells, which are then passaged to obtain the radiotherapy-resistant cell line MC38-RR. Further experiments can be designed to verify the radiotherapy-resistant characteristics of the cell line.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] Most published studies on the construction of radiotherapy-resistant tumor cell lines have adopted the method of repeated in vitro irradiation. In this invention, C57BL / 6 mouse-derived MC38 cells are inoculated subcutaneously into mice to induce tumor formation. Primary cell lines are prepared by repeated radiotherapy in mice, thus constructing a radiotherapy-resistant cell line. Moreover, due to the complex microenvironment in the body, this cell line is more representative of the actual situation in the body than existing radiotherapy-resistant cell lines. Attached Figure Description

[0015] Figure 1 The CCK8 assay was used to verify cell viability.

[0016] Figure 2 The study compared the radiosensitivity of MC38 and MC38-RR in a mouse subcutaneous tumor model in vivo. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0018] I. Establishment of the radiation-resistant cell line MC38-RR

[0019] 1. Cell Culture: The MC38 mouse colon cancer cell line was purchased from Beyotime (#C7399). MC38 cells were seeded in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution in culture flasks and cultured in a 37°C incubator with 5% CO2 and saturated humidity. During the exponential growth phase, cells were digested with trypsin containing 0.25% EDTA, counted, and prepared into a cell suspension of 1×10^6 cells / 100μL. The cell suspension was then mixed 1:1 with matrix gel to prepare a cell-Matrix gel suspension of 5×10^5 cells / 100μL, thoroughly mixed by pipetting, and stored in an ice box.

[0020] 2. Animal Experiments: Female C57BL / 6 mice, aged 6-8 weeks and bred at the Experimental Animal Center of Hangzhou Medical College, Zhejiang Province, were used and placed in the SPF-grade experimental environment of the animal experiment center for one week to acclimatize. 100 μL of cell matrix gel suspension was injected subcutaneously into the mice, and the growth of the subcutaneous tumor was observed and measured every 3 days. The tumors were allowed to grow to 100-150 mm. 3 Mice were given one 10 Gy radiotherapy session. Subcutaneous tumors were observed and measured every 3 days after radiotherapy, and treatment continued until the tumors reached 500 mm. 3 After approximately 1-2 weeks, the mice are euthanized, the tumors are removed, and the tumors are cut into 5-10 mm sections. 3 Small pieces of the tumor were implanted subcutaneously into new mice, and subsequent procedures were the same as before. After five rounds of in vivo passage, the mice were sacrificed and the tumors were removed.

[0021] 3. Irradiation method and dosage: C57BL / 6 mice inoculated with subcutaneous tumors were placed on a plate, and the tumor sites were irradiated with an X-ray beam from a linear accelerator (LINAC, Siemens, Germany), providing a highly uniform and precise radiation dose to the mouse tumors. The X-ray photon beam (6MV) generated by the LINAC had a dose rate of 200mu / min.

[0022] 4. MC38-RR Primary Cell Culture: The removed tumor cells were minced and placed in a 50mL centrifuge tube. 10mL of collagenase IV (1mg / mL) was added. The centrifuge tube was placed on a shaker at 260rpm / min and 37℃ for 30min. After digestion, the cells were centrifuged at 50g for 5min, and the supernatant was collected as a tumor suspension. The suspension was filtered through a 70µM filter into a 50mL centrifuge tube, the filter was rinsed with PBS, and the cells were centrifuged at 1000rpm and 4℃ for 5min. The supernatant was discarded, and the cells were resuspended in 5mL LDM medium to obtain the tumor suspension. The tumor suspension was then transferred to a 6cm culture dish. The medium was changed the next day, and the cells were passaged 5 times. The selected cell line was named MC38-RR. Cells used in subsequent experiments should be passages of 10 or less.

[0023] II. Validation of the radiotherapy-resistant cell line MC38-RR

[0024] 1. CCK8 assay to verify cell viability: The CCK8 assay is a method for detecting cell viability, which is positively correlated with cell viability and cell number, and can also detect cell proliferation. MC38 and MC38-RR cells in the exponential growth phase were seeded at 2000 cells per well in 96-well plates, with three replicates per cell type and dose. Twenty-four hours after seeding, cells were treated with radiotherapy (IR) at doses of 0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy, respectively. The culture medium was changed after IR treatment and the cells were cultured for 7 days. Then, 10 μg of CCK-8 was added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader. Cell viability (%) = OD450 of the radiotherapy group / OD450 of the control group. The results showed that the cell viability of MC38-RR cells was significantly higher than that of MC38 cells under different radiotherapy doses. Figure 1 ).

[0025] 2. Comparison of radiosensitivity of mouse subcutaneous tumor models to MC38 and MC38-RR in vivo: 5×10 5 MC38 and MC38-RR cells were subcutaneously seeded into mice (method as before), and the tumors were allowed to grow to 100-150 mm. 3 After radiotherapy, tumor volume was measured and recorded every 3 days. Twelve days after radiotherapy, mice were sacrificed, tumors were harvested, photographed, and their mass was measured. The volume and mass of MC38-RR tumors after radiotherapy were significantly higher than those of MC38 tumors. Figure 2 ).

[0026] The above results suggest that MC38-RR exhibits a very strong radioresistance effect in vivo. This indicates that the tumor microenvironment does indeed participate in the radioresistance process, and plays a more important role than the tumor cells themselves.

Claims

1. An in vivo mouse radiotherapy-resistant cell line for colorectal cancer, characterized in that, The deposit name is MC38-RR, the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit date is February 26, 2025, and the accession number is CGMCC No. 46328.

2. The cell line as described in claim 1, characterized in that, It originates from MC38 cells.

3. A method for preparing the cell line as described in claim 1, characterized in that, Includes the following steps: 1) Irradiate subcutaneous tumors in mice with X-rays; 2) Continue feeding the mice after radiotherapy until the tumor size is ≥500mm. 3 Then, the tumor was removed and cut into 5-10mm sections. 3 Small pieces were inoculated into the next batch of mice and X-ray irradiation was repeated; 3) Repeat step 2) 5 times, collect primary mouse tumor cells and passage them to obtain the cell line.

4. The method as described in claim 3, characterized in that: The X-ray exposure dose is 10 Gy / time.

Images