Colorectal cancer large segmentation radiotherapy recurrence-resistant cell strain and application thereof

Abstract

The invention discloses a recurrence-resistant cell strain for colorectal cancer large segmentation radiotherapy and application, and relates to the technical field of cell biology. The colorectal cancer large segmentation radiotherapy recurrence-resistant cell strain is preserved in the China Center for Type Culture Collection on January 2, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C202533. By constructing the colorectal cancer large segmentation radiotherapy recurrence-resistant cell strain, a new biological material is provided for studying the radiotherapy recurrence-resistant colorectal cancer. The cell strain is screened in a mouse body with a sound immune system, has better physiological correlation, and is beneficial to research on the influence of an immune microenvironment on tumor cell radiotherapy resistance and the remodeling effect of radiotherapy resistant cells on the immune microenvironment. The cell strain can also monitor tumor recurrence after radiotherapy in real time through fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a colorectal cancer large-fraction radiotherapy recurrence-resistant cell line and application thereof. Background Art

[0002] Colorectal cancer (CRC) is one of the malignant tumors with high morbidity and mortality worldwide. In order to improve the treatment effect of colorectal cancer, radiotherapy (RT) has become one of the important adjuvant treatments for patients with locally advanced or inoperable colorectal cancer, especially in the combination of chemotherapy and immunotherapy.

[0003] The traditional radiotherapy regimen is conventional fractionated radiotherapy (CFRT), the main feature of which is that the total radiation dose is divided into multiple small doses for irradiation. In recent years, hypofractionated radiotherapy (HFRT) has gradually attracted attention as a new radiotherapy method. HFRT reduces the number of treatments through a single large dose of irradiation. Compared with CFRT, HFRT can improve the local control rate in a shorter period of time. However, studies have shown that HFRT's higher control rate of local tumors is also accompanied by a higher risk of resistance and recurrence. HFRT-resistant tumors not only lead to poor treatment effects, but also easily lead to tumor recurrence and metastasis, especially in the treatment of tumors such as colorectal cancer. Radiotherapy resistance is not only related to the repair mechanism of tumor cells themselves, but also closely related to changes in the tumor microenvironment. Immune escape, vascular remodeling, and accumulation of immunosuppressive cells in the tumor microenvironment are important factors in radiotherapy resistance and tumor recurrence. However, most current studies focus only on the transient effects of HFRT's direct killing of tumor cells, and lack systematic studies on changes in the immune microenvironment during tumor resistance to HFRT recurrence, especially the mechanism of HFRT resistance to recurring tumors on the remodeling of the immune microenvironment. In order to better study the role of HFRT in the immune microenvironment and its impact on tumor recurrence, it is very necessary to construct a colorectal cancer cell model with HFRT resistance and immune regulation background.

[0004] The construction of traditional radiotherapy-resistant cell models usually obtains radiotherapy-resistant phenotypes by repeatedly irradiating tumor cells in vitro, but such models fail to effectively reflect the long-term effects of radiotherapy on the tumor immune microenvironment, nor can they simulate the complexity of the tumor microenvironment in clinical treatment. Therefore, the present invention intends to construct a tumor cell model that can both simulate the radiotherapy-resistant phenotype and reflect the remodeling of the immune microenvironment caused by radiotherapy resistance, thereby providing technical support for a deep understanding of the immune mechanism of tumor radiotherapy resistance and recurrence. Summary of the invention

[0005] The purpose of the present invention is to provide a colorectal cancer large-fraction radiotherapy resistant recurrence cell line and application to solve the problems existing in the above-mentioned prior art. The cell line is screened in mice with a healthy immune system, has good physiological relevance, and is helpful to study the influence of the immune microenvironment on the radiotherapy resistance of tumor cells and the remodeling effect of radiotherapy-resistant cells on the immune microenvironment.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line. The colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line was deposited in the China Center for Type Culture Collection on January 2, 2025, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: C202533.

[0008] The present invention also provides the use of the above-mentioned colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line in studying the immune mechanism of colorectal cancer radiotherapy-resistant recurrence.

[0009] The present invention also provides the use of the above-mentioned colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line in screening anti-tumor drugs.

[0010] The present invention also provides the use of the above-mentioned colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line in preparing an animal model of colorectal cancer radiotherapy resistance.

[0011] The present invention also provides a method for constructing an animal model of colorectal cancer resistant to radiotherapy, comprising the steps of inoculating the above-mentioned colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line subcutaneously into an experimental animal to construct the animal model of colorectal cancer resistant to radiotherapy.

[0012] Furthermore, the experimental animal is a mouse.

[0013] Furthermore, the colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line is inoculated subcutaneously in the experimental animal in the form of a cell suspension.

[0014] The present invention also provides an application of an animal model constructed according to the above construction method in studying the immune mechanism of colorectal cancer radiotherapy resistance and recurrence.

[0015] The present invention also provides an application of an animal model constructed according to the above construction method in screening anti-tumor drugs.

[0016] The present invention discloses the following technical effects:

[0017] 1. A colorectal cancer large-fraction radiotherapy-resistant recurrence cell line with an immune background is provided, which can be used to reveal the interaction between radiotherapy and the immune microenvironment. The present invention provides a new biomaterial for studying colorectal cancer with radiotherapy resistance and recurrence by constructing a colorectal cancer large-fraction radiotherapy-resistant recurrence cell line. The cell line is screened in mice with a healthy immune system and has good physiological relevance, which is helpful for studying the effect of the immune microenvironment on the radiotherapy resistance of tumor cells and the remodeling effect of radiotherapy-resistant cells on the immune microenvironment. The cell line can also monitor the recurrence of tumors after radiotherapy in real time by fluorescence imaging.

[0018] 2. Molecular markers that can be used to screen for radiotherapy resistance

[0019] The present invention provides a cell model for screening molecular markers related to radiotherapy resistance and immune microenvironment regulation. The screening of these markers helps to identify and predict radiotherapy-resistant tumors, and further guides the formulation of individualized radiotherapy treatment plans in clinical practice.

[0020] 3. Provide new ideas for the study of radiotherapy resistance mechanism

[0021] The colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line of the present invention can provide a new research idea for exploring the mechanism of radiotherapy resistance. By studying the changes in the immune microenvironment after radiotherapy, the immune escape mechanism and the effect of the immune microenvironment on radiotherapy resistance can be revealed.

[0022] 4. Efficient screening of new anti-tumor drugs

[0023] The present invention provides an experimental platform for developing new radiotherapy combined with immunotherapy and screening radiotherapy resistance-related target drugs. The radiotherapy resistance recurrence model established by the present invention can efficiently screen and evaluate the effects of new anti-tumor drugs and promote the clinical application of radiotherapy combined with immunotherapy.

[0024] In summary, the present invention not only provides a novel research tool for the study of radiotherapy resistance mechanism by constructing a large-fraction radiotherapy-resistant recurrence cell line for colorectal cancer, but also provides technical support for clinical optimization of radiotherapy regimens and development of new anti-tumor drugs, and has high scientific research value and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1Flow chart of the experimental design for establishing a subcutaneous tumor model using CT26 cells and BALB / c immune-competent mice to screen for colorectal cancer cell lines resistant to large-fraction radiotherapy;

[0027] Figure 2 Visible light photographs of the screening process for animal models of tumor recurrence after hypofractionated radiotherapy;

[0028] Figure 3 Schematic diagram of the fluorescence signal detection results of an animal model of tumor recurrence after large-fractionation radiotherapy;

[0029] Figure 4 This is a microscopic image of γ-H2Ax expression detected by immunofluorescence staining after hypofractionated radiotherapy.

[0030] Figure 5 for Figure 4 Statistical graph of the number of fluorescent spots;

[0031] Figure 6 This is the result of flow cytometry analysis of cell apoptosis after hypofractionated radiotherapy;

[0032] Figure 7 It is a statistical graph of the proportion of cell apoptosis;

[0033] Figure 8 This is the result of CCK8 experiment to measure cell proliferation ability;

[0034] Fig. 9 This is the result of the plate cloning experiment;

[0035] Fig.10 It is the SF (survival fraction) statistical graph;

[0036] Fig.11 Flow chart of the in vivo validation experiment of the hypofractionated radiotherapy resistance phenotype of CT26-Luc_HFRT-R;

[0037] Fig.12 Images of subcutaneous tumor fluorescence signals detected for small animal in vivo imaging;

[0038] Fig.13 This is a visible light photograph of the exfoliated tumor tissue;

[0039] Fig.14 It is a statistical graph of tumor weight;

[0040] Fig.15 This is the result of flow cytometry detection of M1 and M2 macrophages in the tumor microenvironment;

[0041] Fig.16 It is a statistical chart of the proportion of M1 and M2 macrophages;

[0042] Fig.17Flow cytometry for detecting CD8 + Typical images of T cell functional markers;

[0043] Fig.18 For CD8 + Statistical graph of T cell function markers;

[0044] Fig.19 This is a flow chart of the experiment to detect the distant metastasis ability of CT26-Luc_HFRT-R in vivo;

[0045] Fig. 20 Fluorescence signal map of liver metastases detected for small animal in vivo imaging;

[0046] Fig.21 This is a visible light photograph of a dissected mouse liver;

[0047] Fig. 22 HE staining of mouse liver sections;

[0048] Fig.23 The volcano plot of differentially expressed genes between the CT26-Luc_HFRT-R cell group and the wild-type (WT) group; genes with log2FC>1 and adj P value<0.05 were selected to reveal the gene expression changes in radiotherapy-resistant cells;

[0049] Fig.24 It is the biological process clustering diagram of differentially expressed genes GO (Gene Ontology);

[0050] Fig.25 This is a clustering diagram of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of differentially expressed genes. DETAILED DESCRIPTION

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0054] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0055] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0056] Example 1

[0057] 1. Materials and Methods

[0058] 1.1 Experimental Materials and Equipment

[0059] The original colorectal cancer cell line was CT26.

[0060] The biological X-ray irradiator is a commercially available RS2000-225 manufactured by RAD SOURCE; the small animal in vivo three-dimensional multi-mode imaging system is a commercially available IVIS SpectrumCT manufactured by PerkinElmer Inc.

[0061] 1.2 Experimental methods

[0062] 1.2.1 Construction of colorectal cancer recurrence-resistant cell lines after hypofractionated radiotherapy

[0063] like Figure 1 As shown, the method for constructing a colorectal cancer large-fraction radiotherapy recurrence-resistant cell line of the present invention is described in detail as follows:

[0064] (1) Construction of CT26-Luc cells stably expressing luciferase

[0065] Use pLenti-CMV-Luc-Puro lentiviral vector and packaging plasmid (psPAX2 and pMD2.G) in a ratio of 5:3.75:1.25, and transfect the plasmid into 293T packaging cells using liposome transfection reagent. Replace the culture medium 24 hours after transfection, collect the viral supernatant 48 hours later, and obtain viral particles after centrifugation and filtration. Mix the viral supernatant with Polybrene and add it to CT26 cells and culture at 37°C for 48 hours. Add 5μg / mL puromycin to select positive cells until the negative cells die completely. Verify the fluorescent signal by chemiluminescence detection system. Amplify and culture the positively screened CT26-Luc cells to ensure that the cells are in good growth condition, resuspend the cells in sterile 1×PBS and count them, and adjust the cell concentration to 1×10 7 / mL, for future use.

[0066] (2) Establishment of animal model

[0067] Culture and collect colorectal cancer CT26-Luc cell lines in the logarithmic growth phase, and add 1×10 6 100 μL suspension of 10 cells was injected subcutaneously into immune-competent BALB / c mice to construct a subcutaneous tumor model. After inoculation, the long and short diameters of the tumor were measured with a caliper every 2-3 days to calculate the tumor volume (V = 0.5 × long diameter × short diameter). 2 On the tenth day after tumor implantation, the tumor volume was 100 ± 20 mm 3 The mice were placed within the range and the radiotherapy experiment started.

[0068] (3) Group processing

[0069] When the subcutaneous tumors of mice reached a specified volume, the mice were randomly divided into two groups:

[0070] Large-fraction radiotherapy group: Lead blocks were used to cover the whole body of the mice, exposing only the subcutaneous tumor. A small animal biological X-ray irradiator (RS2000-225) was used to irradiate the subcutaneous tumor area, while normal tissues were not irradiated. The subcutaneous tumor was irradiated 8 Gy each time, with a 1-day interval in between, for a total of 3 irradiations.

[0071] Surgical resection control group: On the fifteenth day after mice were tumor-bearing (the same day as the end of radiotherapy for the large-fractionation radiotherapy group), subcutaneous tumors were surgically resected and sutured, and the tumor masses were collected and sterilely separated and cultured to obtain control group cells (named CT26-Luc_WT, abbreviated as WT).

[0072] (4) Screening for radiotherapy-resistant recurrent tumors

[0073] The tumor volume was measured every 3 days to observe whether the tumor had significantly shrunk after radiotherapy. The tumor was continuously monitored to record whether it re-increased in volume after radiotherapy was stopped (defined as recurrence). The fluorescence signal intensity of the tumor was detected weekly using a small animal in vivo three-dimensional multimodal imaging system (IVIS system) to monitor tumor growth. Tumors whose fluorescence signals significantly weakened (tumor shrinkage) after radiotherapy and then re-enhanced (recurrence) were defined as radiotherapy-resistant recurrent tumors. On the 21st day after radiotherapy, mice with the largest radiotherapy-resistant recurrent tumor volume in the HFRT treatment group were screened. Figure 2-Figure 3 Diagram of the screening process for animal models of tumor recurrence after hypofractionated radiotherapy.

[0074] (5) Isolation and culture of radiotherapy-resistant recurrent tumors

[0075] Tissue sampling: The mice with the largest radiotherapy-resistant recurrent tumor volume in the HFRT treatment group obtained in step (4) were humanely killed, and the recurrent tumor tissue was isolated under sterile conditions.

[0076] Cell culture: Recurrent tumor tissue was minced (1 mm 3 The cells were cultured in DMEM medium containing 10% fetal bovine serum and passaged for 3 times to obtain radiotherapy-resistant recurrence cell lines.

[0077] (6) Repeat the in vivo screening process

[0078] The radiotherapy-resistant relapse cell line obtained in step (5) was inoculated into BALB / c mice again for a subcutaneous tumor-bearing experiment, and steps (2) to (5) were repeated once. Finally, a cell line with a stable radiotherapy-resistant relapse phenotype was screened and named CT26-Luc_HFRT-R (abbreviated as HFRT-R) cell line.

[0079] 1.2.2 Phenotypic verification

[0080] In vitro experiments: The screened radiotherapy-resistant recurrence cell lines were subjected to immunofluorescence staining, CCK8, clone formation, and cell apoptosis experiments in vitro to verify the radiotherapy resistance of the cell lines.

[0081] In vivo experiments:

[0082] ① Such as Fig.11 As shown, the screened radiotherapy-resistant recurrence cell lines were inoculated subcutaneously into BALB / c mice, and the HFRT treatment experiment was repeated. By comparing with the CT26-Luc_WT cells in the surgical resection group, flow cytometry was used to evaluate the differences in radiotherapy resistance and immune microenvironment regulation of the CT26-Luc_HFRT-R cell line.

[0083] ② If Fig.19As shown, the screened radiotherapy-resistant recurrence cell line was injected through the spleen to construct a liver metastasis mouse model, and the small animal in vivo three-dimensional multimodal imaging system was used to compare it with the CT26-Luc_WT cells in the surgical resection group to evaluate the differences in metastasis and recurrence of the CT26-Luc_HFRT-R cell line.

[0084] 1.2.3 Cell expansion and preservation

[0085] The positively screened CT26-Luc_HFRT-R cells were expanded and cultured to ensure that the cells were in good growth condition, and a portion of the cells were frozen according to standard methods.

[0086] Mouse (Mus musculus) colorectal cancer cells CT26-Luc_HFRT-R were deposited in the China Center for Type Culture Collection (CCTCC) on January 2, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C202533.

[0087] 2. Experimental results

[0088] 2.1 Phenotypic verification results of in vitro experiments

[0089] The results of immunofluorescence staining of radiotherapy-resistant recurrent cell lines in vitro are shown in Figure 4-Figure 5 The results showed that after in vitro radiotherapy, the γH2AX staining signal in the CT26-Luc_HFRT-R cell line was significantly weakened compared with the WT cell line. The statistical results showed that the number of positive foci decreased significantly, indicating that the CT26-Luc_HFRT-R cell line has a strong DNA repair ability when facing radiotherapy.

[0090] Flow cytometry analysis of cell apoptosis after hypofractionated radiotherapy Figure 6-Figure 7 The results showed that the total apoptosis rate (ratio of early plus late apoptotic cells) of CT26-Luc_HFRT-R cell line was significantly lower than that of CT26-Luc_WT cell line after hypofractionated radiotherapy. This indicates that radiotherapy-resistant cell lines can effectively resist radiotherapy-induced cell apoptosis after hypofractionated radiotherapy, which may be closely related to their stronger DNA repair ability and the activation of signaling pathways related to resistance to radiotherapy.

[0091] The results of the CCK8 assay to determine cell proliferation ability are shown in Figure 8 , showing the difference in cell apoptosis before and after radiotherapy treatment. The results showed that the in vitro proliferation rate of the CT26-Luc_HFRT-R cell line remained unchanged compared with the CT26-Luc_WT cell line.

[0092] The results of the plate cloning experiment are shown in Fig. 9, used to verify the proliferation and clone-forming ability of radiotherapy-resistant cells. Compared with the CT26-Luc_WT cell line, the CT26-Luc_HFRT-R cell line was still able to form a larger number of clones after radiotherapy, and the size and number of clones were significantly higher than those of the CT26-Luc_WT cell line; Fig.10 The SF (survival fraction) statistical graph further demonstrates the radiotherapy effect of different treatment groups. In the CT26-Luc_HFRT-R cell line, the survival fraction after radiotherapy treatment was significantly higher than that of the CT26-Luc_WT cell line, and the survival fraction decreased less with the increase of radiotherapy dose.

[0093] 2.2 Phenotypic validation results of in vivo experiments

[0094] The in vivo validation results of the hypofractionated radiotherapy resistance phenotype of CT26-Luc_HFRT-R are shown in Figure 12-Figure 18 By measuring the subcutaneous tumor immune microenvironment of mice, it was found that the infiltration of M2-polarized tumor-associated macrophages (TAMs) in the CT26-Luc_HFRT-R cell line was significantly increased compared with the CT26-Luc_WT cell line, while the tumor-infiltrating CD8 + The cytotoxicity of T cells was significantly inhibited.

[0095] The results of the distant metastasis ability test of CT26-Luc_HFRT-R in vivo are shown in Figure 20-22 By measuring the fluorescent signal of mouse liver metastases, it was found that the CT26-Luc_HFRT-R cell line showed stronger liver metastasis ability than the CT26-Luc_WT cell line, characterized by a shorter tumor latency and larger metastatic foci.

[0096] 2.3 Results of RNA-Seq transcriptome sequencing of CT26-Luc_HFRT-R cells

[0097] Fig.23 The volcano plot of differentially expressed genes compared with the wild-type (CT26-Luc_WT) group, genes with log2FC>1 and adj P value<0.05 were selected, revealing the gene expression changes in radiotherapy-resistant cells; Fig.24 The GO (Gene Ontology) biological process clustering of differentially expressed genes showed the main biological processes involved in radiotherapy resistance. Fig.25 Clustering of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of differentially expressed genes revealed signaling pathways that were significantly enriched in radioresistant cells.

[0098] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A colorectal cancer large-fraction radiotherapy-resistant recurrence cell line, characterized in that: The colorectal cancer large-fraction radiotherapy-resistant recurrence cell line was deposited in the China Center for Type Culture Collection on January 2, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C202533.

2. A use of the colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line as claimed in claim 1 in studying the immune mechanism of colorectal cancer radiotherapy-resistant recurrence.

3. Use of the colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line as claimed in claim 1 in screening anti-tumor drugs.

4. Use of the colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line as claimed in claim 1 in preparing an animal model of colorectal cancer radiotherapy resistance.

5. A method for constructing an animal model of colorectal cancer radiotherapy resistance, characterized in that: The method comprises the steps of inoculating the colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line according to claim 1 subcutaneously into an experimental animal to construct an animal model of colorectal cancer radiotherapy resistance.

6. The construction method according to claim 5, characterized in that: The experimental animals are mice.

7. The construction method according to claim 5, characterized in that: The colorectal cancer large-fractionation radiotherapy-resistant recurrence cell line is inoculated subcutaneously in the experimental animal in the form of a cell suspension.

8. Use of an animal model constructed according to the construction method according to any one of claims 5 to 7 in studying the immune mechanism of colorectal cancer radiotherapy resistance and recurrence.

9. Use of an animal model constructed according to the construction method according to any one of claims 5 to 7 in screening anti-tumor drugs.

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