Colorectal cancer large fractionation radiotherapy resistant recurrence cell line and application thereof

Abstract

This invention discloses a relapse-resistant cell line for hypofractionated colorectal cancer and its applications, relating to the field of cell biology. This relapse-resistant cell line for hypofractionated colorectal cancer was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202533. This invention provides a novel biomaterial for studying radiotherapy-resistant colorectal cancer by constructing a hypofractionated colorectal cancer cell line. The cell line was screened in mice with healthy immune systems, exhibiting good physiological relevance, which is helpful for studying the influence of the immune microenvironment on tumor cell radioresistance and the remodeling effect of radioresistant cells on the immune microenvironment. This cell line can also monitor tumor recurrence after radiotherapy in real time using fluorescence imaging.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, and in particular to a cell line for resisting recurrence in colorectal cancer treated with hypofractionated radiotherapy and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common and deadliest malignant tumors worldwide. To improve the treatment outcomes of CRC, radiotherapy (RT) has become an important adjuvant therapy for patients with locally advanced or inoperable CRC, playing a crucial role, especially in regimens combining chemotherapy and immunotherapy.

[0003] The traditional radiotherapy regimen is conventional fractionated radiotherapy (CFRT), which is characterized by dividing the total radiation dose into multiple smaller doses. In recent years, hypofractionated radiotherapy (HFRT) has gained increasing attention as a novel radiotherapy modality. HFRT reduces the number of treatment sessions by delivering a single high-dose irradiation, and compared to CFRT, it can improve local control rates in a shorter time. However, studies have shown that while HFRT achieves higher local tumor control rates, it also carries a higher risk of resistance and recurrence. HFRT-resistant tumors not only lead to poor treatment outcomes but also increase the likelihood of tumor recurrence and metastasis, especially in the treatment of tumors such as colorectal cancer. Radiotherapy resistance is related not only to the repair mechanisms of tumor cells themselves but also to changes in the tumor microenvironment. Immune escape, vascular remodeling, and the accumulation of immunosuppressive cells in the tumor microenvironment are important factors contributing to radiotherapy resistance and tumor recurrence. However, most current research focuses only on the transient effects of HFRT's direct killing effect on tumor cells, lacking systematic studies on changes in the immune microenvironment during tumor recurrence due to HFRT resistance. In particular, the mechanism of immune microenvironment remodeling in HFRT-resistant recurrent tumors has not been reported. To better investigate the role of HFRT in the immune microenvironment and its impact on tumor recurrence, it is essential to construct a colorectal cancer cell model with HFRT resistance and an immune regulatory background.

[0004] Traditional radiotherapy-resistant cell models are typically constructed by repeatedly irradiating tumor cells in vitro to obtain a radiotherapy-resistant phenotype. However, these models fail to effectively reflect the long-term effects of radiotherapy on the tumor immune microenvironment, nor do they simulate the complexity of the tumor microenvironment in clinical treatment. Therefore, this invention aims to construct a tumor cell model that can both simulate the radiotherapy-resistant phenotype and reflect the remodeling of the immune microenvironment induced by radiotherapy resistance, thereby providing technical support for a deeper understanding of the immune mechanisms underlying tumor radiotherapy resistance and recurrence. Summary of the Invention

[0005] The purpose of this invention is to provide a cell line for resisting recurrence of colorectal cancer treated with large-fractionated radiotherapy and its application, thereby addressing the problems existing in the prior art. This cell line was screened in mice with a healthy immune system, exhibiting good physiological relevance, which is helpful for studying 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 objectives, the present invention provides the following solution:

[0007] This invention provides a cell line resistant to recurrence after hypofractionated radiotherapy for colorectal cancer. The cell line was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202533.

[0008] This invention also provides the application of the above-mentioned relapse-resistant cell line for colorectal cancer in the study of the immune mechanism of relapse resistance to radiotherapy in colorectal cancer.

[0009] The present invention also provides the application of the above-mentioned relapse-resistant cell line for macrofractionated radiotherapy of colorectal cancer in screening antitumor drugs.

[0010] The present invention also provides the application of the above-mentioned large fractionated radiotherapy resistant recurrence cell line of colorectal cancer in the preparation of animal models of radiotherapy resistance in colorectal cancer.

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

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

[0013] Furthermore, the colorectal cancer hypofractionated radiotherapy-resistant recurrence cell line was inoculated subcutaneously into 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 resisting recurrence by radiotherapy.

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

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

[0017] 1. This invention provides a novel biomaterial for studying radiotherapy-resistant recurrence of colorectal cancer with an immune background, specifically a hypofractionated radiotherapy-resistant cell line. The cell line was screened in mice with intact immune systems, demonstrating good physiological relevance and facilitating research on the influence of the immune microenvironment on tumor cell radioresistance and the remodeling effect of radioresistant cells on the immune microenvironment. Furthermore, the cell line allows for real-time monitoring of tumor recurrence after radiotherapy using fluorescence imaging.

[0018] 2. Can be used to screen for molecular markers of radiotherapy resistance.

[0019] This invention provides a cell model for screening molecular markers related to radiotherapy resistance and immune microenvironment regulation. Screening these markers helps identify and predict radiotherapy-resistant tumors, further guiding the development of personalized radiotherapy treatment plans in clinical practice.

[0020] 3. Provides new insights for research on radiotherapy resistance mechanisms.

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

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

[0023] This invention provides an experimental platform for developing novel radiotherapy-immunotherapy regimens and screening drugs targeting radiotherapy resistance. The radiotherapy resistance relapse model established by this invention enables efficient screening and evaluation of the efficacy of novel antitumor drugs, promoting the clinical application of combined radiotherapy and immunotherapy.

[0024] In summary, this invention, by constructing a large-fractionation radiotherapy-resistant cell line for colorectal cancer, not only provides a novel research tool for studying the mechanism of radiotherapy resistance, but also provides technical support for optimizing radiotherapy regimens and developing new anti-tumor drugs in clinical practice, demonstrating high scientific research value and promising clinical application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1Flowchart of experimental design for constructing a subcutaneous tumor model using CT26 cells and BALB / c immunocompetent mice to screen for colorectal cancer cell lines resistant to large fractionation radiotherapy;

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

[0028] Figure 3 A schematic diagram of fluorescence signal detection results in an animal model of tumor recurrence after hypofractionated radiotherapy;

[0029] Figure 4 Microscopic images for detecting γ-H2Ax expression after hypofractionated radiotherapy using immunofluorescence staining;

[0030] Figure 5 for Figure 4 A statistical graph of the number of fluorescent dots;

[0031] Figure 6 Figure showing the results of flow cytometry analysis of cell apoptosis after hypofractionated radiotherapy;

[0032] Figure 7 This is a statistical graph showing the proportion of apoptotic cells.

[0033] Figure 8 The results of the CCK8 assay for cell proliferation were shown in the figure.

[0034] Figure 9 This is a graph showing the results of a plate cloning experiment;

[0035] Figure 10 A statistical chart of SF (Survival Score);

[0036] Figure 11 Flowchart for in vivo validation experiment of CT26-Luc_HFRT-R hypofractionated radiotherapy resistance phenotype;

[0037] Figure 12 Images for detecting fluorescence signals in subcutaneous tumors using in vivo imaging in small animals;

[0038] Figure 13 Visible light photograph of dissected tumor tissue;

[0039] Figure 14 A statistical graph of tumor weight;

[0040] Figure 15 Figure showing the results of flow cytometry analysis of M1 and M2 macrophages in the tumor microenvironment;

[0041] Figure 16 A statistical graph showing the proportions of M1 and M2 macrophages;

[0042] Figure 17For the detection of CD8 in the tumor microenvironment by flow cytometry + A typical diagram of T cell functional markers;

[0043] Figure 18 CD8 + Statistical graph of T cell function markers;

[0044] Figure 19 The flowchart is for the in vivo distant metastasis capability detection experiment of CT26-Luc_HFRT-R;

[0045] Figure 20 Fluorescence signal image for detecting liver metastases in small animals using in vivo imaging;

[0046] Figure 21 Visible light photograph of a dissected mouse liver;

[0047] Figure 22 HE staining image of a mouse liver section;

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

[0049] Figure 24 This is a cluster diagram of differentially expressed genes (GO) biological processes.

[0050] Figure 25 Cluster diagram of the differential gene KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway. Detailed Implementation

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

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0056] Example 1

[0057] 1. Materials and Methods

[0058] 1.1 Experimental Materials and Equipment

[0059] The original colorectal cancer cells were the CT26 cell line.

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

[0061] 1.2 Experimental Methods

[0062] 1.2.1 Construction of a cell line resistant to recurrence after hypofractionated radiotherapy for colorectal cancer

[0063] like Figure 1 As shown, the method for constructing a recurrence-resistant cell line for hypofractionated radiotherapy in colorectal cancer according to the present invention is described in detail below:

[0064] (1) Construct CT26-Luc cells that stably express luciferase.

[0065] The pLenti-CMV-Luc-Puro lentiviral vector and packaging plasmids (psPAX2 and pMD2.G) were mixed at a ratio of 5:3.75:1.25 and transfected into 293T packaging cells using liposome transfection reagent. The culture medium was changed 24 hours after transfection, and the viral supernatant was collected after 48 hours. Viral particles were obtained after centrifugation and filtration. The viral supernatant was mixed with Polybrene and added to CT26 cells, which were then cultured at 37°C for 48 hours. Positive cells were selected by adding 5 μg / mL puromycin until negative cells were completely dead. The fluorescence signal was verified using a chemiluminescence detection system. The positively selected CT26-Luc cells were expanded and cultured to ensure good cell growth. Cells were resuspended in sterile 1×PBS and counted, and the cell concentration was adjusted to 1×10⁻⁶. 7 / mL, for later use.

[0066] (2) Establishment of animal models

[0067] Culture and collect CT26-Luc colorectal cancer cell lines in the logarithmic growth phase, containing 1×10⁻⁶ cells. 6 100 μL of cell suspension was injected subcutaneously into immunocompetent BALB / c mice to establish a subcutaneous tumor model. After inoculation, the long and short diameters of the tumor were measured using calipers every 2-3 days, and the tumor volume was calculated (V = 0.5 × long diameter × short diameter). 2 On the tenth day after tumor implantation, tumors with a volume of 100±20 mm were selected. 3 The mice within the range were then subjected to radiotherapy experiments.

[0068] (3) Grouping

[0069] Once the subcutaneous tumors in the mice reached the prescribed volume, the mice were randomly divided into two groups:

[0070] Hypradiation group: Mice were shielded from the whole body with lead blocks, exposing only the subcutaneous tumors. The subcutaneous tumor areas were irradiated using a small animal biological X-ray irradiator (RS2000-225), while normal tissues were not irradiated. The subcutaneous tumors were treated with 8 Gy per fraction, with a one-day interval between fractions, for a total of three fractions.

[0071] Surgical resection control group: On the fifteenth day after tumor formation in mice (the same day as the end of radiotherapy in the hypofractionated radiotherapy group), subcutaneous tumors were surgically removed and sutured. The removed tumor fragments were collected, aseptically separated, and cultured to obtain control group cells (named CT26-Luc_WT, abbreviated as WT).

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

[0073] Tumor volume was measured every 3 days to observe whether the tumor shrank significantly after radiotherapy. Tumors were continuously monitored, and any recurrence of tumor size increase after radiotherapy cessation was recorded (defined as recurrence). Tumor fluorescence signal intensity was detected weekly using a small animal in vivo three-dimensional multimodal imaging system (IVIS system) to monitor tumor growth. Tumors with significantly decreased fluorescence signal after radiotherapy (tumor shrinkage) followed by re-enhancement (recurrence) were defined as radiotherapy-resistant recurrent tumors. Mice with the largest radiotherapy-resistant recurrent tumor volume in the HFRT treatment group were selected on day 21 after radiotherapy. Figures 2-3 This diagram illustrates the process of screening animal models for tumor recurrence after hypofractionated radiotherapy.

[0074] (5) Radiotherapy-resistant tumor isolation and culture

[0075] Tissue sampling: The mice with the largest radiotherapy-resistant recurrent tumor volume obtained in step (4) were humanely sacrificed, and the recurrent tumor tissue was separated under aseptic conditions.

[0076] Cell culture: Recurrent tumor tissue was minced (1 mm) under aseptic conditions. 3 Small pieces were placed in DMEM culture medium containing 10% fetal bovine serum and cultured. After stable passage three times, a radiotherapy-resistant relapse cell line was obtained.

[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 subcutaneous tumor-bearing experiments. Steps (2) to (5) were repeated once, and finally, a stable radiotherapy-resistant relapse phenotype cell line was obtained and named CT26-Luc_HFRT-R (abbreviated as HFRT-R) cell line.

[0079] 1.2.2 Phenotypic Validation

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

[0081] In vivo experiments:

[0082] ①For example Figure 11 As shown, the selected radiotherapy-resistant relapse-resistant cell lines were subcutaneously inoculated into BALB / c mice, and the HFRT treatment experiment was repeated. The differences in radiotherapy resistance and immune microenvironment regulation between the CT26-Luc_WT cell line and the surgically resected CT26-Luc_HFRT-R cell line were assessed by flow cytometry.

[0083] ②For example Figure 19As shown, the selected radiotherapy-resistant recurrence cell lines were used to construct a mouse model of liver metastasis via spleen injection. The CT26-Luc_HFRT-R cell line was compared with the surgically resected CT26-Luc_HFRT-R cell line using a small animal in vivo three-dimensional multimodal imaging system to evaluate the differences in metastasis and recurrence between the two cell lines.

[0084] 1.2.3 Cell Expansion and Preservation

[0085] The CT26-Luc_HFRT-R cells selected through positive screening were expanded and cultured to ensure good cell growth. A portion of the cells were then cryopreserved according to standard methods.

[0086] The mouse (Mus musculus) colorectal cancer cell line CT26-Luc_HFRT-R was deposited on January 2, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202533.

[0087] 2. Experimental Results

[0088] 2.1 Phenotypic Validation Results from In Vitro Experiments

[0089] The results of immunofluorescence staining of radiotherapy-resistant relapse-resistant cell lines in vitro are shown in the figure. Figures 4-5 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. 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 capacity in the face of radiotherapy.

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

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

[0092] The results of the plate cloning experiment are shown below Figure 9The study was used to verify the proliferation and colony formation capabilities 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 large number of clones after radiotherapy, and the size and number of clones were significantly higher than those of the CT26-Luc_WT cell line. Figure 10 The SF (survival fraction) statistics further illustrate the radiotherapy effects of different treatment groups. In the CT26-Luc_HFRT-R cell line, the survival fraction after radiotherapy was significantly higher than that of the CT26-Luc_WT cell line, and the decrease in survival fraction with increasing radiotherapy dose was smaller.

[0093] 2.2 Phenotypic Validation Results from In Vivo Experiments

[0094] In vivo validation results of the CT26-Luc_HFRT-R hypofractionated radiotherapy resistance phenotype are shown in [link to relevant documentation]. Figures 12-18 By measuring the immune microenvironment of subcutaneous tumors in mice, it was found that the CT26-Luc_HFRT-R cell line, compared with the CT26-Luc_WT cell line, showed a significant increase in the infiltration of M2-polarized tumor-associated macrophages (TAMs), while the tumor-infiltrating CD8+ cells... + The cytotoxicity of T cells was significantly suppressed.

[0095] The results of the in vivo distant metastasis ability detection of CT26-Luc_HFRT-R are shown in the figure. Figures 20-22 By measuring the fluorescence signal of liver metastases in mice, it was found that the CT26-Luc_HFRT-R cell line exhibited stronger liver metastasis ability compared to the CT26-Luc_WT cell line, characterized by a shorter tumor latency period and larger metastatic lesions.

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

[0097] Figure 23 Volcano plots of differentially expressed genes compared to the wild-type (CT26-Luc_WT) group were used to select genes with log2FC>1 and adj P value<0.05, revealing changes in gene expression in radiotherapy-resistant cells; Figure 24 GeneOntology (GO) biological processes were clustered to identify the main biological processes involved in radiotherapy resistance. Figure 25 Clustering of differentially expressed KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed signaling pathways significantly enriched in radiotherapy-resistant cells.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cell line resistant to recurrence after hypofractionated radiotherapy for colorectal cancer, characterized in that, The colorectal cancer cell line resistant to recurrence after hypofractionated radiotherapy was deposited at the China Center for Type Culture Collection (CCTCC) on January 2, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202533.

2. The application of the colorectal cancer hypofractionated radiotherapy-resistant recurrence cell line as described in claim 1 in the screening of antitumor drugs.

3. The application of the colorectal cancer macrofractionated radiotherapy-resistant recurrence cell line as described in claim 1 in the preparation of an animal model of radiotherapy resistance in colorectal cancer.

4. A method for constructing an animal model of radiotherapy resistance in colorectal cancer, characterized in that, The method includes the step of subcutaneously inoculating the colorectal cancer radiotherapy-resistant recurrence cell line of claim 1 into experimental animals to construct the animal model of colorectal cancer radiotherapy resistance.

5. The construction method according to claim 4, characterized in that, The experimental animal was a mouse.

6. The construction method according to claim 4, characterized in that, The colorectal cancer hypofractionated radiotherapy-resistant recurrence cell line was inoculated subcutaneously into the experimental animals in the form of a cell suspension.

7. The application of an animal model constructed according to any one of claims 4-6 in screening antitumor drugs.

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