A drug combination for improving the sensitivity of FLASH radiotherapy in treating peritoneal metastasis of colorectal cancer, a method and application thereof

Abstract

This invention relates to the field of biomedical technology, disclosing a drug combination and its application for improving the sensitivity of FLASH radiotherapy in the treatment of colorectal cancer peritoneal metastases. The combination comprises a therapeutically effective amount of an APOE inhibitor and / or an IL1B agonist. By combining an APOE inhibitor and / or an IL1B agonist, this invention can precisely target and intervene in abnormal gene expression after FLASH radiotherapy, thereby improving the abnormal gene expression state after FLASH radiotherapy, eliminating the negative impact of abnormal gene expression on the efficacy of radiotherapy, significantly improving the therapeutic sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases, achieving radiosensitization, and providing a new targeted strategy for the radiotherapy treatment of colorectal cancer peritoneal metastases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a drug combination, method, and application for improving the sensitivity of FLASH radiotherapy in the treatment of peritoneal metastases of colorectal cancer. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the digestive tract. Peritoneal metastasis is one of the main forms of metastasis in advanced colorectal cancer and a core factor leading to shortened disease-free survival and poor prognosis. Whole-abdominal radiotherapy is an important local treatment for peritoneal metastases of colorectal cancer. X-ray ultrahigh dose rate FLASH irradiation, as a novel ultrahigh dose rate radiotherapy technique, has shown potential advantages in tumor treatment compared to traditional conventional dose rate radiotherapy (CONV). However, the sensitivity of FLASH irradiation to peritoneal metastases of colorectal cancer still needs to be improved, which remains a key issue in clinical treatment.

[0003] Changes in gene expression in the tumor microenvironment after radiotherapy directly affect the efficacy of radiotherapy. Colorectal cancer peritoneal metastases exhibit a unique ascites microenvironment, and abnormal expression of key genes within this microenvironment weakens the anti-tumor effect of FLASH radiotherapy. Currently, the key regulatory genes in the ascites microenvironment of colorectal cancer peritoneal metastases after FLASH radiotherapy that are related to patient survival are not yet clearly identified, and there is a lack of targeted drugs that can improve the sensitivity to FLASH radiotherapy. Therefore, developing drugs that can regulate abnormally expressed genes after radiotherapy and enhance the sensitivity of FLASH radiotherapy for colorectal cancer peritoneal metastases has significant clinical application potential.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] The problem with existing technologies is that changes in gene expression in the tumor microenvironment after radiotherapy directly affect the efficacy of radiotherapy. Colorectal cancer peritoneal metastasis has a unique ascites microenvironment, and abnormal expression of key genes in this microenvironment weakens the anti-tumor effect of FLASH radiotherapy. To address the problems of the existing technologies, this invention provides a drug combination, method, and application for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastasis. By combining APOE inhibitors and / or IL1B agonists, the abnormal expression of APOE and IL1B in the ascites microenvironment after radiotherapy is specifically regulated, reversing the adverse prognostic effects caused by high APOE expression and downregulation of IL1B expression. This improves the abnormal gene expression state after FLASH radiotherapy, eliminates the negative impact of abnormal gene expression on the efficacy of radiotherapy, and significantly improves the therapeutic sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastasis, providing a new targeted strategy for the radiotherapy treatment of colorectal cancer peritoneal metastasis.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a drug combination for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases, comprising a therapeutically effective amount of an APOE inhibitor and / or an IL1B agonist.

[0007] This invention, through a study of FLASH whole-abdominal radiotherapy in mice with peritoneal metastases of colorectal cancer, reveals for the first time that in the FLASH high-dose-rate 21-day group, APOE gene expression was differentially upregulated and IL1B gene expression was differentially downregulated in the ascites microenvironment. Furthermore, high APOE expression was closely associated with significantly worse overall survival (OS) and disease-free survival (DFS) in colorectal cancer patients, while high IL1B expression was closely associated with significantly better OS and DFS. This indicates that while FLASH radiotherapy exerts its therapeutic effect, it unexpectedly induces APOE upregulation and IL1B downregulation, partially offsetting its anti-tumor efficacy and leading to reduced radiosensitivity. Therefore, this invention provides a drug combination that, by using an APOE inhibitor or an IL1B agonist alone or in combination, can target the abnormal expression of APOE upregulation and IL1B downregulation in the ascites microenvironment after FLASH radiotherapy, reverse the adverse prognostic effect mediated by high APOE expression, and enhance the favorable prognostic effect mediated by high IL1B expression. This improves the abnormal gene expression state after FLASH radiotherapy, eliminates the negative impact of abnormal gene expression on the efficacy of radiotherapy, and significantly improves the therapeutic sensitivity of FLASH radiotherapy to peritoneal metastases of colorectal cancer, providing a new targeted strategy for radiotherapy treatment of peritoneal metastases of colorectal cancer.

[0008] In one specific embodiment, the APOE inhibitor is an siRNA, shRNA, antisense oligonucleotide, CRISPR / Cas9 system, anti-APOE antibody, or small molecule compound that targets the APOE gene. The small molecule compound is a chemically synthesized organic compound with a small molecular weight, which is easily absorbed and complementary to the antibody.

[0009] In one specific embodiment, the IL1B agonist is a recombinant IL1B protein, an IL1B gene overexpression vector, or an IL1B mRNA formulation.

[0010] Secondly, the present invention provides the use of the drug combination in the preparation of drugs that enhance the sensitivity of FLASH radiotherapy for colorectal cancer peritoneal metastasis.

[0011] In one specific embodiment, the APOE inhibitor is used to inhibit the upregulated APOE expression after FLASH radiotherapy; the IL1B agonist is used to compensate for the downregulated IL1B expression after FLASH radiotherapy.

[0012] In one specific embodiment, the FLASH radiotherapy is an ultra-high dose rate irradiation with a dose rate ≥40Gy / s and an irradiation dose range of 8-15Gy.

[0013] In one specific embodiment, the peritoneal metastasis of colorectal cancer includes peritoneal metastasis after primary colorectal cancer surgery or peritoneal dissemination of advanced colorectal cancer.

[0014] Thirdly, the present invention provides a method for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases by administering APOE inhibitors and / or IL1B agonists to patients with colorectal cancer peritoneal metastases who are receiving FLASH radiotherapy.

[0015] In one specific embodiment, the administration method includes at least one of intraperitoneal administration, injection administration, or oral administration.

[0016] In one specific embodiment, the APOE inhibitor and / or IL1B agonist are administered before, during, or after FLASH radiotherapy.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention provides a drug combination for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastasis and its application. It is the first discovery of the negative impact of differential upregulation of APOE gene and differential downregulation of IL1B gene on the efficacy of radiotherapy after FLASH treatment. Based on this, a drug combination was developed. When APOE inhibitors and / or IL1B agonists are used in combination during FLASH radiotherapy, the APOE inhibitors reverse the adverse prognostic effect mediated by APOE high expression, and the IL1B agonists improve the downregulation of IL1B expression, enhance the good prognostic effect mediated by IL1B high expression, and eliminate the negative impact of abnormal gene expression on the efficacy of radiotherapy. 2. This invention provides a drug combination and its application for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases. By combining APOE inhibitors and / or IL1B agonists, it can precisely target and intervene in abnormal gene expression after FLASH radiotherapy, thereby improving the abnormal gene expression state after FLASH radiotherapy, eliminating the negative impact of abnormal gene expression on the efficacy of radiotherapy, significantly improving the therapeutic sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases, achieving radiosensitization, and providing a new targeted strategy for radiotherapy treatment of colorectal cancer peritoneal metastases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Representative BLI signals of colorectal cancer peritoneal metastasis in mice before and after radiotherapy (7 days, 14 days and 21 days) in each group of mice provided in the embodiments of the present invention; Figure 2 The mean changes in BLI signals in each group of mice provided in the embodiments of the present invention are shown in the figures. *p<0.05, **p<0.01, ***p<0.001. Figure 3 The changes in abdominal circumference of mice after radiotherapy in each group provided in the embodiments of the present invention are shown in the figures. *p<0.05, **p<0.01, ***p<0.001. Figure 4 The peripheral blood erythrocyte counts of mice in each group provided in the embodiments of the present invention before and after radiotherapy each week, *p<0.05, **p<0.01, ***p<0.001; Figure 5 The peripheral blood hemoglobin levels of mice in each group provided in this embodiment of the invention were measured weekly after radiotherapy. *p<0.05, **p<0.01, ***p<0.001; Figure 6 The peripheral blood leukocyte counts of mice in each group provided in the embodiments of the present invention were calculated weekly after radiotherapy, *p<0.05, **p<0.01, ***p<0.001; Figure 7 The number of peripheral blood neutrophils in each group of mice provided in the embodiments of the present invention was calculated weekly after radiotherapy. *p<0.05, **p<0.01, ***p<0.001; Figure 8 The number of peripheral blood lymphocytes in each group of mice after radiotherapy provided in the embodiments of the present invention, *p<0.05, **p<0.01, ***p<0.001; Figure 9 Liver function indicators (alkaline phosphatase, direct bilirubin, bile acids, albumin) of mice in each group provided in the embodiments of the present invention 21 days after radiotherapy, *p<0.05, **p<0.01, ***p<0.001; Figure 10 The renal function indicators (uric acid, creatinine, blood urea nitrogen) of each group of mice provided in the embodiments of the present invention 21 days after radiotherapy were *p<0.05, **p<0.01, ***p<0.001; Figure 11 The following are the pathological changes in various tissues of mice after radiotherapy in the embodiments of the present invention; H&E staining results for various tissues: (A) Heart H&E section, tissue congestion (green arrow); (B) Liver H&E section, tissue edema (black arrow), vacuolar degeneration (yellow arrow); (C) Spleen H&E section, granulocyte infiltration (blue arrow); (D) Kidney H&E section; (E) Small intestine H&E section, crypt structures (red arrow), lymphocyte infiltration (gray arrow), tissue erosion (brown arrow); (F) Colon H&E section, goblet cells (dark blue arrow); (G) Rectal H&E section. Scale bar, 50μm, 100μm; Figure 12 The differences in the expression levels of cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-10, IL-16 and GM-CSF) in the serum of mice in each group after radiotherapy in the embodiments of the present invention are shown in the figures. *p<0.05, **p<0.01, ***p<0.00. Figure 13 The differences in expression levels of the CXC subfamily of chemokines (CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13 and CXCL16) in the serum of mice in different groups after radiotherapy according to the embodiments of the present invention are shown in the figures. *p<0.05, **p<0.01, ***p<0.001. Figure 14 The differences in the expression levels of the CC subfamily of chemokines (CCL1, CCL3, CCL4, CCL5, CCL7, CCL11, CCL12, CCL17, CCL19, CCL20, CCL24 and CCL27) in the serum of mice in the various groups provided in the embodiments of the present invention after radiotherapy are shown in the figures: *p<0.05, **p<0.01, ***p<0.001. Figure 15 Flow cytometry analysis of mice on day 7 after treatment (inhibiting tumor cell proliferation) provided in this embodiment of the invention; (A) Flow cytometry flowchart for analyzing various immune cells in ascites. Flow cytometry scatter plot of immune cells in ascites of mice on day 7 after irradiation treatment: (B) B cells; (C) T cells; (D) CD45+ cells; (E) CD4+ T cells. Quantitative analysis and comparison of immune cells in ascites of mice after 7 days of treatment were performed based on the flow cytometry scatter plot. (F) B cells; (G) T cells; (H) CD45+ cells; (I) CD4+ T cells. *p<0.05, **p<0.01, ***p<0.001; Figure 16Differences in APOE and IL1B genes in the ascites fluid of mice in each group of mice on day 21 after treatment, as provided in the embodiments of the present invention; Figure 17 This invention provides a Venn diagram of the core hub gene based on the intersection of three Cytohubba algorithms, as shown in the embodiments of the present invention. Figure 18 Survival analysis diagram of colorectal cancer hub genes provided in embodiments of the present invention; Figure 19 OS and DFS diagrams of APOE and IL1B genes in colorectal cancer provided in embodiments of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0021] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0022] Throughout this specification, references to "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases "an embodiment," "an example," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. Moreover, those skilled in the art will understand that the illustrations provided herein are for illustrative purposes and are not necessarily drawn to scale. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Example 1 This invention provides a drug combination to improve the sensitivity of FLASH radiotherapy for colorectal cancer peritoneal metastases, comprising a therapeutically effective amount of an APOE inhibitor and / or an IL1B agonist.

[0024] This invention, through a study of FLASH whole-abdominal radiotherapy in mice with peritoneal metastases of colorectal cancer, reveals for the first time that in the FLASH high-dose-rate 21-day group, APOE gene expression was differentially upregulated and IL1B gene expression was differentially downregulated in the ascites microenvironment. Furthermore, high APOE expression was closely associated with significantly worse overall survival (OS) and disease-free survival (DFS) in colorectal cancer patients, while high IL1B expression was closely associated with significantly better OS and DFS. This indicates that while FLASH radiotherapy exerts its therapeutic effect, it unexpectedly induces APOE upregulation and IL1B downregulation, partially offsetting its anti-tumor efficacy and leading to reduced radiosensitivity. Therefore, this invention provides a drug combination that, by using an APOE inhibitor or an IL1B agonist alone or in combination, can target the abnormal expression of APOE upregulation and IL1B downregulation in the ascites microenvironment after FLASH radiotherapy, reverse the adverse prognostic effect mediated by high APOE expression, and enhance the favorable prognostic effect mediated by high IL1B expression. This improves the abnormal gene expression state after FLASH radiotherapy, eliminates the negative impact of abnormal gene expression on the efficacy of radiotherapy, and significantly improves the therapeutic sensitivity of FLASH radiotherapy to peritoneal metastases of colorectal cancer, providing a new targeted strategy for radiotherapy treatment of peritoneal metastases of colorectal cancer.

[0025] Furthermore, the APOE inhibitor employs siRNA, shRNA, antisense oligonucleotides, CRISPR / Cas9 systems, anti-APOE antibodies, or small molecule compounds that target the APOE gene. The small molecule compounds are chemically synthesized organic compounds with small molecular weights, which are easily absorbed and complementary to the antibodies.

[0026] Furthermore, the IL1B agonist is a recombinant IL1B protein, an IL1B gene overexpression vector, or an IL1B mRNA formulation.

[0027] This invention also provides the application of the drug combination in the preparation of drugs that enhance the sensitivity of FLASH radiotherapy for colorectal cancer peritoneal metastases.

[0028] Furthermore, the APOE inhibitor is used to inhibit the upregulated APOE expression after FLASH radiotherapy; the IL1B agonist is used to compensate for the downregulated IL1B expression after FLASH radiotherapy.

[0029] Furthermore, the FLASH radiotherapy is an ultra-high dose rate irradiation, with a dose rate ≥40Gy / s and an irradiation dose range of 8–15Gy.

[0030] Furthermore, the peritoneal metastasis of colorectal cancer includes peritoneal metastasis after primary colorectal cancer surgery or peritoneal dissemination of advanced colorectal cancer.

[0031] This invention further provides a method for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases by administering APOE inhibitors and / or IL1B agonists to patients with colorectal cancer peritoneal metastases receiving FLASH radiotherapy.

[0032] Furthermore, the administration method includes at least one of intraperitoneal administration, injection administration, or oral administration.

[0033] Furthermore, the APOE inhibitor and / or IL1B agonist are administered before, during, or after FLASH radiotherapy.

[0034] Example 2 This invention provides and constructs a mouse model of colorectal cancer peritoneal metastasis, and conducts a study on whole-abdominal radiotherapy using X-ray ultrahighdose rate FLASH irradiation and conventional dose rate irradiation. Two sequencing groups were set up: a 21-day control group (DZ2) and a 21-day high-dose FLASH group (F2). Ascites cells from both groups of mice were collected for RNA-seq sequencing. Mouse-derived hub genes were screened from differentially expressed genes using the cytohubba and mcode plugins of Cytoscape software. The mouse-derived hub genes were analyzed for Kaplan-Meier disease-free survival on the GEPIA website.

[0035] like Figures 16-19 As shown, in the FLASH high-dose 21-day group, APOE gene was differentially upregulated and IL1B gene was differentially downregulated in the ascites microenvironment. Furthermore, high APOE expression was closely associated with significantly worse disease-free survival in colorectal cancer patients, while high IL1B expression was closely associated with significantly better disease-free survival in colorectal cancer patients.

[0036] Example 3 This invention provides a method for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastasis, specifically as follows: A mouse model of colorectal cancer peritoneal metastasis is constructed, and a study of combined treatment with X-ray ultrahigh dose rate FLASH irradiation, APOE inhibitor, and IL1B agonist (FLASH RT) is conducted, with a control group included. The results are as follows: Figures 1-15 As shown.

[0037] from Figures 1-3 As can be seen from the (Control indicates no FLASH treatment) results, the BLI signal in mice was significantly reduced after treatment with the FLASH RT of this invention, and the abdominal circumference of the mice was significantly reduced after treatment, indicating that the peritoneal metastasis of colorectal cancer in mice was significantly inhibited.

[0038] from Figures 4-10 (Control group represents mice treated with FLASH alone.) As shown in the figure, complete blood counts were performed via tail vein sampling one day before radiotherapy and on days 7, 14, and 21 after radiotherapy. The results showed that hemoglobin (HGB) and red blood cell (RBC) levels in the control group mice continued to decrease, while the decrease in the FLASH RT group was smaller, close to or within the normal range (green dotted line in the figure, RBC: 9.14–10.38 × 10⁻⁶). 12 / L; HGB: 121.65–159.55 g / L), showing a significant difference from the control group ( Figures 2-10 , Figures 2-11 This phenomenon indicates that FLASH RT has a significant therapeutic effect on peritoneal metastasis of colorectal cancer. In the colorectal cancer peritoneal metastasis model, as the tumor continues to develop, it continuously consumes nutrients and energy, inhibiting the production of hemoglobin (HGB) and red blood cells (RBCs); simultaneously, tumor cells release active substances that increase peritoneal capillary permeability, leading to extravasation of HGB and RBCs and the formation of bloody ascites. On day 6 after inoculation (one day before radiotherapy), the number of neutrophils (NEU), white blood cells (WBCs), and lymphocytes (LYMs) in each group of mice significantly increased and exceeded normal values ​​(green dotted line in the figure, NEU: 0.65–1.25 × 10⁻⁶). 9 / L, WBC: 3.15–5.11×10 9 / L, LYM: 2.42–3.85×10 9 / L). After radiotherapy, the NEU, WBC, and LYM levels in the FLASH RT group decreased significantly and stabilized within the normal range, showing a significant difference compared to the control group (P<0.05). Figures 2-12 , Figures 2-13 and Figures 2-14 During tumor growth, a severe inflammatory response may be triggered, leading the immune system to release interleukins (IL-6, IL-8) and tumor necrosis factor (TNF-α), which in turn stimulates the body to produce more NEU, WBC, and LYM. The above results indicate that FLASH RT can effectively inhibit the tumor-induced inflammatory response, resulting in improved blood routine indicators.

[0039] On day 21 post-radiotherapy, mice in each group were sacrificed and serum was collected. Liver and kidney function indicators in the serum were measured using an automated biochemical analyzer. Results showed that, compared with the control group, the treatment group had significantly higher levels of alkaline phosphatase (ALP) and albumin (ALB) (P<0.05), indicating less liver damage in the FLASH RT group. The levels of direct bilirubin (DB) and bile acid (TBA) in the FLASH RT group were significantly lower than those in the control group (P<0.05), suggesting reduced damage to hepatobiliary cells in the treatment group.

[0040] The creatinine (CR) level in the FLASH RT group was significantly lower than that in the control group (P<0.05), while the uric acid (UA) and blood urea nitrogen (UREA) levels were significantly higher in the FLASH RT group than in the control group (P<0.05). Figures 2-16 Elevated CR levels indicate a decreased glomerular filtration rate and are an important indicator of impaired kidney function. Changes in the levels of UA (urine end product) and UREA (protein breakdown product) can also be used to assess the degree of kidney function impairment. These results demonstrate that FLASH RT has a therapeutic effect on peritoneal metastases of colorectal cancer in mice, alleviating liver and kidney function damage caused by tumor development.

[0041] from Figure 11 As can be seen from the HE staining results: Heart: Occasional cardiac congestion was observed in the hearts of mice in both the control group and the FLASH RT group.

[0042] Liver: Edema, vacuolar degeneration, congestion and mild inflammatory cell infiltration were observed in all groups; the degree of hepatocyte edema in the FLASH RT group was slightly less than that in the control group, suggesting that FLASH radiotherapy causes less liver damage.

[0043] Spleen: The white pulp adhesion was most severe in the control group, followed by the FLASH RT group; the degree of white pulp adhesion can reflect irradiation damage, and the results show that FLASH RT caused less damage to the spleen of mice than the control group. Inflammatory cell infiltration was observed in the spleen of mice in all groups, with the most pronounced in the control group, indicating that the combination of radiotherapy and medication can reduce the inflammatory response of the spleen, and that FLASH RT is more effective than the control group alone.

[0044] Kidneys: Congestion was observed in the kidney tissues of all groups, but inflammatory cell infiltration was observed in the control group, indicating that the combination of radiotherapy and medication can reduce kidney inflammation.

[0045] Small intestine: In the control group, the intestinal epithelium of mice was sloughed off and the crypts were loose; no obvious pathological changes were observed in the small intestine of mice in the FLASH RT group, suggesting that FLASH RT can not only inhibit peritoneal metastasis of colorectal cancer in mice and reduce inflammatory response, but also show a good protective effect on the mouse intestine.

[0046] Colon: In the control group, the colonic epithelium of mice was sloughed off, eroded, and accompanied by inflammatory cell infiltration; the colon of mice in the FLASH RT group showed no obvious pathological damage, indicating that FLASH RT has the effect of inhibiting tumor growth and inflammatory response, and does not cause significant damage to colonic tissue.

[0047] Rectum: Mild epithelial cell detachment and mild inflammatory cell infiltration were observed in the control group; no obvious pathological damage was observed in the rectum of mice in the FLASH RT group.

[0048] In summary, X-ray FLASH RT can not only alleviate the pathological changes caused by peritoneal dissemination of colorectal cancer (such as organ congestion, edema, inflammation, etc.), but also effectively protect normal tissues from radiation-induced tissue damage and inflammatory response, and maintain the integrity of intestinal epithelial cells and crypts.

[0049] like Figure 12 As shown, after mice received FLASH radiotherapy combined with drug therapy, the expression levels of cytokines such as IFN-γ, IL-1β, IL-2, IL-6, IL-10, IL-16, TNF-α, and GM-CSF were significantly decreased, showing statistically significant differences compared to the control group (P<0.05). The results suggest that FLASH radiotherapy can effectively inhibit tumor development and inflammatory responses. The specific mechanisms are as follows: IL-1β and TNF-α exacerbate inflammatory responses and induce angiogenesis by activating the NF-κB signaling pathway, accelerating peritoneal metastasis; IL-6 and IL-10 promote tumor growth and upregulate vascular endothelial growth factor levels via the STAT3 pathway, promoting angiogenesis and metastasis; IFN-γ and IL-2, in addition to their pro-inflammatory effects, can also inhibit CD8⁺ T cell function and promote immune escape; GM-CSF recruits neutrophils and macrophages, promotes the release of TNF-α and IL-6, inhibits tumor cell apoptosis, and enhances their invasive ability. FLASH radiotherapy combined with drug therapy reduces the levels of the aforementioned cytokines, thereby blocking tumor progression and metastasis on multiple targets.

[0050] like Figure 13 As shown, regarding the CXC subfamily of chemokines, the levels of CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, and CXCL16 in the control group mice were significantly higher than those in the FLASH radiotherapy group (P<0.05). These chemokines promote peritoneal metastasis through different mechanisms: CX3CL1 accelerates tumor progression by activating CX3CR1⁺ myeloid cells; CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, and CXCL16 bind to receptors such as CXCR2, CXCR3, CXCR4 / CXCR7, CXCR5, and CXCR6, respectively, recruiting immune cells, activating signaling pathways such as MAPK and PI3K / AKT, and inducing epithelial-mesenchymal transition, tumor migration, and angiogenesis. FLASH radiotherapy combined with drug therapy significantly reduced the levels of the above chemokines, effectively inhibiting tumor metastasis. Figure 13 ) like Figure 14As shown, regarding the CC subfamily of chemokines, compared with the control group, the levels of CCL1, CCL3, CCL4, CCL5, CCL7, CCL11, CCL12, CCL17, CCL19, CCL20, CCL24, and CCL27 in the FLASH radiotherapy group were significantly decreased (P<0.05), indicating that tumor growth was inhibited. These CC subfamily chemokines affect the tumor microenvironment by recruiting different immune cells: CCL1, CCL3, CCL4, CCL5, CCL17, CCL20, and CCL27 mainly recruit T cells, natural killer cells, and macrophages; while CCL7, CCL11, CCL12, and CCL24 mainly recruit eosinophils, lymphocytes, and neutrophils. FLASH radiotherapy combined with drug therapy inhibits tumor progression by downregulating these chemokines and reducing the infiltration of pro-tumor immune cells. Figure 14 ) like Figure 15 As shown, single-cell suspensions of mouse ascites fluid from day 7 post-radiotherapy were stained with various staining antibodies, then analyzed using a microarray. Figure 15 Flow cytometry pattern A was used to screen various immune cells in mouse ascites cells. The results showed that the level of intraperitoneal B cells in the FLASHRT group was significantly lower than that in the control group (P<0.05). Figure 15 B cells (B and F), as core effector cells of adaptive immunity, play a crucial role in anti-tumor immune responses. However, in the late-stage tumor microenvironment, B cells can undergo phenotypic and functional remodeling, promoting tumor progression through multiple mechanisms: on the one hand, tumor-infiltrating B cells highly express immune checkpoint molecules such as PD-L1, directly inhibiting T cell activation and cytotoxic function, assisting tumor cells in achieving immune escape; on the other hand, abnormal and persistent activation of the B cell receptor (BCR) signaling pathway can drive abnormal B cell proliferation and survival, and create a microenvironment conducive to tumor growth by secreting pro-inflammatory cytokines and forming immune complexes. These results suggest that reducing B cell infiltration in the tumor microenvironment may be one of the important mechanisms for inhibiting peritoneal tumor progression, and the combination of FLASH radiotherapy and drug therapy demonstrates a significant advantage in B cell regulation.

[0051] The T-cell level in the FLASH radiotherapy + drug combination group was significantly higher than that in the control group (P<0.05). Figure 15(C, G) This suggests that FLASH radiotherapy can effectively improve the tumor immune microenvironment, promote T cell activation and proliferation, and thus continuously enhance the anti-tumor immune response. During tumor development, the tumor microenvironment (TME) typically accumulates immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). These cells directly inhibit T cell proliferation and activation by secreting inhibitory cytokines such as transforming growth factor-β (TGF-β). Furthermore, metabolic stress factors in the TME, such as hypoxia, lactate buildup, and competition for key nutrients, further weaken T cell function. The rapid proliferation of tumor cells competes with T cells for limited energy metabolic substrates, exacerbating nutrient deficiency and inducing T cell exhaustion. FLASH radiotherapy may alleviate these immunosuppressive mechanisms and metabolic stress, thereby relieving the inhibitory effect on T cells and maintaining and enhancing their anti-tumor activity.

[0052] The proportion of CD45+ cells in the FLASH radiotherapy + drug combination group was significantly higher than that in the control group (P<0.05). Figure 15 (D, H) This suggests that FLASH radiotherapy can effectively activate the body's anti-tumor immune response and promote the infiltration of CD45+ immune cells into the tumor microenvironment. In the tumor microenvironment, the increased number and activity of regulatory T cells (Tregs) inhibit the migration, activation, and proliferation of CD45+ immune cells, leading to a decrease in the proportion of CD45+ cells, thereby promoting tumor growth and metastasis. FLASH radiotherapy may enhance the early anti-tumor immune response by inhibiting Treg-mediated immunosuppression, thus relieving functional inhibition of CD45+ cells.

[0053] The CD4+ T cell count in tumor tissue of the FLASH radiotherapy + drug combination group was significantly higher than that of the control group (P<0.05). Figure 15 (E, I) This suggests that FLASH radiotherapy can promote the infiltration of CD4+ T cells into the tumor microenvironment in the early stages. Previous studies have shown that regulatory T cells (Tregs) in the tumor microenvironment can inhibit the proliferation and effector function of CD4+ T cells through direct contact. FLASH radiotherapy promotes the activation and proliferation of CD4+ T cells, thereby enhancing the body's early anti-tumor immune response.

[0054] The above results demonstrate that FLASH radiotherapy combined with drug therapy can effectively treat peritoneal metastases of colorectal cancer.

[0055] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A drug combination for enhancing the sensitivity of FLASH radiotherapy in the treatment of peritoneal metastases of colorectal cancer, characterized in that, This includes therapeutically effective doses of APOE inhibitors and / or IL1B agonists.

2. The drug combination for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases according to claim 1, characterized in that, The APOE inhibitor uses siRNA, shRNA, antisense oligonucleotide, CRISPR / Cas9 system or anti-APOE antibody that targets the APOE gene.

3. The drug combination for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases according to claim 1, characterized in that, The IL1B agonist is a recombinant IL1B protein, an IL1B gene overexpression vector, or an IL1B mRNA formulation.

4. The use of any of the drug combinations described in claims 1-3 in the preparation of drugs that enhance the sensitivity of colorectal cancer peritoneal metastases to FLASH radiotherapy.

5. The application of the drug combination according to claim 4 in the preparation of a drug for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases, characterized in that, The APOE inhibitor is used to suppress the upregulated APOE expression after FLASH radiotherapy; the IL1B agonist is used to compensate for the downregulated IL1B expression after FLASH radiotherapy.

6. The use of the drug combination according to claim 4 in the preparation of a drug for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases, characterized in that, The FLASH radiotherapy is an ultra-high dose rate irradiation, with a dose rate ≥40Gy / s and an irradiation dose range of 8–15Gy.

7. The use of the drug combination according to claim 4 in the preparation of a drug for improving the sensitivity of FLASH radiotherapy to colorectal cancer peritoneal metastases, characterized in that, The peritoneal metastasis of colorectal cancer includes peritoneal metastasis after surgery for primary colorectal cancer or peritoneal dissemination of advanced colorectal cancer.

8. A method for improving the sensitivity of FLASH radiotherapy in the treatment of peritoneal metastases of colorectal cancer, characterized in that, APOE inhibitors and / or IL1B agonists were administered to patients with peritoneal metastases of colorectal cancer who received FLASH radiotherapy.

9. The method for improving the sensitivity of FLASH radiotherapy in treating peritoneal metastases of colorectal cancer according to claim 8, characterized in that, The administration method includes at least one of intraperitoneal administration, injection administration, or oral administration.

10. The method for improving the sensitivity of FLASH radiotherapy for colorectal cancer peritoneal metastases according to claim 8, characterized in that, The APOE inhibitor and / or IL1B agonist are administered before, during, or after FLASH radiotherapy.

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