Application of Ikbkb-inhibited or knocked-out macrophages in preparation of breast cancer radiotherapy sensitizing drugs

Abstract

The invention belongs to the field of biomedicine, and particularly relates to application of Ikbkb inhibited or knocked-out macrophages in preparation of breast cancer radiotherapy sensitizing drugs. The inventor of the invention proves the effect of the macrophage of which the Ikbkb is inhibited or knocked out in breast cancer radiotherapy for the first time. Compared with the strategy of mainly depending on small molecule drugs or antibodies in the prior art, the tumor growth can be more effectively inhibited after the macrophages in the tumor microenvironment are transformed and the Ikbkb knockout engineered macrophages are subjected to combined radiotherapy, and a brand new solution is provided for overcoming breast cancer radiotherapy resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of Ikbkb-inhibited or knocked-out macrophages in the preparation of radiosensitizing drugs for breast cancer. Background Technology

[0002] Radiotherapy (RT) is one of the core methods of comprehensive treatment for breast cancer, with approximately 50% of breast cancer patients requiring RT during treatment. However, primary or acquired radioresistance is a major cause of treatment failure and tumor recurrence. Traditional research has focused on the DNA damage repair (DDR) mechanisms of tumor cells themselves, but increasing evidence suggests that the tumor microenvironment (TME), particularly its immune cells, plays a decisive role in mediating radioresistance. While radiotherapy kills tumor cells, it induces a series of complex immune responses, which may either activate anti-tumor immunity (immunosensitization) or exacerbate immunosuppression. The final outcome depends on the dynamic balance of various immune cells within the TME. Therefore, reshaping the immunosuppressive TME after radiotherapy is a key breakthrough for synergistically sensitizing radiotherapy and improving long-term efficacy.

[0003] TAMs (tumor endothelial cells) are the most important immune cell component of the tumor microenvironment (TME), exhibiting high phenotypic and functional plasticity. They are generally classified into the pro-inflammatory, anti-tumor M1 type and the anti-inflammatory, pro-tumor M2 type. In most solid tumors, TAMs primarily exhibit an M2-like phenotype, suppressing T cell function by secreting cytokines such as IL-10 and TGF-β; and aiding tumor growth and metastasis by promoting angiogenesis and tissue remodeling. After radiotherapy, damaged tumor cells release "find-me" and "eat-me" signals, recruiting a large number of macrophages to infiltrate the tumor area. However, these macrophages are often "tamed" by the TME into the M2 type. Instead of effectively clearing dead tumor cells, they participate in the damage repair process, secreting pro-angiogenic and pro-fibrotic factors, ultimately weakening the long-term efficacy of radiotherapy and even promoting tumor recurrence. Therefore, reprogramming TAMs from "accomplices" (M2 type) to "killers" (M1 type) is a key strategy for breaking radiotherapy resistance and enhancing immunotherapy efficacy.

[0004] IκB kinase beta (Ikbkb, also known as IKKβ) is a core kinase in the classical NF-κB signaling pathway. This pathway plays a central role in regulating inflammatory responses, immune responses, cell proliferation, and apoptosis. Traditionally, IKKβ activation of the NF-κB pathway is thought to primarily promote inflammation and immune activation. However, the function of this pathway is highly context-dependent. In the tumor microenvironment, persistent, chronic NF-κB activation is associated with immunosuppression and tumor progression. Although some studies have suggested that the NF-κB pathway is related to TAMs (tumor chemoradiosensitizers), whether Ikbkb directly regulates macrophage responses to ionizing radiation and determines their final phenotype and function in the post-radiotherapy TME remains unknown. Currently, there is a lack of direct evidence revealing its potential as a radiosensitizing target. Summary of the Invention

[0005] In view of this, to fill the aforementioned technological gaps in this field, the purpose of this invention is to provide the application of Ikbkb-inhibited or knocked-out macrophages in the preparation of radiosensitizing drugs for breast cancer. Radiotherapy is one of the core methods of comprehensive treatment for breast cancer. However, the immunosuppressive properties of the tumor microenvironment (TME), especially the dominant role of pro-tumor M2 tumor-associated macrophages (TAMs), are key factors leading to radioresistance and treatment failure. Based on previous innovative whole-genome CRISPR-Cas9 unbiased screening, this invention has for the first time identified IκB kinase β (Ikbkb) as a core target regulating the radiosensitivity and functional phenotype of macrophages. The inventors discovered that knocking out Ikbkb can confer dual beneficial properties on macrophages: on the one hand, it significantly enhances their own radioresistance, enabling them to survive and continue to function in the immunosuppressive microenvironment after radiotherapy; on the other hand, it reprograms them from the pro-tumor M2 phenotype to the anti-tumor M1-like phenotype, thereby enhancing their ability to phagocytose tumor cells and activate adaptive immunity.

[0006] Based on the above findings, the present invention first provides an engineered macrophage.

[0007] The Ikbkb gene of the engineered macrophage is knocked out, or the biological function of the Ikbkb gene product of the macrophage is suppressed.

[0008] The Ikbkb gene (IκB kinase β gene) is a key target for regulating radiosensitivity and immune function in mouse macrophages. Its coding sequence can be found in NCBI accession number NM_010546.

[0009] Furthermore, the engineered macrophages are obtained by knocking out the Ikbkb gene of the target macrophages, which are the RAW264.7 cell line.

[0010] The present invention further provides a method for preparing the above-mentioned engineered macrophages.

[0011] The preparation method includes knocking out the Ikbkb gene of the target macrophage to obtain the engineered macrophage.

[0012] Furthermore, the method involves knocking out the Ikbkb gene of the target macrophage using the CRISPR-Cas9 method.

[0013] When knocking out the Ikbkb gene using the CRISPR-Cas9 method, the target sequences of the sgRNA combination used are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0014] The present invention also provides the application of the above-mentioned engineered macrophages in the preparation of radiosensitizing drugs for breast cancer.

[0015] The engineered macrophages of this invention can be used as "cell sensitizers" in combination with radiotherapy to treat tumors, particularly breast cancer. When cultured in vitro at a certain cell concentration, the engineered macrophages exhibit the expected anti-breast cancer tumor functional characteristics.

[0016] This invention demonstrates through a series of in vitro and in vivo experiments that, compared with wild-type macrophages, the Ikbkb knockout engineered macrophages of this invention can more effectively inhibit tumor growth after combined radiotherapy.

[0017] This invention also provides tool materials for constructing the above-mentioned engineered macrophages and their applications.

[0018] That is, the application of substances that knock out the Ikbkb gene or inhibit the expression of the Ikbkb gene in the preparation of the engineered macrophages.

[0019] The substance preferably includes: A combination of sgRNAs targeting the Ikbkb gene, comprising sgRNAs with sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; or, a recombinant lentiviral vector containing a DNA sequence encoding the combination of said sgRNAs.

[0020] The significant advantages of this invention compared to the prior art are as follows: The inventors have demonstrated for the first time the role of macrophages with inhibited or knocked-out Ikbkb in the radiotherapy of breast cancer. Compared with existing strategies that mainly rely on small molecule drugs or antibodies, this invention, by modifying macrophages in the tumor microenvironment, utilizes Ikbkb knockout engineered macrophages, which, when combined with radiotherapy, can more effectively inhibit tumor growth, providing a novel solution for overcoming radiotherapy resistance in breast cancer. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 Western blotting results of IKBKB protein in wild-type and Ikbkb-KORAW264.7 macrophages; Figure 2 The results of observation of 4T1 cells after 0 h and 72 h in different culture media under different conditions; Figure 3 EdU flow cytometry results of 4T1 breast cancer cells after treatment with different culture media; Figure 4 The results of apoptosis detection of 4T1 breast cancer cells after treatment with different macrophage conditioned media are shown in Figure A. Figure A is a scatter plot of apoptosis of 4T1 cells in a single flow cytometry analysis in different treatment groups, where Q1-LL region represents live cells, Q1-LR region represents early apoptotic cells, Q1-UR region represents late apoptotic cells, and Q1-UL region represents necrotic cells; total apoptosis rate = LR + UR; Figure B is a bar chart of the statistical results of multiple replicate experiments of apoptosis rate of 4T1 cells in different treatment groups. Figure 5 CCK8 proliferation curves of 4T1 breast cancer cells under different culture conditions; Figure 6 This is a tumor growth curve in a tumor-bearing mouse model. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in multiple embodiments of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0025] Unless otherwise specified, specific techniques or conditions in the embodiments described herein shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available products.

[0026] Radiotherapy is one of the core methods of comprehensive treatment for breast cancer. However, the immunosuppressive properties of the tumor microenvironment (TME), especially the tumor-promoting function of tumor-associated macrophages (TAMs), is a key bottleneck leading to radiotherapy resistance. Developing new strategies that can reverse TAM function and synergistically enhance radiosensitization is a major challenge in clinical practice.

[0027] In the inventors' earlier research, a CRISPR-Cas9 knockout library covering the entire mouse genome was first constructed, and a high-fidelity screening platform was established. By applying 10 Gy of ionizing radiation to the library cells, it was found that the sgRNA targeting the IκB kinase β (Ikbkb) gene was most significantly enriched in the cell population that survived and dominated under radiation pressure.

[0028] Crucially, in a more stringent dynamic analysis (comparing cell populations 2 weeks and 3 days post-irradiation), Ikbkb ranked first among all candidate genes by an absolute advantage, indicating that its loss of function most effectively drives the long-term survival and clonal expansion of macrophages after irradiation.

[0029] Further mechanistic investigations revealed that radiation triggered extensive transcriptional reprogramming centered on the p53 pathway and DNA damage repair. Although the transcriptional level of Ikbkb itself remained unchanged, its protein function as a core kinase in the NF-κB pathway played a crucial "checkpoint" role in this stress network, determining cell fate.

[0030] To verify the clinical relevance of this finding, the inventors analyzed peripheral blood single-cell sequencing data from patients who received thoracic radiotherapy. The results revealed a significant upregulation of IKBKB transcription levels in classical monocytes (macrophage precursors) in the peripheral blood of patients after radiotherapy. This, combined with the in vitro functional screening results, forms a perfect logical loop, strongly suggesting that IKBKB is a key intrinsic pathway mediating macrophage radiation damage and a highly promising intervention target.

[0031] Based on the above findings, this invention proposes to knock out the Ikbkb gene in macrophages using gene editing technology, which can simultaneously confer significant autoradioresistance on macrophages and reprogram them from the pro-tumor M2 phenotype to the anti-tumor M1 phenotype, thereby achieving a dual effect of synergistic radiosensitization.

[0032] To specifically verify and implement this technical solution, the following examples will elaborate on the construction method, in vitro function, and in vivo anti-breast cancer tumor effect of the engineered macrophages in combination with radiotherapy.

[0033] As used herein, "macrophage" refers to innate immune cells derived from myeloid progenitor cells, which have functions such as phagocytosis, antigen transduction, and immune regulation. The "engineered macrophage" of this invention refers to macrophages in which the expression of the Ikbkb gene is knocked out or its function is lost through gene editing technology.

[0034] Ikbkb Gene and Target Selection: IκB kinase β (Ikbkb, also known as IKKβ) is a core kinase in the classical NF-κB signaling pathway, which is traditionally considered to primarily promote inflammation and survival responses. In some embodiments, the Ikbkb gene is mouse-derived, and its coding sequence can be found in NCBI accession number NM_010546. In some embodiments, the Ikbkb gene is human-derived, and its coding sequence can be found in NCBI accession number NM_001556.

[0035] Example 1: Construction and validation of engineered macrophages with Ikbkb knockout or suppressed function 1. Experimental Materials and Methods 1.1 Cell lines The mouse macrophage cell line used in the experiment was RAW264.7, purchased from ATCC, and cultured in medium containing 10% fetal bovine serum glucose. 1.2 sgRNA Design and Synthesis: Using CRISPR-Cas9 technology, four specific sgRNAs were designed and synthesized targeting the coding sequence of the mouse Ikbkb gene, based on gene sequence specificity and off-target risk screening. The sequences are as follows: sgRNA1:5'-TGTCGGGTGTTTGAGTTCAG-AGG-3' (SEQIDNO:1); sgRNA2:5'-CCTCTTCTATGCCTTCTTTG-GGG-3' (SEQIDNO:2); sgRNA3:5'-AATGGAGATAGGACCCTTTA-AGG-3' (SEQIDNO:3); sgRNA4:5'-GCCCGTATTCAGCCTTCTTG-TGG-3' (SEQ IDNO: 4); Among them, sgRNA1, sgRNA2 and sgRNA3 are the sense strands targeting the mouse Ikbkb gene, and sgRNA4 is the antisense strand targeting the mouse Ikbkb gene. The synthesis was commissioned to Cyagen (Suzhou) Biotechnology Co., Ltd., which constructed sgRNA1-Cas9, sgRNA2-Cas9, sgRNA3-Cas9 and sgRNA4-Cas9 and cloned them into lentiviral vectors.

[0036] 1.3 Lentiviral Packaging and Cell Infection: Lentiviral cells containing sgRNA1-Cas9, sgRNA2-Cas9, sgRNA3-Cas9 and sgRNA4-Cas9 were added to macrophages (Raw264.7) for infection. After monoclonal screening and Sanger sequencing verification, macrophages with the Ikbkb gene knocked out (Ikbkb-KO) were obtained.

[0037] 1.4 Detection Indicators and Methods Western blot analysis was used to further validate the protein expression of the Ikbkb gene. Total cellular protein was extracted, and Western blotting analysis was performed using anti-Ikbkb antibody and anti-β-actin antibody to confirm that Ikbkb protein expression was effectively knocked out.

[0038] Figure 1 Western blotting results of IKBKB protein in wild-type and Ikbkb-KORAW264.7 macrophages; Figure 1 The results showed that, with β-actin as an internal control, a clear IKBKB protein band could be detected in wild-type cells; while the IKBKB protein band completely disappeared in Ikbkb-KO cells, indicating that the expression of the Ikbkb gene at the protein level was effectively blocked, further verifying the successful construction of Ikbkb knockout or functionally inhibited macrophages.

[0039] Example 2: Effects of Ikbkb knockout on the in vitro antitumor function and radiosensitivity of macrophages 1. Experimental Materials and Methods 1.1 Cell lines and laboratory animals Cell line: Mouse breast cancer cell line 4T1; Macrophages: Ikbkb gene knockout macrophages (Ikbkb-KO) and wild-type mouse macrophages (RAW264.7) obtained in Example 1.

[0040] Experimental animals: 6-8 week old female BALB / c mice, housed in an SPF-grade environment.

[0041] 1.2 Preparation of conditioned medium Wild-type mouse macrophages (RAW264.7) and Ikbkb gene knockout macrophages were seeded at the same density, replaced with fresh complete culture medium, and cultured for another 72 hours. The supernatant was obtained by centrifugation and used as conditioned medium, which was then frozen for later use.

[0042] 1.3 Grouping and processing in in vitro experiments 4T1 cells were seeded in appropriate culture plates and irradiated with a single dose of 8 Gy of X-rays. Immediately after irradiation, the culture media were replaced with the following conditioned media: WT-MФ-CM: replaced with WT macrophage conditioned media; Ikbkb-MФ-CM group: replaced with Ikbkb-KO macrophage conditioned media; Control group: 4T1 cells without any treatment were set up as a control.

[0043] 1.4 Detection Indicators and Methods Cell morphology and density: After in vitro treatment for 0 h and 72 h, the morphological and density changes of 4T1 cells were observed and photographed using a fluorescence microscope. Cell proliferation: After 48 hours of in vitro treatment, the incorporation of EdU (5-ethynyl-2'-deoxyuridine) into 4T1 cells was detected by flow cytometry using an EdU (5-ethynyl-2'-deoxyuridine) kit to analyze cell proliferation activity. EDU (5-ethynyl-2'-deoxyuridine) is a thymidine analog that can be taken up by proliferating cells and integrated into newly synthesized DNA. EDU-labeled cells emit fluorescent signals, and flow cytometry distinguishes between proliferating (S phase) and non-proliferating cells by detecting fluorescence intensity. EDU-positive cells (high fluorescence signal) correspond to cells in the S phase. Apoptosis: After 48 hours of in vitro treatment, the apoptosis rate of 4T1 cells was quantitatively analyzed by flow cytometry using an Annexin V-FITC / PI double staining apoptosis detection kit. The total apoptosis rate was the sum of the percentages of early apoptotic (LR, Annexin V⁺ / PI⁻) and late apoptotic (UR, Annexin V⁺ / PI⁺) cells. Cell viability: After in vitro treatment for 0, 24, 48, and 72 hours, the cell viability was assessed using a CCK-8 assay kit. CCK-8 solution was added to the culture wells, and the absorbance was measured using a microplate reader to calculate the relative cell viability, thus evaluating the inhibitory effect of the conditioned medium on tumor cell growth.

[0044] 2. Experimental Results Figure 2 The results of observation of 4T1 cells after 0 h and 72 h in different culture media under different conditions; Figure 3 The results of EdU flow cytometry detection of 4T1 breast cancer cells after treatment with different culture media are shown. In the histogram, the horizontal axis (ECD-A) represents the EDU fluorescence intensity. The stronger the fluorescence, the more EDU the cells take up and the more active the proliferation. The vertical axis (Count) represents the number of cells. The P3 region is a pre-defined statistical interval for EDU-positive cells (proliferating cells).

[0045] Figure 4 The results of apoptosis detection of 4T1 breast cancer cells after treatment with different macrophage conditioned media are shown in Figure A. Figure A is a scatter plot of apoptosis of 4T1 cells in a single flow cytometry analysis in different treatment groups, where Q1-LL region represents live cells, Q1-LR region represents early apoptotic cells, Q1-UR region represents late apoptotic cells, and Q1-UL region represents necrotic cells; total apoptosis rate = LR + UR; Figure B is a bar chart of the statistical results of multiple replicate experiments of apoptosis rate of 4T1 cells in different treatment groups. Figure 5 CCK8 proliferation curves of 4T1 breast cancer cells under different culture conditions; Figure 2 The results showed that after 72 hours of conditioned medium treatment, the density of 4T1 cells in the Ikbkb-KO-MФ-CM group was significantly lower than that in the WT-MФ-CM group, indicating that the conditioned medium of Ikbkb-KO macrophages exhibited significantly stronger tumor-suppressive activity.

[0046] Figure 3The results showed that after 48 hours of treatment with conditioned medium, the histogram (single-experiment data) indicated that the proportion of EDU-positive (proliferating) cells in the Control group of 4T1 cells reached 60.23%, indicating that normally cultured 4T1 cells proliferated actively. In the WT-MΦ-CM group treated with wild-type macrophage conditioned medium, the EDU positivity rate decreased to 29.56%; while in the Ikbkb-KO-MΦ-CM group, the EDU positivity rate further decreased to 17.45%, showing a more significant trend of proliferation inhibition. The quantitative bar chart below represents the statistical average of multiple biological replicates: the EDU positivity rate was 58.91% in the Control group, 26.95% in the WT-MΦ-CM group, and 19.24% in the Ikbkb-KO-MΦ-CM group, consistent with the trend shown in the histogram data, and the differences between groups were statistically significant. It was observed that after 48 hours of treatment, the proliferation activity (EDU positivity rate) of 4T1 cells in the Ikbkb-KO macrophage conditioned medium treatment group was significantly lower than that in the WT-MΦ-CM group, indicating that the conditioned medium for Ikbkb knockout macrophages can more effectively inhibit the proliferation of 4T1 tumor cells, demonstrating stronger tumor proliferation inhibitory activity.

[0047] Figure 4 The results showed that after 48 hours of treatment with conditioned medium, the scatter plot above (single experimental data) showed that in the Control group: Q1-LL (live cells) accounted for 95.24%, Q1-LR (early apoptotic cells) 1.83%, and Q1-UR (late apoptotic cells) 2.50%, indicating a high proportion of live cells and a low level of apoptosis; in the WT-MΦ-CM group: Q1-LL (live cells) accounted for 93.33%, Q1-LR (early apoptotic cells) 3.33%, and Q1-UR (late apoptotic cells) 2.91%, indicating a decrease in the proportion of live cells and an increase in the proportion of apoptotic cells compared to the Control group; in the Ikbkb-KO-MΦ-CM group: Q1-LL (live cells) accounted for 90.22%, Q1-LR 4.63% (early apoptotic cells), and Q1-UR 4.56% (late apoptotic cells), indicating a further decrease in the proportion of live cells and a significant increase in the proportions of early and late apoptotic cells. The quantitative bar chart below represents the statistical average of multiple biological replicate experiments: the apoptosis rate shows a consistent trend with the scatter plot data. Compared with the WT-MФ-CM group and the Control group, the apoptosis rate (LR+UR) of 4T1 cells in the Ikbkb-KO-MФ-CM group was significantly increased, indicating that the conditioned medium of Ikbkb-KO macrophages can exert stronger tumor suppressive activity by significantly promoting 4T1 cell apoptosis.

[0048] Figure 5The results showed that after 48 hours of treatment, the conditioned medium for wild-type macrophages (WT-Mφ-CM) could inhibit the proliferation of 4T1 cells to a certain extent; while the conditioned medium for Ikbkb knockout macrophages (Ikbkb-KO-Mφ-CM) had a significantly stronger inhibitory effect on the proliferation of 4T1 cells, indicating that Ikbkb knockout macrophages have stronger tumor suppressor activity.

[0049] Example 3: Therapeutic effect of Ikbkb knockout engineered macrophages combined with radiotherapy in a tumor-bearing mouse model 1. Experimental Materials and Methods 1.1 Cell lines and laboratory animals The cell lines and experimental animals used in this embodiment are the same as those in Embodiment 2.

[0050] 1.2 Establishment of tumor-bearing model 4T1 cells in the logarithmic growth phase were collected and inoculated at a dose of 1×10^4 cells per BALB / c mouse. 5 Injected subcutaneously into the right buttock of mice at a density of 1; Every two days, the long diameter (L) and short diameter (W) of the tumor were measured using calipers, the tumor volume was calculated (V=L×W² / 2), and the mouse weight was monitored.

[0051] 1.3 Grouping and Treatment When the tumor volume grew to about 100 mm³, the mice were randomly divided into the following 3 groups: Vehicle group, WTMφ group and Ikbkb-KOMφ group; All three groups received radiotherapy, and in addition to radiotherapy, they received different cell therapies, including: Vehicle group: Intratumoral injection of PBS; WTMφ: Intratumoral injection of 5×10^ 6 One wild-type macrophage; Ikbkb-KOMφ group: Intratumoral injection of 5×10^ 6 Knockout of the ikbkb gene in macrophages.

[0052] Combined treatment regimen: Intratumoral injection of cells or PBS was performed on days 0, 3, and 6. On day 1 after each injection (i.e., days 1, 4, and 7), local radiotherapy was administered to the tumor sites of mice in all groups, with a dose of 5 Gy per injection and a dose rate of 2 Gy / min.

[0053] 1.4 Detection Indicators and Methods Starting from day 0, the tumor volume was measured once a day until the end of the experiment.

[0054] 2. Experimental Results Figure 6 This is a tumor growth curve in a tumor-bearing mouse model. Figure 6 The results showed that in tumor-bearing mice treated with Ikbkb-KO macrophages after radiotherapy, the tumor size gradually decreased, showing a significant difference compared to the control group. This indicates that Ikbkb-KO macrophage therapy has a good effect when used as an adjunct to radiotherapy.

[0055] Most importantly, this combination regimen effectively controlled tumor progression under moderate-dose (5 Gy) radiotherapy conditions, demonstrating the function of Ikbkb-ko macrophages as radiosensitizers. This provides solid preclinical experimental evidence for reducing radiotherapy dose while ensuring efficacy in future clinical practice.

[0056] In summary, this embodiment demonstrates through in vitro and in vivo experiments that Ikbkb knockout macrophages secrete factors (conditioned medium) that synergistically inhibit radiotherapy in vitro, more effectively suppressing breast cancer cell proliferation, inhibiting cell activity, and inducing apoptosis. Intratumoral infusion of Ikbkb-KO engineered macrophages as a "cell sensitizer," combined with local radiotherapy, produces a significant synergistic antitumor effect in tumor-bearing mice, achieving excellent tumor regression even under low-to-medium dose (5 Gy) radiotherapy.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of macrophages with Ikbkb gene inhibition or knockout in preparation of a breast cancer radiotherapy sensitization drug.

2. Use according to claim 1, characterized in that, The macrophages are obtained by knocking out the Ikbkb gene of target macrophages.

3. Use according to claim 2, characterized in that, The target macrophages are RAW264.7 cell lines.

4. Use according to claim 2, characterized in that, The Ikbkb gene of the target macrophages is knocked out by a CRISPR-Cas9 gene editing method.

5. Use according to claim 4, characterized in that, In the CRISPR-Cas9 gene editing method, the target sequence targeting the Ikbkb gene is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

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