Use of ikbkb-inhibited or knocked-out macrophages in preparation of breast cancer radiotherapy sensitizing drugs

Abstract

The present application belongs to the field of biomedicine, and particularly relates to application of Ikbkb-inhibited or knocked-out macrophages in preparation of a breast cancer radiotherapy sensitization drug. The present inventors first prove the role of Ikbkb-inhibited or knocked-out macrophages in breast cancer radiotherapy. Compared with the strategy in the prior art which mainly relies on small molecule drugs or antibodies, the present application modifies the macrophages in the tumor microenvironment, and the Ikbkb-knocked-out engineered macrophages can more effectively inhibit tumor growth after combined radiotherapy, thereby providing a brand-new solution for overcoming breast cancer radiotherapy resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular, it relates to the application of Ikbkb-inhibited or knocked-out macrophages in the preparation of a breast cancer radiotherapy sensitization drug. BACKGROUND

[0002] Radiotherapy (RT) is one of the core means of comprehensive treatment of breast cancer, and about 50% of breast cancer patients need to receive radiotherapy during treatment. However, primary or acquired radiotherapy resistance is the main reason for treatment failure and tumor recurrence. Traditional researches are mostly focused on the DNA damage repair (DDR) mechanism of tumor cells themselves, but increasing evidence shows that the tumor microenvironment (TME), especially the immune cells therein, plays a decisive role in mediating radiotherapy resistance. Radiotherapy can induce a series of complex immune responses while killing tumor cells, which can either activate anti-tumor immunity (immunosensitizing effect) or exacerbate immune suppression, and the final result depends on the dynamic balance of various immune cells in the TME. Therefore, reshaping the immunosuppressive TME after radiotherapy is a key breakthrough for synergistic sensitization of radiotherapy and improvement of long-term efficacy.

[0003] TAMs are the most important immune cell components in the TME, and their phenotypes and functions are highly plastic. They are usually divided into M1 type that is pro-inflammatory and anti-tumor, and M2 type that is anti-inflammatory and pro-tumor. In most solid tumors, TAMs mainly present as M2-like phenotype, inhibit T cell function by secreting cytokines such as IL-10 and TGF-β, and help tumor growth and metastasis by promoting angiogenesis and tissue remodeling. After radiotherapy, damaged tumor cells release “find-me” and “eat-me” signals, and a large number of macrophages are recruited to infiltrate the tumor area. However, these macrophages are often “domesticated” by the TME into M2 type, and they not only cannot effectively clear the dead tumor cells, but also participate in the damage repair process, secrete pro-angiogenic and pro-fibrotic factors, and ultimately weaken the long-term efficacy of radiotherapy and even promote tumor recurrence. Therefore, reprogramming TAMs from “accomplice” (M2 type) to “killer” (M1 type) is a key strategy to break radiotherapy resistance and enhance immunotherapy efficacy.

[0004] IκB kinase beta (Ikbkb, also known as IKKβ) is the core kinase of the canonical NF-κB signaling pathway. This pathway plays a central role in regulating inflammatory responses, immune responses, cell proliferation and apoptosis. In traditional cognition, IKBKB activates the NF-κB pathway mainly to promote inflammation and immune activation. However, the function of this pathway is highly context-dependent. In the tumor microenvironment, persistent and chronic activation of NF-κB is associated with immune suppression and tumor progression. Although previous studies have suggested that the NF-κB pathway is related to the function of TAMs, whether Ikbkb directly regulates the response of macrophages to ionizing radiation and determines their final phenotype and function in the TME after radiotherapy is still an unknown field. There is currently a lack of direct evidence to reveal its potential as a radiosensitization target. SUMMARY

[0005] In view of this, in order to make up for the above technical gaps existing in the current field, the purpose of the present application is to provide the use of Ikbkb-inhibited or -knocked-out macrophages in the preparation of a breast cancer radiosensitization drug. Radiotherapy is one of the core means of comprehensive treatment of breast cancer. However, the immunosuppressive properties of the tumor microenvironment (TME), especially the dominant role of tumor-promoting M2-type tumor-associated macrophages (TAMs) therein, are key factors leading to radioresistance and treatment failure. Based on previous innovative unbiased genome-wide CRISPR-Cas9 screening, the present application first identifies IκB kinase beta (Ikbkb) as a core target that regulates the radiosensitivity and functional phenotype of macrophages. The inventors found that knocking out Ikbkb can endow macrophages with dual beneficial properties: on the one hand, it significantly enhances its own radioresistance, enabling it to survive and continue to function in the immunosuppressive microenvironment after radiotherapy; on the other hand, it reprograms it from the tumor-promoting M2 phenotype to the anti-tumor M1-like phenotype, thereby enhancing its ability to phagocytose tumor cells and activate adaptive immunity.

[0006] Based on the above findings, the present application 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 inhibited.

[0008] The Ikbkb gene (IκB kinase beta gene) is a key target that regulates radiosensitivity and immune function in mouse macrophages, and its coding sequence can refer to NCBI accession number NM_010546.

[0009] Further, the engineered macrophage is obtained by knocking out the Ikbkb gene of the target macrophage, and the target macrophage is a RAW264.7 cell line.

[0010] The application further provides a preparation method of the engineered macrophage.

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

[0012] Further, the Ikbkb gene of the target macrophage is knocked out by a CRISPR-Cas9 method in the method.

[0013] When the Ikbkb gene is knocked out by 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 application also provides an application of the engineered macrophage, and the application of the engineered macrophage in preparing a breast cancer radiotherapy sensitization drug.

[0015] The engineered macrophage of the application can be used as a "cell sensitization agent" in combination with radiotherapy for treating tumors, in particular breast cancer. When cultured in vitro at a certain cell concentration, the engineered macrophage exhibits expected anti-breast cancer tumor function characteristics.

[0016] The application proves through a series of in vitro and in vivo experiments that, compared with wild-type macrophages, the Ikbkb-knocked-out engineered macrophage of the application can more effectively inhibit tumor growth after radiotherapy.

[0017] The application also provides a tool substance for constructing the engineered macrophage and an application thereof.

[0018] That is, the application of the substance for knocking out the Ikbkb gene or inhibiting the expression of the Ikbkb gene in preparing the engineered macrophage.

[0019] Preferably, the substance comprises:

[0020] An sgRNA combination targeting the Ikbkb gene, the combination being composed of sgRNAs of sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; or a recombinant lentivirus vector comprising a DNA sequence encoding the sgRNA combination.

[0021] The application has the following advantages compared with the prior art:

[0022] The present inventors first prove the role of macrophages with inhibited or knocked out Ikbkb in the radiotherapy of breast cancer. Compared with the strategy in the prior art mainly relying on small molecule drugs or antibodies, the present application can more effectively inhibit tumor growth after combined radiotherapy by modifying macrophages in the tumor microenvironment, and provides a brand new solution to overcome radiotherapy resistance of breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 IKBKB protein Western Blot detection results of wild type and Ikbkb-KO RAW264.7 macrophages;

[0025] Figure 2 Observation results of 4T1 cells after 0h and 72h in different conditioned media;

[0026] Figure 3 EdU flow cytometry detection results of 4T1 breast cancer cells after treatment in different conditioned media;

[0027] Figure 4 Apoptosis detection results of 4T1 breast cancer cells after treatment in different macrophage conditioned media; Fig. A is the scatter plot of apoptosis flow cytometry of single 4T1 cells in different treatment groups, wherein Q1-LL region is live cells, Q1-LR region is early apoptotic cells, Q1-UR region is late apoptotic cells, and Q1-UL region is necrotic cells; total apoptosis rate = LR+UR; Fig. B is a statistical column chart of apoptosis rate of 4T1 cells in different treatment groups in multiple repeated experiments;

[0028] Figure 5 CCK8 proliferation curve of 4T1 breast cancer cells treated in different conditioned media;

[0029] Figure 6 Tumor growth curve in tumor-bearing mouse model. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0031] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in a number of embodiments of the application. The appearance of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily mutually exclusive or alternative embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0032] In the embodiments described herein, the specific techniques or conditions not specified are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used, if not specified by the manufacturer, are conventional products that can be purchased through regular channels. The experimental methods in the embodiments, if not specifically stated, are conventional methods. The test materials used in the following examples, if not specifically stated, are commercially available products.

[0033] Radiotherapy is one of the core means of comprehensive treatment of breast cancer. However, the immunosuppressive characteristics of the tumor microenvironment (TME), especially the tumor-associated macrophage (TAM) pro-tumor function, is a key bottleneck leading to radiotherapy resistance. Developing new strategies that can reverse the function of TAMs and synergistically enhance radiotherapy is a major challenge in clinical practice.

[0034] In the author's previous research, a CRISPR-Cas9 knockout library covering the whole mouse genome was first constructed, and a high-fidelity screening platform was established. By applying 10Gy ionizing radiation to the library cells, it was found that the sgRNA targeting IκB kinase β (Ikbkb) gene was most significantly enriched in the cell population that survived under radiation stress.

[0035] What is particularly critical is that in a more rigorous dynamic analysis (comparing the cell population 2 weeks after radiation with 3 days after radiation), Ikbkb ranked first among all candidate genes with an absolute advantage, indicating that its functional deletion can most effectively drive the long-term survival and clonal expansion of macrophages after radiation.

[0036] Further mechanism exploration showed that radiation triggered extensive transcriptional reprogramming centered on the p53 pathway and DNA damage repair. Although the transcription level of Ikbkb itself did not change, its protein function as a core kinase of the NF-κB pathway played a "checkpoint" role in this stress network, determining the cell fate.

[0037] To verify the clinical relevance of this finding, the inventors analyzed the peripheral blood monocyte sequencing data of patients who received thoracic radiotherapy. The results revealed that the IKBKB transcription level in the classical monocytes (macrophage precursors) of the patients' peripheral blood was significantly up-regulated after radiotherapy. This perfectly logically closed loop with the in vitro functional screening results collectively strongly suggests that Ikbkb is a key intrinsic pathway that mediates the radio-damage of macrophages and is a highly potential intervention target.

[0038] Based on the above findings, the present application proposes that knocking out the Ikbkb gene in macrophages through gene editing technology can simultaneously endow macrophages with significant self-radioresistance and reprogram them from the pro-tumor M2 phenotype to the anti-tumor M1 phenotype, thereby achieving the dual effect of synergistic radiosensitization.

[0039] To specifically verify and implement this technical solution, the construction method of the engineered macrophages, their in vitro functions, and their in vivo anti-breast cancer tumor effects in combination with radiotherapy will be described in detail below through examples.

[0040] As used herein, "macrophages" refer to innate immune cells derived from myeloid precursor cells, which have functions such as phagocytosis, antigen delivery, and immune regulation. The "engineered macrophages" of the present application refer to macrophages in which the expression of the Ikbkb gene is knocked out or functionally lost through gene editing technology.

[0041] The Ikbkb gene and target selection, IκB kinase beta (Ikbkb, also known as IKKβ) is the core kinase of the classical NF-κB signaling pathway, which is traditionally believed to mainly promote inflammatory and survival responses. In some embodiments, the Ikbkb gene is of mouse origin, and its coding sequence can be referred to NCBI accession number NM_010546. In some embodiments, the Ikbkb gene is of human origin, and its coding sequence can be referred to NCBI accession number NM_001556.

[0042] Example 1: Construction and verification of engineered macrophages with Ikbkb knockout or functionally inhibited

[0043] 1. Experimental materials and methods

[0044] 1.1 Cell lines

[0045] The mouse macrophage cell line used in the experiment is RAW264.7, purchased from ATCC, and cultured in a 10% fetal bovine serum glucose medium;

[0046] 1.2 sgRNA design and synthesis:

[0047] Using CRISPR-Cas9 technology, four specific sgRNAs were designed and synthesized according to the sequence specificity and off-target risk of the coding sequence of the mouse Ikbkb gene. The sequences are as follows:

[0048] sgRNA1: 5'-TGTCGGGTGTTTGAGTTCAG-AGG-3' (SEQ ID NO: 1);

[0049] sgRNA2: 5'-CCTCTTCTATGCCTTCTTTG-GGG-3' (SEQ ID NO: 2);

[0050] sgRNA3: 5'-AATGGAGATAGGACCCTTTA-AGG-3' (SEQ ID NO: 3);

[0051] sgRNA4: 5'-GCCCGTATTCAGCCTTCTTG-TGG-3' (SEQ ID NO: 4);

[0052] 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.

[0053] Synthetic commission race industry (Suzhou) biotechnology Co., Ltd. was carried out, sgRNA1-Cas9, sgRNA2-Cas9, sgRNA3-Cas9 and sgRNA4-Cas9 were constructed and cloned into lentiviral vector.

[0054] 1.3 Lentivirus packaging and cell infection:

[0055] Macrophages (Raw264.7) were infected with lentivirus containing sgRNA1-Cas9, sgRNA2-Cas9, sgRNA3-Cas9 and sgRNA4-Cas9, and monoclonal screening was carried out. Sanger sequencing verification obtained Ikbkb gene knockout macrophages (Ikbkb-KO).

[0056] 1.4 Detection index and method

[0057] Western Blot was used to further verify the expression of Ikbkb gene at the protein level:

[0058] Total protein was extracted, and Western Blot analysis was performed using anti-Ikbkb antibody and anti-β-actin antibody to confirm that Ikbkb protein expression was effectively knocked out.

[0059] Figure 1The results of Western Blot detection of IKBKB protein of wild type and Ikbkb-KO RAW264.7 macrophages;

[0060] Figure 1 The results show that, taking β-actin as the internal reference, clear IKBKB protein bands can be detected in wild type cells; the IKBKB protein bands of Ikbkb-KO cells completely disappear, indicating that the expression of Ikbkb gene at the protein level is effectively blocked, further verifying that the Ikbkb knockout or functionally inhibited macrophage is successfully constructed.

[0061] Example 2: Effect of Ikbkb knockout on the in vitro anti-tumor function and radiosensitivity of macrophages

[0062] 1. Experimental materials and methods

[0063] 1.1 Cell lines and experimental animals

[0064] Cell line: mouse breast cancer cell line 4T1;

[0065] Macrophages: Ikbkb gene knockout macrophages (Ikbkb-KO) and wild type mouse macrophages (RAW264.7) obtained in Example 1.

[0066] Experimental animals: 6-8 week old female BALB / c mice, raised in SPF level environment.

[0067] 1.2 Preparation of conditioned medium

[0068] Wild type mouse macrophages (RAW264.7) and Ikbkb gene knockout macrophages were inoculated at the same density, and fresh complete medium was replaced for further culture for 72 hours. The supernatant was obtained by centrifugation, which was used as the conditioned medium and stored in the freezer.

[0069] 1.3 In vitro experimental grouping and treatment

[0070] 4T1 cells were inoculated in appropriate culture plates and subjected to single dose of 8Gy X-ray irradiation; after irradiation, the following groups of conditioned medium were immediately replaced for culture: WT-MФ-CM: replaced with WT macrophage conditioned medium; Ikbkb-MФ-CM group: replaced with Ikbkb-KO macrophage conditioned medium; Control group: 4T1 cells without any treatment were set as control.

[0071] 1.4 Detection index and method

[0072] Cell morphology and density: after in vitro treatment for 0h and 72h, the morphology and density changes of 4T1 cells were observed and photographed using a fluorescence microscope;

[0073] Cell proliferation: After 48h in vitro treatment, the cell proliferation activity was analyzed by flow cytometry using EdU (5-ethynyl-2'-deoxyuridine) kit to detect the incorporation of EdU in 4T1 cells. EDU (5-ethynyl-2'-deoxyuridine) is a thymidine analogue that can be taken up by proliferating cells and integrated into newly synthesized DNA. EDU-labeled cells emit a fluorescent signal, 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 S phase;

[0074] Cell apoptosis: After 48h in vitro treatment, the apoptosis rate of 4T1 cells was quantitatively analyzed by flow cytometry using Annexin V-FITC / PI double staining apoptosis detection kit. The total apoptosis rate is the sum of the percentages of early apoptotic (LR, AnnexinV⁺ / PI⁻) and late apoptotic (UR, AnnexinV⁺ / PI⁺) cells:

[0075] Cell viability: After 0h, 24h, 48h and 72h in vitro treatment, CCK-8 kit was used for detection. CCK-8 solution was added to the culture well, and the absorbance value was detected using a microplate reader. The relative viability of cells was calculated to evaluate the inhibitory ability of conditioned medium on tumor cell growth.

[0076] 2. Experimental results

[0077] Figure 2 Observation results of 4T1 cells after 0h and 72h in different conditioned media;

[0078] Figure 3 EdU flow cytometry detection results of 4T1 breast cancer cells after treatment with different conditioned media. 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).

[0079] Figure 4 Apoptosis detection results of 4T1 breast cancer cells after treatment with different macrophage conditioned media. Figure A is a scatter plot of apoptosis flow cytometry of 4T1 cells in different treatment groups. Q1-LL region is live cells, Q1-LR region is early apoptotic cells, Q1-UR region is late apoptotic cells, and Q1-UL region is necrotic cells. Total apoptosis rate = LR + UR. Figure B is a bar chart of apoptosis rate of 4T1 cells in different treatment groups from multiple repeated experiments;

[0080] Figure 5CCK8 proliferation curve of 4T1 breast cancer cells under different conditioned medium treatment;

[0081] Figure 2 The results show that after 72 hours of conditioned medium treatment, the 4T1 cell density in the Ikbkb-KO-MΦ-CM group is significantly lower than that in the WT-MΦ-CM group, indicating that the conditioned medium of Ikbkb-KO macrophages exhibits significantly stronger tumor inhibition activity.

[0082] Figure 3 The results show that after 48 hours of conditioned medium treatment, the histogram (single experiment data) shows that the EDU positive (proliferation) cell proportion of 4T1 cells in the Control group reaches 60.23%, indicating that the normal cultured 4T1 cells are active in proliferation; the EDU positive rate of the WT-MΦ-CM group treated with wild type macrophage conditioned medium decreases to 29.56%; while the EDU positive rate of the Ikbkb-KO-MΦ-CM group further decreases to 17.45%, and the proliferation inhibition trend is more significant. The lower quantitative column chart is the statistical average value of multiple biological repeat experiments: the EDU positive rate of the Control group is 58.91%, that of the WT-MΦ-CM group is 26.95%, and that of the Ikbkb-KO-MΦ-CM group is 19.24%, which is consistent with the change trend of the histogram data, and the difference between groups has statistical significance. It can be seen that after 48 hours of treatment, the proliferation activity (EDU positive rate) of 4T1 cells in the Ikbkb-KO macrophage conditioned medium treatment group is significantly lower than that in the WT-MΦ-CM group, indicating that the conditioned medium of Ikbkb knockout macrophages can more effectively inhibit the proliferation of 4T1 tumor cells, and reflects stronger tumor proliferation inhibition activity.

[0083] Figure 4The results show that after 48 hours of conditioned medium treatment, the upper scatter plot (single experiment data) shows that the Control group: Q1-LL (live cells) accounts for 95.24%, Q1-LR (early apoptotic cells) accounts for 1.83%, and Q1-UR (late apoptotic cells) accounts for 2.50%. The proportion of live cells is high, and the apoptosis level is low. The WT-MΦ-CM group: Q1-LL (live cells) accounts for 93.33%, Q1-LR (early apoptotic cells) accounts for 3.33%, and Q1-UR (late apoptotic cells) accounts for 2.91%. The proportion of live cells decreases, and the proportion of apoptotic cells increases compared with the Control group. The Ikbkb-KO-MΦ-CM group: Q1-LL (live cells) accounts for 90.22%, Q1-LR (early apoptotic cells) accounts for 4.63%, and Q1-UR (late apoptotic cells) accounts for 4.56%. The proportion of live cells further decreases, and the proportions of early and late apoptotic cells significantly increase. The lower quantitative column chart is the statistical average of multiple biological repeat experiments: the apoptosis rate is consistent with the trend of the scatter plot data. Compared with the WT-MΦ-CM group and the Control group, the apoptosis rate (LR+UR) of 4T1 cells in the Ikbkb-KO-MΦ-CM group significantly increases, indicating that the conditioned medium of Ikbkb-KO macrophages can significantly promote 4T1 cell apoptosis and exert stronger tumor inhibition activity.

[0084] Figure 5 The results show that after 48 hours of treatment, the wild-type macrophage conditioned medium (WT-MΦ-CM) can inhibit 4T1 cell proliferation to a certain extent; and the conditioned medium of Ikbkb knockout macrophages (Ikbkb-KO-MΦ-CM) has a significantly stronger inhibitory effect on 4T1 cell proliferation, indicating that Ikbkb-knockout macrophages have stronger tumor inhibition activity.

[0085] Example 3: Therapeutic effect of Ikbkb-knockout engineered macrophages combined with radiotherapy in tumor-bearing mouse models

[0086] 1. Experimental materials and methods

[0087] 1.1 Cell lines and experimental animals

[0088] The cell lines and experimental animals used in this example are the same as those in Example 2.

[0089] 1.2 Establishment of tumor-bearing models

[0090] Logarithmic growth phase 4T1 cells were taken, and 1×10^ 5 cells were injected into the right hip of each BALB / c mouse subcutaneously;

[0091] Tumor length (L) and width (W) were measured every 2 days using a vernier caliper, and tumor volume (V = L x W2 / 2) was calculated, and the body weight of mice was monitored.

[0092] 1.3 Grouping and treatment

[0093] 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;

[0094] Three groups were given radiotherapy, in addition to radiotherapy, different cell treatment was given, wherein:

[0095] Vehicle group: intratumoral injection of PBS;

[0096] WTMφ: intratumoral injection of 5 x 10^ 6 Wild type macrophages;

[0097] Ikbkb-KOMφ group: intratumoral injection of 5 x 10^ 6 Ikbkb gene knockout macrophages.

[0098] Combined treatment regimen: intratumoral injection of cells or PBS on days 0, 3, and 6. On the first day after each injection (i.e. days 1, 4, and 7), the tumor sites of all groups of mice were treated with local radiotherapy, with a dose of 5 Gy per irradiation and a dose rate of 2 Gy / min.

[0099] 1.4 Detection index and method

[0100] From day 0, the tumor volume was measured once a day until the end of the experiment.

[0101] 2. Experimental results

[0102] Figure 6 Tumor growth curve graph for tumor-bearing mice model;

[0103] Figure 6 The results showed that after receiving radiotherapy, the tumor size of the mice treated with Ikbkb-KO macrophages gradually decreased, which was significantly different from the control group. This indicates that Ikbkb-ko macrophage therapy assisted radiotherapy has good effect.

[0104] What is particularly important is that this combined regimen can effectively control tumor progression under the condition of medium dose (5 Gy) radiotherapy, confirming the function of Ikbkb-ko macrophages as a radiosensitizer, and providing a solid preclinical experimental basis for reducing the radiotherapy dose while ensuring efficacy in future clinical practice.

[0105] In summary, the present embodiment is confirmed by in vitro and in vivo experiments that Ikbkb knockout macrophages, which secrete factors (conditioned medium), can synergize with radiotherapy in vitro to more effectively inhibit breast cancer cell proliferation, inhibit activity and induce apoptosis. The Ikbkb-KO engineered macrophage as a "cell sensitizer" is intratumorally infused, combined with local radiotherapy, which can produce a significant synergistic anti-tumor effect in tumor-bearing mice, even at a low dose (5 Gy) of radiotherapy. Excellent tumor regression effect can be achieved.

[0106] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of Ikbkb gene-inhibited or knocked-out macrophages in the preparation of radiosensitizing drugs for breast cancer.

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

3. The application according to claim 2, characterized in that, The target macrophages were the RAW264.7 cell line.

4. The application according to claim 3, characterized in that, The Ikbkb gene of the target macrophages was knocked out using the CRISPR-Cas9 gene editing method.

5. The application according to claim 4, characterized in that, The sgRNA sequences used in the CRISPR-Cas9 gene editing method are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

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