Application of SPP1 as target spot in preparation of large segmentation radiotherapy sensitizer for lung cancer

Abstract

The invention belongs to biological medicine, and relates to a sensitizer, in particular to application of SPP1 as a target spot in preparation of a lung cancer large-segmentation radiotherapy sensitizer, and the lung cancer large-segmentation radiotherapy sensitizer is prepared by inhibiting the activity of SPP1 protein, inhibiting the expression of SPP1 coding genes, reducing the expression of MIF, inhibiting the activity of MIF signal channels and enhancing the recruitment of CD8 + T cells and tumor immune activation. The sensitivity of lung cancer patients to radiotherapy can be improved, so that the lung cancer radiotherapy effect is improved. Moreover, the invention discloses lung cancer radiotherapy resistance markers SPP1 and MIF signal pathways, and clarifies a new mechanism of lung cancer radiotherapy resistance, which is of great significance for exploring the relationship between SPP1 and lung cancer radiotherapy sensitivity and improving radiotherapy curative effect and patient prognosis.

Description

Technical Field

[0001] The present invention belongs to biomedicine and relates to a sensitizer, and in particular to use of SPP1 as a target in the preparation of a sensitizer for large-fractionation radiotherapy of lung cancer. Background Art

[0002] Lung cancer is a common and fatal malignancy worldwide. Among them, non-small cell lung cancer (NSCLC) accounts for approximately 80%-85% of all lung cancer cases. Radiotherapy (RT) is the basic therapy for the treatment of NSCLC. Its principle is to exert anti-tumor effects through ionizing radiation-induced DNA damage and tumor cell death. However, the efficacy of RT is often limited by immunosuppression in the tumor microenvironment (TME). Recent studies have shown that RT can reshape the TME and affect local immune responses. Modulating this immune environment can further increase the sensitivity of tumors to RT and enhance its anti-tumor efficacy. Therefore, combining RT with immunomodulatory strategies can improve clinical efficacy by enhancing anti-tumor immunity.

[0003] Secreted phosphoprotein 1 (SPP1) is a multifunctional cytokine-like glycoprotein involved in inflammation, tissue remodeling, and immune regulation. Although studies have shown that SPP1 can be used as a biomarker for lung cancer diagnosis, there is currently a lack of research on the correlation between SPP1 and radiotherapy sensitivity.

[0004] Based on this background, exploring the relationship between SPP1 and radiosensitivity of non-small cell lung cancer is of great significance for improving the efficacy of radiotherapy and patient prognosis. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use of SPP1 as a target in the preparation of a lung cancer hypofractionated radiotherapy sensitizer, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned and other related purposes, the present invention first provides a use of SPP1 as a target in the preparation of a hypofractionated radiotherapy sensitizer for lung cancer.

[0007] The present invention also provides use of an SPP1 gene or protein inhibitor in preparing a lung cancer radiotherapy sensitizer.

[0008] The present invention also provides a method for improving the radiotherapy effect of lung cancer, comprising administering an SPP1 gene or protein inhibitor to a subject.

[0009] The present invention also provides a sensitizer for improving the radiotherapy effect of lung cancer, wherein the effective substance in the sensitizer is an SPP1 gene or protein inhibitor.

[0010] As described above, the use of SPP1 as a target in the preparation of a lung cancer radiotherapy sensitizer of the present invention has the following beneficial effects:

[0011] The SPP1 gene or protein inhibitors described herein are particularly suitable as lung cancer radiosensitizers for use in hypofractionated radiotherapy regimens. When used within this regimen, SPP1 inhibitors exhibit significant synergistic effects with radiotherapy. These sensitizers can effectively enhance the sensitivity of tumor cells to radiation, significantly improving the tumor-killing efficacy of single, hypofractionated doses. Their core value lies in their potential to maintain or enhance the anti-tumor efficacy of hypofractionated radiotherapy while reducing the required absolute radiation dose or the number of exposures, thereby effectively mitigating radiation damage to surrounding normal tissues and reducing complications caused by radiotoxicity.

[0012] Compared with the existing technology, the present invention combined with radiotherapy (especially in the large fractionation mode) shows a significant positive technical effect: it not only effectively inhibits the activity of SPP1 protein and the expression of its encoding gene, but also significantly inhibits the activity of the downstream MIF signaling pathway. More importantly, this combined strategy significantly enhances the CD8 + The recruitment and activation of T cells promotes anti-tumor immune responses. These combined effects constitute the molecular and immunological basis for the sensitization effect of SPP1 inhibitors in radiotherapy, especially hypofractionated radiotherapy.

[0013] The sensitizer provided by the present invention is SPP1 + Macrophages are specific targets, and this targeting minimizes the impact on normal cells, which is crucial for protecting normal tissues within and adjacent to the high-dose irradiation field of large-fraction radiotherapy, further improving the safety of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagrams illustrating the construction of the conditional knockout mouse and disease models of the present invention. (A) Schematic diagram of the hybridization of LysCre mice with SPP1fl / fl mice to generate LysCre-SPP1fl / fl mice. (B) PCR verification demonstrates the presence of Lys Cre (left) and SPP1 loxP (right) alleles in the knockout mice. (C) Experimental timeline for LLC cell injection, RT treatment (8 Gy x 3), and tumor resection on day 24 in SPP1fl / fl and LysCre-SPP1fl / fl mice.

[0015] Figure 2 Shown are changes in local genes, immune cell infiltration, and SPP1 and CD68 expression before and after RT. (A) Comparison of gene expression in lung cancer tumors before and after RT. (B) CIBERSORT analysis shows an increase in macrophage numbers after RT. (C) Immunofluorescence co-staining shows upregulation of both SPP1 and CD68 after RT.

[0016] Figure 3 Shown are the expression of SPP1 in various cell types and phagocyte subsets. (A) SPP1 expression in various cell types. (B) Macrophage subsets based on functional gene expression patterns. (C) SPP1 expression in different macrophage subsets. (D) M1 / M2 polarization scores of macrophage subsets. (E) Differentially expressed genes in SPP1-IR_Macs and SPP1-IR_Macs.

[0017] Figure 4 The expression levels of MIF in different macrophage subsets of the present invention are shown. (A) The dot matrix shows SPP1 + The probability of communication between IR_Macs and other immune cells. (B) Highlighting SPP1 + MIF signaling pathway network affected by IR_Macs. (C) Violin plot comparing MIF expression levels in macrophage subsets. (D) SPP1 + Comparison of MIF expression in SPP1-IR_Macs.

[0018] Figure 5 Figure 1 shows in vivo experiments demonstrating that SPP1 knockdown increases radiosensitivity in lung cancer. (A) Tumors in different groups of mice. (B) Tumor volume growth curves in different groups. (C) HE staining of tumor morphology. (D) Immunofluorescence staining of PCNA in tumors. (E) Quantification of PCNA-positive cells. (F) Immunofluorescence staining of MIF in tumors. (G) Quantification of MIF expression. (H) Immunofluorescence staining of CD8+ T cells in tumors. (I) Quantification of CD8+ T cells. DETAILED DESCRIPTION

[0019] Based on a large number of studies, it was first discovered that SPP1 is a key factor upregulated after RT, which is mainly expressed by tumor-associated macrophages (TAMs) and inhibits CD8 + T cell infiltration contributes to tumor progression, angiogenesis, and invasion. Therefore, high expression of SPP1 is associated with poor prognosis in various cancers, including lung cancer.

[0020] The present invention first provides the use of SPP1 as a target in the preparation of a lung cancer hypofractionated radiotherapy sensitizer.

[0021] In radiotherapy, "sensitizers" are substances that increase the sensitivity of tumor cells to radiation. They can improve the efficacy of radiotherapy and reduce damage to normal tissues.

[0022] Hypofractionated radiotherapy refers to single-dose radiation therapy with a dose greater than 2 Gy, typically with significantly fewer doses than conventional fractionated radiotherapy. The specific number of treatments is determined based on tumor type, location, volume, and the technical approach. Hypofractionated radiotherapy can be categorized into the following types based on dose and frequency: 1) Moderately hypofractionated radiotherapy: Each treatment dose ranges from 2.0 to 4.0 Gy; and 2) ultra-hypofractionated radiotherapy: Each treatment dose exceeds 5 Gy.

[0023] The present invention also provides use of an SPP1 gene or protein inhibitor in preparing a lung cancer radiotherapy sensitizer.

[0024] In certain embodiments of the present invention, the lung cancer is non-small cell lung cancer.

[0025] In the present invention, the SPP1 gene can be selected from SPP1 genes of different species, such as SPP1 genes of different species in the SCBI database, specifically selected from mice, rats, and rabbits.

[0026] In certain embodiments of the present invention, the SPP1 gene or protein inhibitor is selected from one or more of nucleic acid molecules, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0027] In certain embodiments of the present invention, the SPP1 gene or protein inhibitor is an agent that specifically knocks out exons 2 to 8 of the SPP1 gene through gene editing technology.

[0028] Furthermore, the gene editing technology is a conditional gene knockout technology, and the conditional gene knockout technology is a Cre-LoxP conditional gene knockout technology, wherein the 5' end of exon 2 and the 3' end of exon 8 of the SPP1 gene are respectively inserted with the same direction LoxP sites.

[0029] Furthermore, the nucleotide sequence of exons 2 to 8 is shown in SEQ ID NO.1.

[0030] In certain embodiments of the present invention, the SPP1 gene or protein inhibitor as a sensitizer has any of the following effects:

[0031] 1) Inhibit the activity of SPP1 protein;

[0032] 2) inhibit the expression of the gene encoding SPP1;

[0033] 3) reduce the expression of MIF;

[0034] 4) Inhibit the activity of the MIF signaling pathway;

[0035] 5) Strengthen CD8 + T cell recruitment and tumor immunity activation.

[0036] Inhibiting the activity of the SPP1 protein specifically refers to reducing the activity of the SPP1 protein. Preferably, the activity of the SPP1 protein is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and even more preferably by at least 90%, compared to the level before inhibition. Most preferably, the activity of the SPP1 protein is reduced to approach or reach the level of a healthy individual.

[0037] Inhibiting the expression of the SPP1 encoding gene refers to inhibiting the transcription and translation of the SPP1 encoding gene. Specifically, it may refer to reducing the transcriptional activity of the SPP1 encoding gene or reducing the translation level of the SPP1 encoding gene.

[0038] Those skilled in the art can use conventional methods to regulate the expression of the SPP1 encoding gene, such as gene knockout, homologous recombination, interfering RNA, etc.

[0039] The inhibition of SPP1 encoding gene expression can be verified by PCR and Western Blot detection of expression levels.

[0040] Preferably, compared with before radiotherapy, the expression of the SPP1 encoding gene is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and even more preferably by at least 90%. Optimally, the expression of the SPP1 encoding gene is close to or reaches the level of a healthy individual.

[0041] Reducing MIF expression means that compared to before radiotherapy, MIF expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and even more preferably by at least 90%. Most preferably, MIF expression approaches or reaches the level of a healthy individual.

[0042] Inhibiting the MIF signaling pathway means that compared to before radiotherapy, the activity of the MIF signaling pathway is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and even more preferably by at least 90%. Optimally, the activity of the MIF signaling pathway approaches or reaches the level of a healthy individual.

[0043] Enhance CD8 + T cell recruitment refers to: being able to enhance CD8 + The ability of T cells to enter tumor cells, i.e. more CD8 + T cells can reach the tumor site and promote tumor immune activation. Compared with before radiotherapy, CD8 + The recruitment capacity of T cells is increased by at least 10%, preferably by at least 30%, even better by at least 50%, even better by at least 70%, and even better by at least 90%. +The recruitment capacity of T cells is close to or reaches the level of healthy individuals.

[0044] The sensitizer must include an SPP1 gene or protein inhibitor, or use the SPP1 gene or protein inhibitor as an effective ingredient for the aforementioned efficacy.

[0045] The active ingredient in the sensitizer that exerts the aforementioned function may be solely the SPP1 gene or protein inhibitor, or may contain other molecules that exhibit the aforementioned function. In other words, the SPP1 gene or protein inhibitor is the sole active ingredient or one of the active ingredients in the sensitizer. The sensitizer may be a single-component substance or a multi-component substance.

[0046] The form of the sensitizer is not particularly limited and can be in the form of solid, liquid, gel, semi-fluid, aerosol or other substances.

[0047] The sensitizer is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0048] The sensitizers include but are not limited to drugs, health products, foods, etc.

[0049] The present invention also provides a method for improving the radiotherapy effect of lung cancer, comprising administering an SPP1 gene or protein inhibitor to a subject.

[0050] The subject can be a mammal or lung tissue of a mammal. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like. The primate is preferably a monkey, an ape, or a human. The lung tissue can be isolated lung tissue.

[0051] The subject may be a patient suffering from non-small cell lung cancer or an individual expecting treatment for non-small cell lung cancer, or the subject may be ex vivo lung tissue of a patient suffering from non-small cell lung cancer or an individual expecting treatment for non-small cell lung cancer.

[0052] The present invention also provides a sensitizer for improving the radiotherapy effect of lung cancer, wherein the effective substance in the sensitizer is an SPP1 gene or protein inhibitor.

[0053] The sensitizer also contains a pharmaceutically acceptable carrier or excipient.

[0054] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0055] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0056] In the present invention, unless otherwise specified, the dosage form of the sensitizer is not particularly limited and can be prepared into injections, oral solutions, tablets, capsules, dripping pills, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the mode of administration.

[0057] The sensitizer may be a pharmaceutical composition.

[0058] When the sensitizer is used to enhance the effects of radiotherapy, an effective dose of the sensitizer is administered to a subject. Using this method, the level of SPP1 in lung tissue is suppressed. Furthermore, the level of SPP1 in the lung tissue is suppressed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0059] The form of the sensitizer is not particularly limited and can be in the form of solid, liquid, gel, semi-fluid, aerosol or other substances.

[0060] The sensitizer is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0061] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0063] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0064] 1. Experimental Methods

[0065] 1.1 Experimental subjects:

[0066] C57BL / 6 male mice (6-8 weeks old) were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). LysCre-SPP1fl / fl and control SPP1fl / fl C57BL / 6 mice were purchased from Cyagen Biosciences Inc. All mice were housed under SPF standards at a temperature of 24°C ± 2°C, a relative humidity of 40%-70%, and a 12-h light and 12-h dark cycle. All animal experiments were performed according to protocols approved by the Fudan University Animal Care and Use Committee.

[0067] LLC lung cancer cells from the American Type Culture Collection

[0068] 1.2 Methods for constructing conditional gene knockout mouse models

[0069] The present invention constructs a conditional gene knockout mouse model for specifically knocking out exons 2 to 8 of the SPP1 gene.

[0070] The specific method is as follows:

[0071] Select parental mice: Lys CreThe SPP1fl / fl mice and SPP1fl / fl mice. In the SPP1fl / fl mice, exons 2 to 8 of the SPP1 gene have been pre-translationally inserted with LoxP sites. LysCre mice carry the Cre recombinase gene driven by the lysozyme promoter and specifically express the Cre recombinase.

[0072] Hybridization process: Lys Cre Mice were mated with SPP1fl / fl mice ( Figure 1 A) Among hybrid offspring, approximately 50% of mice will carry both Lys Cre and SPP1fl / fl genes, and the genotypes of the offspring mice were identified by PCR. The PCR products were analyzed by electrophoresis, and the genotypes of the mice were determined according to the fragment size to screen out double-positive mice, that is, mice carrying both LysCre and SPP1fl / fl genes ( Figure 1 B) In SPP1 mice, Cre recombinase is specifically expressed under the drive of the Lys promoter. It recognizes and targets the LoxP sites flanking exons 2 to 8 of the SPP1 gene. Through Cre-LoxP-mediated site-specific recombination, it specifically deletes exons 2 to 8 of the SPP1 gene, thereby achieving conditional knockout of the SPP1 gene in specific tissues. The nucleotide sequence of exons 2 to 8 is shown in SEQ ID NO. 1.

[0073] 1.3 Construction of disease model

[0074] 1×10 6 / 100μL LLC cells. The mice were divided into 4 groups: SPP1fl / fl group, LysCre-SPP1fl / fl group, SPP1fl / fl+RT group and LysCre-SPP1fl / fl+RT group. On the 9th day, SARRP3 X-ray biological irradiator (Xtrahl, USA) was used for irradiation at a dose rate of 3.96Gy / min. The single dose was 8Gy, and the irradiation was continuous for 3 days. On the 10th day, the tumor volume of the mice was measured. Thereafter, the body fluid volume was monitored every three days. On the 24th day, the mice were sacrificed ( Figure 1 C). After tumor resection, the tumors were photographed, weighed, fixed in formalin for 24 hours, dehydrated using a graded ethanol series, embedded in paraffin, and sectioned for further staining.

[0075] The formula for calculating tumor volume is: tumor volume = (1 / 2×long diameter)×(short diameter)2.

[0076] 1.4 Establishment of subcutaneous lung cancer mouse model

[0077] LLC lung cancer cells (1×10 6 / 100μL) was injected subcutaneously into the inner left lower limb of 8-week-old male C57BL / 6 mice. 3 The SARRP3 X-ray biological irradiator (Xtrahl, USA) was used for irradiation at a dose rate of 3.96 Gy / min. The single dose was 8 Gy and the irradiation was continued for 3 consecutive days.

[0078] 1.5 Immunofluorescence staining

[0079] Tumor tissue blocks were cut into 4-6 μm thick sections and mounted on glass slides. Sections were deparaffinized in xylene and then rehydrated in a graded ethanol series. Antigen retrieval was performed using citrate buffer in a microwave oven. After cooling to room temperature, sections were incubated with 5% BSA for 1 hour to block nonspecific binding. The corresponding primary antibody was applied and incubated overnight at 4°C. After rinsing with PBS, sections were incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature in the dark. Cell nuclei were stained with DAPI, and slides were mounted with antifade medium. Fluorescence images were acquired using a fluorescence microscope.

[0080] 1.6 HE staining

[0081] Tissue sections were deparaffinized in xylene and rehydrated in a series of decreasing ethanol concentrations. They were then stained with hematoxylin for 5 minutes, washed in running tap water for 5 minutes, briefly differentiated in 1% acidic alcohol, and then blued with 0.2% ammonia or saturated lithium carbonate solution. After rinsing in tap water, sections were counterstained with eosin for 2 minutes. Stained sections were dehydrated in sequential ethanol solutions, washed in xylene, and mounted with coverslips using synthetic resin. Sections were examined and captured using a light microscope.

[0082] 2. Results

[0083] 2.1 Changes in local genes, immune cell infiltration, and SPP1 and CD68 before and after RT

[0084] To investigate the expression of local genes in lung cancer patients before and after RT, we compared the gene expression profiles of local tumors in lung cancer patients before and after RT and found that SPP1 was significantly upregulated after RT ( Figure 2 A). Immune cell infiltration analysis based on CIBERSORT and ssGSEA showed that the amount of macrophage infiltration increased significantly after RT, and SPP1 expression was positively correlated with macrophage infiltration ( Figure 2 B).

[0085] By establishing a subcutaneous lung cancer mouse model and conducting immunofluorescence co-staining experiments on SPP1 and the macrophage marker CD68, it was confirmed that the expression of both increased synchronously after RT. Further analysis found that SPP1 showed high expression characteristics in macrophages, especially in macrophages in tumor tissues, with the expression intensity and number of positive cells being the most significant ( Figure 2 C).

[0086] 2.2 The role of SPP1 in the IR_Macs subset and its impact on the tumor immune microenvironment

[0087] Macrophages were divided into four subgroups based on functional gene expression patterns: immune-related macrophages (IR_Macs), T cell receptor-positive macrophages (TCR_Macs), lipid metabolism-related macrophages (LM_Macs) and mitochondria-related macrophages (MT_Macs). It was found that SPP1 was specifically highly expressed in the IR_Macs subgroup, and this subgroup also had the highest anti-inflammatory (M2) phenotype score, suggesting that SPP1 may be involved in regulating the immunosuppressive function of macrophages. Further, SPP1 was compared with + Differentially expressed genes between IR Mac and SPP1-IR Mac were found, and the immunosuppression-related gene IL4I1 was found to be significantly expressed in SPP1 + The gene showed a significant high expression feature in the subgroup, and it is known that it is closely related to T cell immunosuppression response ( Figure 3 ).

[0088] Then, we focused on SPP1 in tumor tissue. + The interaction between IR_Macs, SPP1-IR_Macs and T cell subsets revealed that they formed a specific communication pattern through the MIF-CD74-CD44 / CXCR4 signaling axis. MIF is an immunosuppressive factor known to be associated with poor prognosis in various cancers and can drive tumor immune escape and progression. This signal is mainly mediated by SPP1. + IR_Macs secrete GZMK, which has strong cytotoxicity and tumor immune surveillance functions. + CD8 + T cells are the main receptor targets. Further analysis showed that compared with SPP1-IR_Macs, SPP1 + MIF expression levels were significantly elevated in IR_Macs, suggesting a potential cooperative role of these two factors in forming an immunosuppressive and tumor-promoting microenvironment ( Figure 4 ).

[0089] 2.3 In vivo experiments confirmed that SPP1 knockdown increased radiosensitivity in lung cancer

[0090] To validate targeting SPP1 +Macrophages can significantly improve the efficacy of RT. By constructing a LysCre-SPP1fl / fl mouse model, the mice were divided into four groups: SPP1fl / fl control group, LysCre-SPP1fl / fl group, SPP1fl / fl+RT group and LysCre-SPP1fl / fl+RT combined treatment group.

[0091] The results are as follows Figure 5 As shown in Figure 3, macrophage SPP1 gene knockout or RT alone can inhibit tumor growth, and the combined treatment group has the most significant anti-tumor effect ( Figure 5 AB), HE staining and PCNA immunofluorescence staining further confirmed the anti-tumor effect of the combined therapy (5C-E). + Macrophages, MIF, and CD8 + The relationship between T cells. Knocking out SPP1 in macrophages significantly reduced MIF expression and increased CD8 expression. This effect became more pronounced after RT (5F-I). These findings suggest that targeting SPP1 + Macrophages combined with RT can promote CD8 + Local recruitment of T cells and enhanced anti-tumor immune responses.

[0092] Sequences involved in this application:

[0093] SEQ ID NO.1:

[0094]

[0095]

[0096] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. The use of SPP1 as a target in the preparation of a large-fractionation radiotherapy sensitizer for lung cancer.

2. Use of SPP1 gene or protein inhibitors in the preparation of lung cancer radiotherapy sensitizers.

3. The use according to claim 2, wherein the radiotherapy is hypofractionated radiotherapy.

4. The use according to claim 2, characterized in that The lung cancer is non-small cell lung cancer.

5. The use according to claim 2, characterized in that The SPP1 gene or protein inhibitor is a reagent that specifically knocks out exons 2 to 8 of the SPP1 gene through gene editing technology; preferably, the gene editing technology is a conditional gene knockout technology; more preferably, the conditional gene knockout technology is a Cre-LoxP conditional gene knockout technology; Preferably, the nucleotide sequence of exons 2 to 8 is shown as SEQ ID NO.

1.

6. The use according to claim 2, characterized in that SPP1 gene or protein inhibitors as sensitizers have any of the following effects: 1) Inhibit the activity of SPP1 protein; 2) inhibit the expression of the gene encoding SPP1; 3) reduce the expression of MIF; 4) Inhibit the activity of the MIF signaling pathway; 5) Strengthen CD8 + T cell recruitment and tumor immunity activation.

7. The use according to claim 1 or 2, characterized in that The form of the sensitizer is selected from one or more of solid, liquid, gel, semi-fluid or aerosol.

8. The use according to claim 1 or 2, characterized in that The sensitizer is selected from one or more of drugs, health products or foods; preferably, when the sensitizer is a drug or a health product, the sensitizer further includes a pharmaceutically acceptable carrier or excipient.

9. A sensitizer for improving the radiotherapy effect of lung cancer, characterized in that: The effective substances in the sensitizer are SPP1 gene or protein inhibitors and pharmaceutically acceptable carriers or excipients.

10. The sensitizer according to claim 9, characterized in that The dosage form of the sensitizer is selected from any one or more of injection, oral solution, tablet, capsule, pill or spray.

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