Application of inhibiting expression of GPAA1 or CD24 for enhancing radiotherapy sensitivity and activating radiotherapy remote isolation effect and application of combined radiotherapy in tumor treatment

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to application of inhibiting expression of GPAA1 or CD24 for enhancing radiotherapy sensitivity and activating a radiotherapy remote isolation effect and application of combined radiotherapy in tumor treatment. The preparation for detecting the CD24 expression level is applied to detection of tumor immunocompetence, meanwhile, the application of inhibiting CD24 in assisting tumor radiotherapy treatment is provided, and mainly, the radiotherapy sensitivity is enhanced, and the radiotherapy remote isolation effect can be activated. In addition, the invention also provides an application of inhibiting GPAA1 in assisting tumor radiotherapy, which is mainly used for enhancing the radiotherapy sensitivity and activating the radiotherapy remote isolation effect. In the disclosure, ablation of GPAA1 or CD24 significantly enhances the local anti-tumor effect of radiotherapy, the deletion of CD24 can stimulate the remote isolation effect and inhibit the growth of non-irradiated tumors, a new perspective is provided for radiation-induced immune escape, and a substitution strategy for improving the curative effect of radiotherapy is proposed.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to the use of inhibiting the expression of GPAA1 or CD24 to enhance radiotherapy sensitivity and activate radiotherapy abscopal effect and combined radiotherapy in treating tumors. Background Art

[0002] Radiation therapy has a long-standing role in cancer treatment, with approximately 50% of cancer patients receiving radiation during their treatment. While its direct cytotoxicity is well characterized, emerging evidence highlights its role as an "in situ tumor vaccine." This immunostimulatory capacity stems from radiation-induced immunogenic cell death (ICD), which releases damage-associated molecular patterns (DAMPs) such as calreticulin (CRT; an "eat me" signal) and extracellular ATP (a "find me" signal). Antigen-presenting cells (APCs) phagocytose irradiated tumor cells, which then promote APC maturation and cross-presentation of tumor-associated antigens (TAAs), eliciting adaptive T cell responses. This cascade has the potential to induce systemic antitumor responses against non-irradiated tumor deposits, termed the abscopal effect. However, clinically significant abscopal effects remain extremely rare. To enhance systemic antitumor responses, studies have explored combining radiation with immune checkpoint blockade (ICB) targeting PD-1 / PDL1 or CTLA-4. However, these attempts have yielded limited clinical benefit, suggesting that immune evasion mechanisms beyond the T cell inhibitory pathway have not yet been addressed. One plausible explanation is insufficient antigen presentation, leading to insufficient T cell activation.

[0003] Antigen-presenting cells (APCs) serve as a critical bridge between innate and adaptive immunity. Among them, macrophages—a predominant immune population in solid tumors—perform dual antitumor functions: direct phagocytic elimination and antigen presentation. Thus, while macrophage responsiveness to radiation upregulates "eat me" and "find me" signals, triggering antitumor immune cascades, their dysfunction abrogates immune stimulation and promotes immune evasion following radiation. However, tumor cells employ multiple mechanisms to evade macrophage surveillance, most notably through "don't eat me" signals such as CD47, PD-L1, β2M, and CD24. While CD47 is a prototypical innate immune checkpoint, its ubiquitous expression on normal cells and limited efficacy in solid tumors limit its clinical application. CD24, a glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), has recently been described as a tumor-restricted "don't eat me" signal that interacts with the macrophage Siglec-10 receptor to inhibit phagocytosis. The paradoxical immune evasion after radiotherapy suggests that CD24 is a plausible target for radioimmunotherapy combinations and has prompted critical investigation of the potential regulation of CD24 by radiation. Elucidating the post-irradiation dynamics of this innate immune checkpoint may provide a rationale for combination therapy and hopefully reveal new therapeutic vulnerabilities.

[0004] Previously, we also comprehensively analyzed several related research reports and initially speculated that CD24 may indirectly affect the "abscopal effect" through local immunosuppression (such as inhibition of macrophage function). However, we could not confirm whether CD24 is the key factor in directly blocking the abscopal effect signaling pathway. We only know that CD24 is one of the "negative regulators" of the abscopal effect, not the sole determinant. Based on this, we cannot clearly determine whether CD24 hinders the "abscopal effect." In addition, during the study, we also found that knocking out CD24 alone had no effect on tumor suppression, indicating that CD24 may not directly affect the growth of tumor tissue. Summary of the Invention

[0005] The present invention provides the use of inhibiting the expression of GPAA1 or CD24 to enhance radiotherapy sensitivity and activate the abscopal effect of radiotherapy and combined with radiotherapy in treating tumors, mainly to solve the problems that there is currently no effective means to activate the abscopal effect of treatment and the means to enhance radiotherapy sensitivity need to be improved.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of CD24 in enhancing the therapeutic efficacy of tumors. CD24 is primarily used as a target for enhancing the therapeutic efficacy of tumors. As a target, it plays a role in the following aspects:

[0008] One of these involves the use of a preparation for detecting CD24 expression levels in the preparation of a product for detecting immune activity during tumor radiotherapy, TIME. CD24 expression levels are negatively correlated with TIME immune activity. Each condition is further selected as follows: any feature of any condition can be selected individually: 1) The preparation for detecting CD24 expression levels primarily involves detecting CD24 protein expression levels, which can be specifically implemented using existing technologies; 2) TIME immune activity represents an immune microenvironment characterized by immune activation. Increased CD24 expression indicates an activated immune microenvironment, which is more conducive to radiotherapy. In other words, increasing CD24 expression levels indicate a higher degree of immune microenvironment activation. In specific applications, CD24 expression levels can be used to screen cancer patients suitable for immunotherapy (the use of preparations for detecting CD24 expression levels in the preparation of a product for screening cancer patients suitable for immunotherapy). Lower CD24 expression levels indicate stronger TIME immune activity, making the patient more suitable for immunotherapy.

[0009] The second one is the use of a preparation for detecting CD24 expression levels in the preparation of a product for predicting overall survival rate after tumor radiotherapy or immunotherapy; wherein, low CD24 expression indicates a high overall survival rate after tumor radiotherapy. Each condition is further selected as follows: any feature of any condition can be selected separately: 1) The preparation for detecting CD24 expression levels is mainly a product for detecting CD24 protein expression levels, which can be specifically implemented using existing technologies; 2) The overall survival rate after tumor radiotherapy is the survival rate after tumor radiotherapy or immunotherapy, and when the CD24 expression level decreases, the survival rate after tumor radiotherapy or immunotherapy is high; 3) The expression level of CD24 can be used as a biomarker to predict the prognosis of immunotherapy (such as checkpoint inhibitors), providing an important basis for patient stratification for ICI therapy.

[0010] The third of these is the use of agents that inhibit CD24 expression in combination with radiotherapy for the treatment of tumors. This article mainly focuses on the combined use of agents that inhibit CD24 expression with radiotherapy for the treatment of tumors, which can be manifested by directly using agents that inhibit CD24 expression as adjuvant agents for radiotherapy. This study found that knocking out CD24 alone in tumor cells in vitro was unable to effectively inhibit tumor growth. Our research confirmed that knocking out CD24 can improve the immune microenvironment, and thus, when combined with radiotherapy, it significantly enhances the killing effect on tumor cells. Therefore, knocking out CD24 has a significant synergistic effect on radiotherapy. The specific manifestations can also be seen in the fourth and fifth items of the next two paragraphs.

[0011] The fourth of these is the use of a preparation that inhibits CD24 expression in the preparation of a product that enhances the abscopal effect of tumor radiotherapy. Each of the conditions is further selected as follows: any feature of any condition can be selected separately: 1) The preparation that inhibits CD24 expression is primarily a product that inhibits CD24 protein expression, which can be specifically implemented using existing technologies such as shRNA and siRNA; 2) Enhancing the abscopal effect of tumor radiotherapy involves both enhancing the abscopal effect of tumor radiotherapy and activating the abscopal effect of tumor radiotherapy. When CD24 expression is inhibited, the abscopal effect after tumor radiotherapy is activated, thereby enhancing the tumor radiotherapy effect.

[0012] The fifth of these is the use of preparations that inhibit CD24 expression in the preparation of products that enhance radiotherapy sensitivity. This article mainly applies to preparations that inhibit CD24 expression for the preparation of products that enhance radiotherapy sensitivity. Enhanced radiotherapy sensitivity can be manifested as accelerated tumor regression, shortened symptom relief time, accelerated oxygenation and immune response, and activation of the immune microenvironment.

[0013] With regard to the above, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer; more preferably, the non-small cell lung cancer is lung adenocarcinoma or lung squamous cell carcinoma.

[0014] A second aspect of the present invention provides the use of GPAA1 in enhancing the therapeutic effect of tumors. GPAA1 primarily serves as a target for enhancing the therapeutic effect of tumors. As a target, it primarily functions in the following ways:

[0015] One of them is the application of preparations that inhibit GPAA1 expression in combination with radiotherapy in the treatment of tumors; this article mainly focuses on the combination of preparations that inhibit GPAA1 expression and radiotherapy to treat tumors, and its manifestation can be the direct use of preparations that inhibit GPAA1 expression as radiotherapy adjuvant preparations.

[0016] The second of these is the use of agents that inhibit GPAA1 expression in the preparation of products that enhance radiosensitivity. This article mainly focuses on the use of agents that inhibit GPAA1 expression in products that enhance radiosensitivity. Enhanced radiosensitivity can be manifested as accelerated tumor regression, shortened symptom relief time, accelerated oxygenation and immune response, and activated immune microenvironment.

[0017] The third of these involves the use of an agent that inhibits GPAA1 expression in the preparation of a tumor cell model that low-expresses CD24. Each condition is further selected such that any feature of any condition can be selected individually: 1) a tumor cell model that low-expresses CD24, in which the CD24 expression level is low, thereby constructing a specific cell model to provide a biological model for specific research; 2) the tumor cell model that low-expresses CD24 is an in vitro irradiated tumor cell model; 3) the tumor cells are KPC cells (other similar cells) with GPAA1 knockout, and the agent that inhibits GPAA1 expression is shGPAA1 or siGPAA1.

[0018] Fourth, the use of agents that inhibit GPAA1 expression in the preparation of products that enhance the abscopal effect of tumor radiotherapy. This study has confirmed that CD24 KO amplifies the abscopal effect of radiotherapy and that GPAA1 is an important regulatory gene of CD24. Therefore, inhibiting GPAA1 can achieve similar effects as inhibiting CD24. Each condition is further selected as follows: any feature of any condition can be selected separately: 1) Agents that inhibit GPAA1 expression levels are mainly products that inhibit GPAA1 protein expression levels. This can be specifically achieved using existing technologies such as shRNA and siRNA; 2) Enhancing the abscopal effect of tumor radiotherapy involves both enhancing the abscopal effect of tumor radiotherapy and activating the abscopal effect of tumor radiotherapy. When GPAA1 expression is inhibited, the abscopal effect after tumor radiotherapy is activated, thereby enhancing the tumor radiotherapy effect.

[0019] Fifthly, the agent that inhibits GPAA1 expression can also be used in combination with the agent that inhibits CD24 expression for all of the above.

[0020] With regard to the above, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer; more preferably, the non-small cell lung cancer is lung adenocarcinoma or lung squamous cell carcinoma.

[0021] A third aspect of the present invention provides the use of a preparation for detecting the expression level of CD24 or GPAA1 in the preparation and analysis of a product for the prognosis of tumor radiotherapy; wherein the CD24 expression level is negatively correlated with the prognosis of tumor radiotherapy; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further is any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer; more preferably, the non-small cell lung cancer is lung adenocarcinoma or lung squamous cell carcinoma.

[0022] The fourth aspect of the present invention provides a preparation for detecting the expression level of CD24 or GPAA1 in the preparation and analysis of M1 macrophages, CD8 + T cells, CD8 +The invention relates to an application of the present invention in a product that can detect the infiltration level of any effector memory T cell; wherein the CD24 expression level is negatively correlated with the infiltration level and negatively correlated with the macrophage T cell subset; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further is any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer; more preferably, the non-small cell lung cancer is lung adenocarcinoma or lung squamous cell carcinoma.

[0023] Low expression, reduced expression, and inhibition are all relative to normal levels. When abnormal, the risk changes accordingly. A negative correlation indicates a corresponding change. Alternatively, if necessary, a normal range can be defined based on clinical needs, and the test results can be compared with normal values ​​to provide a reference for clinical work. This expression level can be assessed at both mRNA and protein levels. The aforementioned radiotherapy and immunotherapy are at least currently established methods, and further extensions should also be included, while remaining within the scope of radiotherapy and immunotherapy.

[0024] In the present disclosure, irradiated tumor cells upregulate CD24 surface expression through the ANAPC5 / GPAA1 axis, inducing phagocytic resistance and immune escape. Mechanistically, radiation inhibits the late promoting complex / cycle body (APC / C), reduces ANAPC5-mediated ubiquitination of GPAA1 at position 111 lysine (K111); as the catalytic subunit of glycosylphosphatidylinositol (GPI) transamidase, the subsequent accumulation of GPAA1 promotes GPI anchoring, thereby enhancing CD24 membrane localization. A variety of preclinical models have made it clear that ablation of GPAA1 or CD24 significantly enhances the local anti-tumor effect of radiotherapy (this effect depends on T cells and macrophages). It is worth noting that CD24 deficiency can stimulate remote effects and inhibit the growth of non-irradiated tumors. By clarifying the regulatory mechanism of radiotherapy on the innate immune checkpoint CD24, a new perspective is provided for radiation-induced immune escape, and an alternative strategy to improve the efficacy of radiotherapy is proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Despite the induction of immunogenic signals, radiation failed to enhance macrophage phagocytosis. a, Single-cell RNA sequencing (scRNA-seq) analysis of immunogenic markers on tumor cells from control or irradiated LLC tumors (n = 3). b, c, Flow cytometric histograms and mean fluorescence intensity (MFI) of CRT on control or irradiated H460 (b) and PANC-1 (c) cells (n = 3). d, Immunofluorescence images of CRT (red) on control or irradiated H460 cells. e, Flow cytometric histograms and MFI of CRT on tumor cells from control or irradiated LLC tumor models (n = 6). f, g, Medium HMGB1 levels measured by ELISA in control or irradiated H460 (f) and PANC-1 (g) cells (n = 3). h, i, Extracellular ATP release measured by ELISA in control or irradiated H460 (h) and PANC-1 (i) cells (n = 3). j, scRNA-seq-derived M1 signature scores of tumor-associated macrophages (TAMs) from irradiated or control LLC tumors (n = 3). k, Flow cytometry histograms and quantification of phagocytosis of control or irradiated H460 cells by THP-1 macrophages (n = 3). l, Immunofluorescence images and quantification of phagocytosis of control or irradiated H460 cells (green) by THP-1 macrophages (red), arrows indicate phagocytic events (n = 6). Scale bar, 50 μm. m, Flow cytometry dot plots and quantification of phagocytosis of tumor cells by TAMs from control or irradiated LLC tumors (n = 6). All data are presented as mean ± standard error and compared using a two-tailed Student's t-test. NS, not significant. Source data are provided as source data files.

[0026] Figure 2 Radiation enhances the tumor cell "don't eat me" signal CD24. a, Uniform manifold approximation and projection (UMAP) map of 10 pancreatic cancer clusters, NCBI Sequence Read Archive: GSE281288, with CD24 expression overlaid on the UMAP. b, Immunofluorescence images of CD24 (red) on control or irradiated H460 cells. Scale bars, 25 μm, 5 μm. c, d, Flow cytometric histograms and MFI of CD24 surface expression on control or irradiated H460 (c) and PANC-1 (d) cells (n = 3). e, CD24-MFI of tumor cells from a control or irradiated LLC tumor model (n = 6). f, Flow cytometric histograms and MFI of CD24 surface expression on H460 cells 48 hours after 2 to 18 Gy of radiation (n = 3). All data are presented as mean ± standard error. Two-sided Student's t-tests were performed for (ce). (F) was subjected to one-way ANOVA-Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0027] Figure 3 CD24 inhibition combined with radiation promotes macrophage phagocytosis and activation. a, b, Flow cytometric histograms of CD24 surface expression on H460 (a) and PANC-1 (b) cells transfected with CD24-targeting siRNA (siCD24), negative control siRNA (siNC), or isotype control. c, d, Flow cytometric histograms and quantification of phagocytosis of H460 (c) and PANC-1 (d) cells by THP-1 macrophages (n = 3). e, Immunofluorescence images and quantification of phagocytosis of H460 cells by THP-1 macrophages (red) in response to the indicated treatments (green); arrows indicate phagocytic events (n = 6). Scale bar, 50 μm. f, RT-PCR analysis of Siglec10 in THP-1 macrophages transfected with siSiglec10 or siNC (n = 3). g, Flow cytometric histograms and quantification of phagocytosis of irradiated H460 cells by THP-1 macrophages (n = 3). hj, Flow cytometry histograms and MFI of CD80 (h), CD86 (i), and PDL1 (j) in tumor cells cocultured with the indicated treatments on BMDM (n = 3). All data are presented as mean ± standard error. One-way ANOVA with Tukey's multiple comparison test was performed for (b, d, e, gj). Two-sided Student's t-test was performed for (f). NS, not significant. Source data are provided as source data file.

[0028] Figure 4 Radiation enhances CD24 membrane trafficking via GPI anchoring. a, b, Western blot analysis of CD24 in whole-cell lysates of control or irradiated H460 (a) and PANC-1 (b). c, Schematic diagram of GPI anchoring. d, Western blot analysis of CD24 in membrane and cytoplasmic lysates of control or irradiated H460 cells. e, Proteomic analysis heatmap of differentially expressed proteins in control or irradiated H1299 cells. f, g, Western blot analysis of GPAA1 in whole-cell lysates of control or irradiated H460 (f) and PANC-1 (g). h, Flow cytometric histogram of CD24 surface expression on H460 cells with the indicated treatments or isotype control (n = 3). i, j, Flow cytometric histograms and quantification of macrophage phagocytosis of H460 (i) and KPC (j) cells with the indicated treatments (n = 3). All data are presented as mean ± standard error and compared using one-way ANOVA with Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0029] Figure 5Irradiation disrupts APC / C-mediated GPAA1 ubiquitination at K111. a, Schematic diagram of the IP-MS method. b, GO enrichment analysis (Biological Process) of predicted GPAA1-interacting proteins from IP-MS. c, d, Western blot analysis of CHX chase assays in H460 cells treated with MG132 (c) or irradiation (d). e, Western blot analysis and IP evaluation of GPAA1 ubiquitination in the indicated treatment groups. f, Western blot analysis of GPAA1 in H460 cells treated with Apcin. g, Flow cytometry histogram of CD24 surface expression on H460 cells treated with Apcin or isotype control (n = 3). h, Schematic diagram of screening for GPAA1 binding to APC / C subunits. i, IP analysis of the GPAA1-ANAPC5 interaction. j, Western blot analysis followed by IP evaluation of GPAA1 ubiquitination in H460 cells transfected with siANAPC5 or siNC. k, Western blot analysis of ANAPC5 and GPAA1 in H460 cells transfected with siANAPC5 or siNC. l, Flow cytometry histogram of CD24 surface expression on H460 cells transfected with siANAPC5, siNC, or an isotype control (n = 3). m, Western blot analysis and IP assessed ubiquitination of GPAA1 in 293T cells transfected with the various mutants. All data are presented as mean ± standard error. One-way ANOVA with Tukey's multiple comparison test was performed for (g). Two-sided Student's t-test was performed for (k). Source data are provided as source data file.

[0030] Figure 6To target CD24 to sensitize tumors to radiotherapy and induce abscopal effects in vivo. a, Schematic diagram of radiotherapy planning in animal models. Tumor growth curves (b) and Kaplan-Meier survival plots (c) of the Hepa1-6 subcutaneous tumor model with the indicated treatment were created using BioRender.com.b, c (n=8). d, e, Tumor growth curves (d) and tumor weights (e) of the KPC subcutaneous tumor model with the indicated treatment (n=6). f, Immunohistochemistry images and quantification of apoptotic cells in tumor tissue (n=6). g, h, Tumor growth curves (g) and Kaplan-Meier survival plots (h) of the KPC subcutaneous tumor model with the indicated treatment (n=8). i, Schematic diagram of abscopal effect assessment in animal models. Orthotopic and abscopal effect tumor growth curves (j) and Kaplan-Meier survival plots (k) of the KPC subcutaneous tumor model described in (i) were created using BioRender.com.j (n=6). All data are expressed as mean ± standard error. Two-way ANOVA with Tukey's multiple comparison test was performed for (b, d, g, j). One-way ANOVA with Tukey's multiple comparison test was performed for (e, f). Log-rank test was performed for (c, h, k). NS, not significant. Source data are provided as source data file.

[0031] Figure 7 Macrophages and T cells are required for CD24i-RT combination therapy. ag, Flow cytometric analysis of macrophage and T cell subsets in KPC tumor tissues (n=6). hk, Immunofluorescence images and activated TAMs (h, i) and CD8 + Quantification of T cells (j, k) (n = 6). Scale bar, 100 μm. l, Schematic diagram of the immune cell depletion scheme in the animal model. Created using BioRender.com.mo, tumor growth curves (m), tumor images (n), and tumor weights (o) of the KPC subcutaneous tumor model with the indicated treatments (n = 6). All data are presented as mean ± standard error. One-way ANOVA with Tukey's multiple comparison test was performed for (ag, i, k, o). Two-way ANOVA with Tukey's multiple comparison test was performed for (m). NS, not significant. Source data are provided as source data files.

[0032] Figure 8Low CD24 expression is associated with increased immune cell infiltration and prolonged survival across cancer types. ae, Immunofluorescence images (a) and correlation analysis of CD24 mean fluorescence intensity (AFI) with CD8 (b), CD68 (c), CD86 (d), and PDL1 (e) expression in independent HGSOC arrays (n = 44). Scale bars, 200 μm, 50 μm. fg, CD24 expression levels in LUSCs correlate with M1 macrophages (f), CD8 + T cells (g) and CD8 + Correlation analysis of infiltration levels of effector memory T cells (h). Graphed by TIMER 2.0. i, Overall survival of PRAD patients (n=77) receiving radiotherapy stratified by CD24 expression level based on the TCGA database. jk, Overall survival of LIHC patients (n=370) stratified by CD24 (j) or GPAA1 (k) expression level based on the Pan-Cancer Database of Kaplan-Meier plotter. lm, Overall survival of patients with non-small cell lung cancer (n=1044) stratified by CD24 (l) or GPAA1 (m) expression level based on the SurveExpress lung meta-repository. Pearson correlation test was performed for (be). R squared, coefficient of determination. Log-rank test was performed for (im). Hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated using the Cox proportional hazards regression model. Source data are provided as source data files.

[0033] Figure 9 Irradiation alone does not enhance phagocytosis. a, b, Flow cytometric histograms and MFI of CRT on control or irradiated LLC (a) and KPC (b) cells (n = 3). c, Flow cytometric gating strategy for in vivo CRT analysis. d, e, HMGB1 levels in the culture medium of control or irradiated LLC (d) and KPC (e) cells measured by ELISA (n = 3). f, g, Extracellular ATP release from control or irradiated LLC (f) and KPC (g) cells measured by ELISA (n = 3). h, Flow cytometric dot plots and quantification of phagocytosis of control or irradiated LLC cells by BMDM (n = 3). i, Immunofluorescence images and quantification of phagocytosis of control or irradiated LLC cells (green) by BMDM (red); arrows indicate phagocytic events (n = 6). j, Flow cytometric gating strategy for in vivo phagocytosis assay. All data are presented as mean ± standard error and compared using a two-tailed Student's t test. NS, not significant. The source data is provided as source data files.

[0034] Figure 10Radiation enhances the "don't eat me" signal CD24 on tumor cells. a, Uniform manifold approximation and projection (UMAP) maps of pancreatic cancers from three patients with primary advanced cancer, showing cells stained by cluster identity. NCBI Sequence Read Archive: GSE281288. b, c, d, CD47 (b), PDL1 (c), and B2M (d) expression overlaid on the UMAP. eh, Flow cytometry histograms and MFI of CD24 surface expression on control or irradiated H1299 (e), BxPC-3 (f), LLC (g), and KPC (h) cells (n=3). i, Flow cytometric gating strategy for CD24 in vivo. j, Flow cytometric histograms and MFI of CD24 surface expression on KPC cells 48 hours after 2 to 18 Gy irradiation (n=3). k, Flow cytometric histograms and MFI of CD24 surface expression on KPC cells 96 hours after 10 Gy irradiation (n=3). All data are presented as mean ± standard error. Two-sided Student's t-test was performed for (eh, k). One-way ANOVA-Tukey's multiple comparison test was performed for (j). NS, not significant. Source data are provided as source data file.

[0035] Figure 11 CD24 knockout combined with irradiation promotes macrophage uptake. a, b, c, Flow cytometric histograms of CD24 surface expression on CD24-knockout LLC (a), KPC (b), and Hepa1-6 (c) cell lines (sgCD24), negative control cell lines (sgVector), or isotype controls. d, e, f, Flow cytometric dot plots and quantification of BMDM phagocytosis of LLC (d), KPC (e), and Hepa1-6 (f) cells with the indicated treatments (n = 3). g, Immunofluorescence images and quantification of LLC cells phagocytosed by BMDM with the indicated treatments (green); arrows indicate phagocytic events (n = 6). All data are presented as mean ± standard error and compared using one-way ANOVA with Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0036] Figure 12Radiation-enhanced GPAA1 promotes CD24 membrane trafficking. a, b, Western blot analysis of CD24 in whole-cell lysates of control or irradiated H1299 (a) and BxPC-3 (b). c, RT-qPCR analysis of CD24 in control or irradiated H460, H1299, PANC-1, and BxPC-3 cells. d, Plasmid maps for overexpression of CD24 and a C-terminal Flag tag. e, Western blot analysis of CD24 in H460 cells treated with the indicated treatments. f, g, Western blot analysis of GPAA1 in whole-cell lysates of control or irradiated H1299 (f) and BxPC-3 (g). h, Western blot analysis of GPAA1 and CD24 in H460 cells transfected with siGPAA1 or siNC. i, Flow cytometry histogram of CD73 surface expression on H460 cells with the indicated treatments or isotype control (n = 3). All data are presented as mean ± standard error. (A) Two-sided Student's t-test was performed. (I) One-way ANOVA-Tukey multiple comparison test was performed. NS, not significant. Source data are provided as source data file.

[0037] Figure 13 Irradiation inhibits APC / C-mediated GPAA1 ubiquitination. a, RT-qPCR analysis of GPAA1 in control or irradiated H460, H1299, PANC-1, and BxPC-3 cells. b, GO enrichment analysis of GPAA1-interacting proteins predicted from IP-MS analysis (cellular components). c, Western blot analysis and IP assessment of GPAA1 ubiquitination in H460 cells treated with Apcin. d, Flow cytometric histogram of CD73 surface expression on H460 cells treated with Apcin or an isotype control (n = 3). e, Flow cytometric histogram of CD73 surface expression on H460 cells transfected with siANAPC5, siNC, or an isotype control (n = 3). f, Western blot analysis of GPAA1 in irradiated H460 cells. All data are presented as mean ± standard error. Two-sided Student's t-test was performed for (a, e). (C) was subjected to one-way ANOVA-Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0038] Figure 14 GPAA1 prefers to bind to the ANAPC5 subunit of the APC / C. a, Protein-protein docking structural model of the GPAA1-APC / C subunit binding. b, Electrostatic surface analysis of the GPAA1-APC / C subunit binding. c, Complex structure prediction and Interchain PredictionTM (ipTM) score for the GPAA1-APC / C subunit binding.

[0039] Figure 15 CD24 knockout has limited efficacy in CD24-low tumor models. a, MFI of CD24 surface expression across cell lines. b, c, Tumor growth curves (b) and Kaplan-Meier survival plots (c) for LLC subcutaneous tumor models treated with the indicated treatments (n = 8). All data are presented as mean ± standard error. ANOVA with Tukey's multiple comparison test was performed for (b). Log-rank test was performed for (c). NS, not significant. Source data are provided as source data files.

[0040] Figure 16 GPAA1 inhibition combined with radiation promotes macrophage uptake and is abrogated by CD24 overexpression. a, Flow cytometric histograms and MFI of CD24 surface expression on KPC cells treated with the indicated treatments or isotype control (n=3). b, Flow cytometric dot plots and quantification of macrophage phagocytosis of KPC cells treated with the indicated treatments (n=3). All data are presented as mean ± standard error and compared using one-way ANOVA with Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0041] Figure 17 Targeting CD24 does not sensitize radiotherapy in vitro. Cell survival of ac, LLC (a), Hepa1-6 (b), and KPC (c) sgCD24 or sgVector cell lines was assessed at different radiation doses. All data are presented as mean ± standard error and compared using one-way ANOVA with Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0042] Figure 18 Activation of macrophages and T cells for CD24 combined radiation therapy. a, Flow cytometric gating strategy for detecting macrophages and T cells in KPC subcutaneous tumors. bd, Flow cytometric analysis of CD4 T cells in KPC tumor tissues after indicated treatment. + T cell subsets (n=6). e, f, Macrophages (e) and CD8 T cells in KPC tumor tissues under the indicated treatments + Quantification of T cells (f) (n = 6). Scale bar, 100 μm. All data are presented as mean ± standard error and compared using one-way ANOVA with Tukey's multiple comparison test. NS, not significant. Source data are provided as source data file.

[0043] Figure 19 To verify the efficiency of immune cell depletion, Figure 7Related. a, b, Flow cytometric analysis of immune cells in peripheral blood and spleen of mice treated with the indicated immune cell depletion therapies (n = 3). All data are presented as mean ± standard error and compared using a two-tailed Student's t-test. NS, not significant. Source data are provided as source data files. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to specific research examples.

[0045] 1. Experimental Methods

[0046] Cell lines and culture: All cell lines were purchased from the American Type Culture Collection. NCI-H460, NCI-H1299, and THP-1 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. PANC-1, BxPC-3, KPC, Hepa1-6, and LLC were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. All cell lines were maintained in a humidified incubator at 37°C with 5% CO2 and routinely tested for the presence of mycoplasma.

[0047] Radiation: For external irradiation, cells were exposed to the indicated doses using a linear accelerator (6 MV beam; 6 Gy / min). For internal radiotherapy, the tumor was locally irradiated with 8 Gy (6 MV beam; 6 Gy / min). Radiation dosimetry was verified in situ using a thermoluminescent dosimeter (TLD).

[0048] Animal model and efficacy evaluation: C57BL / 6J mice aged between 6 and 8 weeks were purchased from SLB Laboratory Animal Company (Hunan, China). All animals were housed in individually ventilated cages (IVC) at the Cancer Center of Wuhan Union Hospital. All animal studies were performed in accordance with protocols approved by the Hubei Provincial Animal Care and Use Committee and in compliance with the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology (Wuhan, China). Before all surgeries, mice were anesthetized with 1% sodium pentobarbital. Tumor cells (1 × 10 6 / 100μL) to establish a tumor model. Mice were randomly divided into different groups. When the average tumor volume reached 50-100mm 3 The mice in the radiotherapy group received 8 Gy × 3 doses of radiotherapy. The length (L) and width (W) of the tumor were measured every two days, and the tumor volume (V) was calculated according to V = (L × W2) / 2. At the end point (tumor volume ≥ 1000 mm 3 )Euthanize the mice.

[0049] Single-cell RNA sequencing analysis: scRNA-seq data from three primary pancreatic tumor samples were obtained from GSE131907. To control data quality, cells with more than 20% mitochondrial gene expression, unique molecular identifier (UMI) counts outside the 100-150,000 range, or gene counts exceeding 200-10,000 were excluded. The UMI counts for each cell were then log-normalized to transcripts per million (TPM) for data normalization. During feature selection, genes detected in less than 0.1% of cells in each batch were filtered. For dimensionality reduction and clustering, genes with a quantile-normalized variance greater than 0.5 and a mean expression between 0.0125 and 3 were selected for principal component (PC) analysis. Cell clustering was performed using Seurat v2.3.4, and significant PCs were identified. Cell clusters were visualized using UMAP and annotated based on canonical marker expression. Expression patterns of predefined genes of interest in annotated cell subpopulations were displayed on the UMAP plots. To quantify the expression differences of each target gene between cell clusters, the Wilcoxon rank-sum test was applied. P values ​​were adjusted for multiple testing using the Benjamini-Hochberg method, and an adjusted P value of less than 0.05 was defined as statistically significant. All analyses were performed in R v4.5.

[0050] Immunofluorescence staining: Cells were seeded in 24-well plates containing cell slides. After the indicated treatments, slides were fixed with 4% paraformaldehyde for 30 minutes at 4°C, washed three times with 1× PBS, and then blocked with 10% bovine serum albumin for 1 hour at room temperature. Slides were then incubated with primary antibodies overnight at 4°C, followed by incubation with fluorescently conjugated secondary antibodies for 1 hour at 4°C. Stained cells were visualized using a laser scanning confocal microscope.

[0051] In vitro phagocytosis assay: For flow cytometry-based phagocytosis assays, CFSE-labeled tumor cells (target cells) and macrophages (effector cells) were co-cultured at a 1:1 ratio in a humidified incubator at 37°C with 5% CO2. After 4 hours of co-culture, cells were harvested and stained with F4 / 80 or CD11b. Phagocytosis was defined as F4 / 80 + or CD11b + F4 / 80 in macrophages + CFSE + or CD11b + CFSE + For immunofluorescence-based phagocytosis assays, GFP-expressing tumor cells were used as target cells. Phagocytosis was defined as the percentage of F4 / 80 + or CD11b + Co-localization of macrophages (red) and GPF-expressing tumors (green).

[0052] In vivo phagocytosis assay: A subcutaneous tumor model was established using GFP-expressing tumor cells. After the indicated treatments, tumors were harvested and digested with collagenase and hyaluronidase for 1 hour at 37°C, followed by erythrocyte lysis to obtain single-cell suspensions. Cells were stained with a Zombie Violet fixative viability kit and then stained with CD45, CD11b, and F4 / 80 antibodies for 30 minutes at 4°C according to the manufacturer's concentration. Phagocytosis was assessed by flow cytometry and defined as CD11b + f / 4 / 80 + GFP + CD11b + V4 / 80 + The percentage of macrophages.

[0053] Real-time quantitative polymerase chain reaction (RT-qPCR): Total RNA was extracted using TRIzol reagent and quantified using a Thermo NanoDrop ND-1000. One microgram of total RNA was reverse-transcribed into cDNA using HiScript III RT SuperMix according to the manufacturer's protocol. RT-qPCR was performed on a StepOnePlus system using ChamQ SYBR qPCR Master Mix. β-actin was used to normalize gene expression.

[0054] Western Blot and Co-immunoprecipitation: Total protein was extracted using RIPA buffer containing 1% protease and phosphatase inhibitors. TM Membrane proteins were extracted using the ELISA Plus kit. Proteins were then quantified using a bicinchoninic acid detection kit. For western blot analysis, proteins were boiled at 100°C for 10 minutes in 5x loading buffer, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature, incubated with the primary antibody overnight at 4°C, washed with TBST, and then incubated with an HRP-conjugated secondary antibody for 1 hour at room temperature. Signals were visualized using ECL reagents. For co-immunoprecipitation experiments, cells were lysed with IP lysis solution containing 1% protease and phosphatase inhibitors. The lysate was incubated with protein A / G agarose beads and incubated overnight at 4°C. The beads were washed five times with NETN buffer, then eluted in 1x loading buffer and boiled at 100°C for 10 minutes. The samples were used for subsequent western blot analysis.

[0055] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis: Cells were lysed with IP lysis buffer. Lysates were incubated with protein A / G agarose beads and anti-GPAA1 antibody or IgG control overnight at 4°C. LC-MS / MS analysis was performed using a ThermoScientific Ultimate 3000RSLC system and a Q Exactive Plus high-resolution mass spectrometer provided by SpecAlly Life Technology Co., Ltd. (Wuhan, China). Data were searched using MaxQuant software and the Andromeda algorithm. The UniProt human proteome database was used as a reference, and proteins and peptides were filtered using a false discovery rate (FDR) of 1%.

[0056] Computational structural analysis using AlphaFold3: The APC / C structure is publicly available in the Protein Data Bank (PDB) with the identifier PDB ID: 4UI9. GPAA1 has been structurally characterized and is documented in PDB ID: 7W72. For pairwise protein-protein docking using ClusPro, 10 conformations were generated (GPAA1 as the receptor protein and APC / C as the ligand protein), which were then screened based on interface characteristics, and the most promising conformations were selected for further analysis. All selected docking configurations were evaluated for geometric plausibility and spatial accessibility. CDC16, CDC27, ANAPC7, ANAPC2, and ANAPC5 were identified as promising binding subunits. For electrostatic surface analysis, the electrostatic properties of the interaction interfaces were analyzed using PyMOL and the APBS plugin. The interaction interfaces between GPAA1 and CDC16, ANAPC7, and ANAPC5 exhibited good electrostatic complementarity. For structure prediction, AlphaFold3 was used to further predict the complex structures of GPAA1 and the CDC16, ANAPC7, and ANAPC5 subunits. Each prediction task was performed using 10 independent runs initialized with different random seeds to enhance conformational diversity. All predicted conformations were evaluated for geometric plausibility and spatial accessibility, and the best conformations were selected for further comparison. The Interchain PredictionTM score (ipTM) metric from AlphaFold3 was used to estimate the global structural similarity between the predicted interface and the true interface of two protein chains. ANAPC5 had the highest interface confidence score and the most accurate single-chain prediction.

[0057] Flow cytometric analysis of the tumor immune landscape: Tumors were harvested and digested with collagenase and hyaluronidase for 1 hour at 37°C, followed by erythrocyte lysis to obtain single-cell suspensions. To exclude dead cells, single-cell suspensions were stained with a Zombie Violet fixable viability kit. To analyze macrophages, cells were stained with antibodies against CD45, CD11b, F4 / 80, CD80, and CD86 for 30 minutes at 4°C, according to the manufacturer's concentrations. To analyze T cells, a portion of the cell suspension was first stimulated with PMA (100 ng / mL), ionomycin (100 ng / mL), and monensin (1 μg / mL) for 5 hours at 37°C with 5% CO2, and then stained with antibodies against CD45, CD3, CD4, and CD8. Subsequently, cells were fixed and permeabilized, and then stained with antibodies against IFN-γ, Grzmb, and FoxP3 for 30 minutes at 4°C, according to the manufacturer's concentrations.

[0058] Macrophage and T cell depletion: For macrophage depletion studies, clodronate liposomes were injected intraperitoneally at a dose of 150 ml per mouse every 4 days. + For T cell depletion studies, mouse anti-CD8 antibody diluted in PBS was injected intraperitoneally every 4 days at a dose of 100 mg per mouse. Flow cytometry was used to verify the depletion effect.

[0059] Statistical analysis: GraphPad Prism 10.0 was used for statistical analysis. All quantitative data are expressed as mean ± standard error. Differences between two or more groups were analyzed using a two-sided Student's t-test or a one-way analysis of variance with Tukey's multiple comparison test. Tumor growth curves were analyzed using a two-way analysis of variance with Tukey's multiple comparison test. Kaplan-Meier survival analysis was performed using the log-rank test. Pearson's test was used to assess correlations. P < 0.05 was considered statistically significant.

[0060] Although radiation induces immunogenic signals, it fails to enhance macrophage phagocytosis: To systematically investigate the mechanisms of immune escape after radiation, we analyzed single-cell RNA sequencing (scRNA-seq) data from a Lewis lung carcinoma (LLC) subcutaneous tumor model in mice treated with radiation (IR, 8 Gy × 3) and control (Ctrl). Tumor cell clusters showed significant enrichment of transcripts associated with "eat me" and "find me" signaling, ICD, and antigen processing pathways ( Figure 1 These signals are crucial for recruiting immune effectors such as macrophages to recognize and eliminate damaged cells. Cross-tumor validation confirmed that CRT (a typical pro-phagocytic cell signal) is upregulated in lung and pancreatic cancer cells from humans and mice after radiation exposure ( Figure 1Middle bd and Figure 9 Flow cytometric analysis of CRT in a mouse model further confirmed these in vivo findings ( Figure 1 Zhongehe Figure 9 Meanwhile, radiation-induced increases in HMGB1 and ATP release act as pro-inflammatory DAMPs and “find me” signals in multiple tumor cell lines ( Figure 1 Zhongfi and Figure 9 dg). Collectively, these results suggest that radiotherapy has the potential to initiate remodeling of the tumor immune microenvironment (TIME). Next, we explored whether immune cells could effectively respond to these radiation-induced immunogenic signals. Macrophage-mediated tumor cell phagocytosis and antigen presentation are central to activating antitumor immunity. However, gene set variation analysis (GSVA) ​​of macrophage clusters based on scRNA-seq data showed no significant differences in the antitumor M1 phenotypic signature score after irradiation ( Figure 1 To assess the functional status of macrophages, we co-cultured human THP-1 monocyte-derived macrophages with CFSE-labeled H460 cells. Flow cytometry and immunofluorescence-based quantitative analysis showed that phagocytic clearance of irradiated tumor cells was comparable to that of control tumor cells ( Figure 1 Consistent results were obtained using mouse bone marrow-derived macrophages (BMDM) phagocytosis assays ( Figure 9 Based on in vitro observations, in vivo tumor cell uptake remained equivalent between control and irradiated tumor cells ( Figure 1 Chinese Figure 9 These findings suggest that although radiation induces tumor-intrinsic immunogenic signals, it does not enhance macrophage phagocytosis. Macrophage dysfunction implies activation of counteracting mechanisms after radiation, hindering overall therapeutic efficacy.

[0061] Radiation enhances the "don't eat me" signal CD24 on tumor cells: While intrinsic "eat me" signals contribute to immune surveillance, cancer cells often overexpress "don't eat me" signals to evade macrophage-mediated clearance. To identify clinically relevant innate immune checkpoints, we interrogated the cellular expression profile of "don't eat me" signals using a human cancer scRNA-seq dataset ( Figure 2 This systematic comparison revealed that CD24 is the primary therapeutic target because it has higher tumor specificity than CD47, PD-L1, or B2M ( Figure 2 A and Figure 10 Next, we assessed the surface levels of CD24 in human and mouse tumor cell lines. Immunofluorescence and flow cytometry analysis showed that surface CD24 was significantly upregulated 24 hours after irradiation ( Figure 2 c, d and Figure 10 Consistent with in vitro observations, in vivo flow cytometric analysis of irradiated LLC subcutaneous tumors demonstrated elevated surface CD24 levels compared with non-irradiated controls ( Figure 2 Zhongehe Figure 10 Time course and dose response experiments further confirmed that CD24 upregulation was radiation dose-dependent and persisted for at least four days after irradiation ( Figure 2 Medium f and Figure 10 These results redefine CD24 as a radiation-responsive innate immune checkpoint, where enhanced CD24 signaling after irradiation limits antitumor clearance by the innate immune system, thereby impairing the efficacy of radiotherapy.

[0062] Targeting CD24 combined with radiation promotes macrophage phagocytosis and activation: To address the paradoxical immune escape mediated by CD24 after radiation, we first used siRNA-mediated CD24 knockdown in H460 and PANC-1 cells, and flow cytometry confirmed that the silencing efficiency was over 80% ( Figure 3 Flow cytometry and immunofluorescence-based phagocytosis assays demonstrated that CD24 inhibition increased macrophage-mediated tumor cell uptake, and combined CD24 silencing and irradiation further enhanced phagocytic clearance compared with either treatment alone ( Figure 3 To confirm these findings, we generated stable CD24 knockout (CD24-KO) cell lines in Lewis lung carcinoma (LLC), pancreatic ductal adenocarcinoma (KPC), and hepatocellular carcinoma (Hepa1-6) in mice using CRISPR-Cas9 ( Figure 11 ac). CD24 knockout combined with radiation significantly enhanced BMDM-mediated phagocytosis ( Figure 11 Silencing the antiphagocytic receptor sialic acid-binding Ig-like lectin 10 (Siglec-10) in THP-1-derived macrophages enhanced the uptake of irradiated tumor cells. However, this enhancement was not further enhanced when combined with CD24 inhibition, indicating that radiation-upregulated CD24 signaling also acts through the canonical CD24-Siglec-10 axis ( Figure 3 In addition to phagocytosis, macrophages play a key role in antigen presentation and T cell activation. Next, we investigated the expression of co-stimulatory molecules on BMDM using a phagocytosis assay. BMDM co-cultured with irradiated CD24 KO tumor cells showed significantly increased expression of CD80, CD86, and PDL1, indicating enhanced maturation and activation ( Figure 3Collectively, these findings suggest that targeting CD24 combined with radiotherapy can reverse phagocytic resistance and promote the functional maturation and activation of macrophages through the canonical CD24–Siglec-10 interaction pathway.

[0063] Radiation enhances CD24 membrane trafficking via GPI anchoring: To characterize CD24 dynamics after radiation, we first assessed total CD24 protein and transcript levels after radiation. Interestingly, Western blot and RT-qPCR analysis failed to detect alterations in CD24 expression ( Figure 4 a, b and Figure 12 This suggests that radiation enhances membrane trafficking of CD24 rather than its biosynthesis. CD24 is a highly glycosylated protein that requires post-translational glycosylation for proper subcellular localization and functional activity. The pre-protein of GPIAP has a C-terminal signal peptide that can be recognized, cleaved, and replaced with a pre-assembled GPI by a GPI transamidase, thereby generating a nascent GPIAP ( Figure 4 These newly formed nascent GPIAPs then undergo a series of remodeling processes before being transported to and anchored on lipid rafts within the cell membrane. To explore the GPI anchoring that is crucial for CD24 membrane trafficking and to elucidate the mechanism behind radiation-induced CD24 regulation, we established an H460 cell line that stably overexpresses C-terminally Flag-tagged CD24 (H460 OE). Figure 12 Western blot analysis of isolated membrane and cytoplasmic fractions revealed that irradiation increased the abundance of membrane GPI-anchored CD24, as detected by anti-CD24 antibody, while decreasing the levels of cytoplasmic uncleaved CD24 proprotein, as detected by anti-Flag antibody ( Figure 4 (middle d). These findings suggest that GPI-dependent membrane localization of CD24 is enhanced. We further screened candidates including GPI transamidase subunits and other reported CD24 interactors to identify regulatory factors that control radiation-induced CD24 surface accumulation. GPAA1, a key subunit of GPI transamidase responsible for catalyzing the amide bond formation between the proprotein and GPI-ethanolamine (EtN of GPI), showed significant upregulation after irradiation ( Figure 4 Western blot confirmed the elevation of GPAA1 protein in different tumor types ( Figure 4 f, g and Figure 12 f, g). siRNA-mediated knockdown of GPAA1 reduced the surface enrichment of GPI-anchored proteins, including CD24 and CD73, but did not alter their total protein expression. Irradiation treatment restored the surface levels of GPI-anchored proteins, reaffirming the key role of GPAA1 in radiation-enhanced GPI anchoring ( Figure 4 Zhong h and Figure 12 Functionally, genetic inhibition of GPAA1 recapitulated the phenotypic effects of CD24 ablation and resulted in enhanced phagocytic uptake when combined with radiation therapy (RT). Figure 4 These results establish that GPAA1-mediated GPI anchoring is a molecular bridge connecting radiation exposure to CD24-dependent phagocytic resistance.

[0064] Radiation impairs APC / C-mediated ubiquitination of GPAA1 at lysine 111: Based on our identification of GPAA1 as a key regulator of GPI-anchored CD24, we next sought to dissect the molecular mechanism of GPAA1 accumulation after radiation. To this end, we used immunoprecipitation coupled with mass spectrometry (IP-MS) to analyze the GPAA1 interacting proteins ( Figure 5 (a) Gene ontology (GO) enrichment analysis of candidate interactors showed that the protein K11-linked ubiquitination pathway was significantly enriched, suggesting that GPAA1 is post-translationally regulated through the ubiquitin-proteasome system ( Figure 5 Cyclohexylamine (CHX) chase analysis showed that the half-life of GPAA1 was prolonged after proteasome inhibition (MG132 treatment), confirming the ubiquitin-dependent degradation of GPAA1 ( Figure 5 (C). Irradiation did not alter GPAA1 transcription ( Figure 13 Conversely, it abolishes the effects of CHX-mediated transcriptional arrest by reducing GPAA1 ubiquitination ( Figure 5 d), thereby stabilizing the protein ( Figure 5 In-depth analysis of the IP-MS dataset revealed that 11 of the 41 candidate proteins are components of the posterior phase facilitating complex / cyclosome (APC / C), a multi-subunit E3 ubiquitin ligase complex, indicating that APC / C is the primary E3 ligase controlling GPAA1 ubiquitination ( Figure 13 Inhibition of APC / C E3 ligase activity (Apcin treatment) reproduced the radiation-induced effects, resulting in a decrease in GPAA1 ubiquitination levels ( Figure 13 c), GPAA1 protein levels increased ( Figure 5 f), and promoted the membrane localization of GPI-anchored proteins ( Figure 5 Medium g and Figure 13 To pinpoint direct interactors of GPAA1, we combined IP-MS data with computational analyses, including protein docking, electrostatic surface analysis, and AlphaFold3 multimeric structure predictions. Through this integrated approach, we systematically refined our initial pool of 11 candidate subunits and identified ANAPC5 as the primary binding partner of GPAA1 ( Figure 5 Zhong h and Figure 14 Endogenous co-IP experiments support the ANAPC5-GPAA1 interaction ( Figure 5 Middle i). siRNA-mediated knockdown of ANAPC5 reduced GPAA1 ubiquitination ( Figure 5 j), stabilized GPAAl( Figure 5 k), thereby enhancing the transport of GPI-APs to the membrane ( Figure 5 Neutral Figure 13 Notably, ANAPC5 protein levels decreased rapidly after radiation ( Figure 13 f), indicating that radiation mediates the accumulation of GPAA1 through APC / C disruption. AlphaFold3 structural predictions locate three lysine residues (K82, K83, and K111) as potential ubiquitination sites on GPAA1. To verify their functional relevance, we performed exogenous IP with site-specific mutants of GPAA1 (K82R, K83R, and K111R) and identified K111 as a key ubiquitination site ( Figure 5 Collectively, these findings delineate a radiation-responsive ubiquitination cascade whereby radiation impedes ANAPC5-mediated ubiquitination of GPAA1 at K111, leading to enhanced CD24 membrane localization.

[0065] Targeting CD24 enhances local and abscopal tumor control with radiotherapy: Having elucidated the dynamics of CD24 after irradiation, we focused on exploiting this radiation-responsive immune checkpoint to address immune evasion after radiotherapy. Flow cytometric analysis demonstrated that CD24 is overexpressed in Hepa1-6 and KPC cell lines ( Figure 15 Therefore, we used CD24 - Knockout or control tumor cells were subcutaneously implanted into C57BL / 6J mice to establish mouse hepatocellular carcinoma and cancer tumor models, and then the cells were cultured when the tumor volume reached 50-100 mm. 3 Then radiotherapy (8Gy×3) ( Figure 6 In the CD24-high Hepa1-6 tumor model, CD24 knockout alone delayed tumor progression, and its combination with radiotherapy achieved significant tumor growth inhibition and prolonged survival ( Figure 6 Consistent therapeutic synergy was observed in the KPC tumor model, with CD24 deficiency enhancing radiation-induced tumor regression and reducing late-stage tumor burden ( Figure 6 Immunohistochemistry revealed that the proportion of apoptotic cells was highest in radioactive CD24 knockout tumors, demonstrating the therapeutic efficacy of this combination in immunologically "cold" pancreatic cancer ( Figure 6Middle f). CD24 knockout alone was beneficial in high-expressing tumors, while there was no significant difference in low-expressing tumors. All models showed synergistic sensitization effects after combined radiotherapy ( Figure 15 Given the key role of GPAA1 in regulating CD24 membrane trafficking after radiation, we constructed a GPAA1 knockout (shGPAA1) KPC cell model for subsequent functional experiments. GPAA1 inhibition significantly reduced CD24 levels and overcame the in vitro phagocytic resistance effect after radiation, while exogenous CD24 overexpression (shGPAA1+oeCD24) reversed these effects ( Figure 16 Further in vivo efficacy evaluation showed that GPAA1 knockout alone did not inhibit tumor growth, but its combination with radiotherapy significantly inhibited tumor progression and prolonged survival. This synergistic effect was also abolished by CD24 overexpression ( Figure 6 g, h). The above results jointly verified in vitro and in vivo the key mechanism by which GPAA1 mediates post-irradiation phagocytic resistance by regulating CD24, highlighting the therapeutic potential of targeting GPAA1 to block CD24 signaling. Clinical reports have confirmed the existence of an "abscopal effect" in radiotherapy, that is, local irradiation of a tumor lesion can induce an anti-tumor response in distant non-irradiated lesions. To further explore the efficacy of combined treatment of CD24 interference (CD24i) and radiotherapy (RT) on distal tumors, we constructed the following experimental model: After the primary tumor was inoculated on the left ventral side of mice, they were divided into three groups for treatment - irradiation group (receiving local radiotherapy), sgCD24 group (CD24 gene knockout by CRISPR) and sgVector group (empty vector control). One week later, a secondary tumor was implanted on the right ventral side (not irradiated, sgVector group) ( Figure 6 The results showed that only the CD24-deficient primary tumor group showed delayed growth of non-irradiated secondary lesions ( Figure 6 (j, k) This indicates that combined CD24i and radiotherapy can induce an abscopal effect and achieve regression of distant metastases. Radiotherapy only exhibits a significant abscopal effect after in situ inhibition of CD24, and CD24 inhibition has no beneficial effect on the suppression of non-irradiated tumors.

[0066] Enhanced macrophage and T cell responses orchestrate CD24i RT synergy: Based on our previous findings that targeting CD24 can enhance the efficacy of radiotherapy, we further investigated the underlying mechanism. Notably, in vitro cell viability assays showed that CD24 knockout tumor cells had no intrinsic enhanced radiosensitivity ( Figure 17 In view of this, we performed flow cytometry-based immune analysis on KPC tumor tissues from previously treated models ( Figure 18CD24 deficiency significantly enhanced macrophage infiltration ( Figure 7 In addition, macrophages in the combination treatment group exhibited an anti-tumor activation state, characterized by high expression of CD80, CD86, and PDL1 ( Figure 7 These findings are consistent with our in vitro phagocytosis assay and together demonstrate that the CD24i-RT combination improves the functional activation of macrophages. It has been reported that the antibody effect depends on the adaptive immune system, especially tumor-associated antigen cross-priming and cytotoxic CD8 + T cell activation. Guided by this understanding, we went on to examine T cell subsets within TIME. Combination therapy significantly enhanced T cell infiltration within tumors ( Figure 7 e), specifically enhancing the activity and number of cytotoxic T lymphocytes (CTL; ​​CD8 + IFNγ + or GrzmB + ), indicating that CD8 + Enhanced T cell effector capacity ( Figure 7 In addition, we observed immunosuppressive regulatory T cells (Tregs; CD4 + FoxP3 + )reduce( Figure 18 b), while T helper type 1 cells (Th1; CD4 + IFNγ + ) ratio and CD4 + / CD8 + The ratio remains unchanged ( Figure 18 Multispectral immunofluorescence analysis confirmed these observations, revealing that macrophages and CD8 + T cell infiltration and enhanced effector activity in tumors following combined therapy ( Figure 7 Zhonghong and Figure 18 To explore whether the efficacy of CD24i-RT combination depends on the treatment of reactive macrophages and CD8 + T cells, we depleted these cell populations using clodronate liposomes or anti-CD8 antibodies in a CD24-deficient KPC model ( Figure 7 Neutral Figure 19 Notably, depletion of either subset significantly impaired the therapeutic effect of radiotherapy, indicating that both macrophages and T cells are essential for the CD24i-RT combination ( Figure 7 in mo).

[0067] Low CD24 expression predicts favorable time to and improved clinical outcomes: Based on preclinical evidence linking CD24 to an immunosuppressive microenvironment, we sought to validate its clinical relevance using multicohort analyses. CD24 may function as a dominant innate immune checkpoint in ovarian cancer. Therefore, we first interrogated the relationship between CD24 expression and time to outcome using multiplex immunofluorescence staining on a tissue microarray containing 44 high-grade serotype carcinoma (HGSOC) tissue samples. Figure 8 Quantitative analysis showed that there was a negative correlation between CD24 levels and the TIME signature of activation, with decreased CD24 corresponding to increased CD8 + Increased T cell density, increased macrophage infiltration (CD68), and increased expression of co-stimulatory (CD86) and immune checkpoint (PD-L1) molecules ( Figure 8 Analysis of lung squamous cell carcinoma (LUSC) patient data from The Cancer Genome Atlas (TCGA) database confirmed the pan-cancer correlation, showing that CD24 expression was associated with M1 macrophages, CD8 + T cells and CD8 + There is a conserved negative correlation between effector memory T cell infiltration characteristics ( Figure 8 In the fh), high CD24 expression was negatively correlated with macrophage T cell subsets that exert antitumor functions. Given the role of CD24 as a radiation-responsive innate immune checkpoint, we evaluated the clinical outcomes of prostate cancer patients receiving radiotherapy. Low CD24 expression predicted improved overall survival ( Figure 8 GPAA1 is a key molecule that mediates CD24 membrane localization after irradiation. Parallel analysis of patients with hepatocellular carcinoma (LIHC) and non-small cell lung cancer (NSCLC) confirmed that CD24 and GPAA1 are independent prognostic factors ( Figure 8 These analyses confirmed that reduced CD24 expression is a biomarker of an immune-competent tumor microenvironment (TIME) and favorable survival outcomes in multiple malignancies.

[0068] 3. Analysis

[0069] Although the immunostimulatory effects of radiotherapy have long been recognized, clinically significant systemic responses beyond the irradiated area remain extremely rare. This discrepancy suggests that radiotherapy-induced immune activation is limited by concurrent immunosuppressive signals. Based on this understanding, extensive efforts to combine radiotherapy with PD-1 / PD-L1 inhibitors aim to restore post-irradiation T cell function and achieve radiosensitization. However, this combination therapy does not consistently produce synergistic antitumor effects, suggesting that immune escape mechanisms beyond adaptive immunity hinder radiation-induced systemic tumor control.

[0070] Our studies identify macrophage phagocytosis and activation as key prerequisites for converting radiotherapy cytotoxicity into systemic immunity and identify CD24 as a radiation-responsive innate immune checkpoint that blocks this important pathway. CD24 is a recently discovered "don't eat me" signal that mediates anti-phagocytosis by interacting with Siglec-10 on macrophages. We demonstrate that CD24 exhibits superior tumor specificity compared with established innate immune checkpoints CD47, B2M, and PD-L1. Targeting CD24 may have the advantage of reducing off-target effects, bypassing the "antigen pool" associated with CD47 blockade, and potentially reducing the risk of toxicity. CD24Fc has demonstrated a favorable safety profile in Phase II / III clinical trials for the prevention of graft-versus-host disease and the treatment of severe acute pneumonia caused by COVID-19. Multiple anti-CD24 antibodies are entering clinical trials for advanced malignant solid tumors. Our findings, in preclinical models, indicate that CD24 controls both local and systemic responses to radiotherapy, expanding the therapeutic scope of this target. Our animal model studies and patient cohort analyses showed that CD24 was negatively correlated with immune activity TIME, indicating that high CD24 expression is associated with a poor immune microenvironment. This suggests that CD24 expression levels may serve as a biomarker for predicting prognosis in response to immunotherapy (such as checkpoint inhibitors), providing an important basis for patient stratification for ICI therapy. In summary, our study provides comprehensive clinical guidance for targeting CD24 as a therapeutic agent and for utilizing CD24 as a predictive biomarker in cancer management.

[0071] While investigating the dynamic changes of CD24 after radiation, our research revealed a novel mechanism regulating the GPI-anchoring process: radiation promotes the accumulation of the GPI transamidase subunit GPAA1, thereby enhancing the membrane localization of GPI-anchored proteins (GPI-APs). Inhibition of GPAA1 effectively reverses the membrane trafficking of CD24 after radiation, promoting macrophage phagocytosis of tumor cells and improving the efficacy of radiotherapy. Our preclinical data validate the clinical therapeutic potential of targeting GPAA1 in cancer immunotherapy. Although multiple CD24 antibodies that block the CD24-Siglec10 axis have entered clinical trials, small molecule therapies offer advantages in terms of cost, ease of administration, and avoidance of Fc-mediated immunogenicity, making them high-priority translational targets. It is important to emphasize that radiation-enhanced GPI-anchoring is not limited to CD24: this study also double-validated this mechanism using CD73, another GPI-anchored protein involved in adenosine-mediated immunosuppression. Other GPI-anchored proteins (such as mesothelin MSLN, carcinoembryonic antigen CEA / CD66e, and complement regulatory proteins CD55 / CD59) are also overexpressed in tumors and are associated with tumor occurrence, progression, or an immunosuppressive tumor microenvironment, making GPI anchoring a targetable weak link for combined radiotherapy. However, the indispensable role of GPI-anchored proteins in normal physiology poses a major challenge: defects in GPI biosynthesis may lead to neurological deficits, T cell immune dysfunction, and hemolytic diseases, so biosafety must be ensured through strictly targeted delivery. Targeting the GPI pathway remains an underexplored direction in cancer treatment and requires in-depth preclinical and clinical research verification.

[0072] We investigated radiation-induced immune evasion through the lens of innate immunity, reframing CD24 as a radiation-responsive "don't eat me" signal. Radiation enhances CD24 membrane trafficking via a GPI anchor regulated by the ANAPC5 / GPAA1 axis, thereby mediating phagocytic evasion. GPAA1 or CD24 inhibition enhanced the local antitumor efficacy induced by radiotherapy in multiple mouse models. Combination therapy with CD24 inhibition and radiotherapy (CD24iRT) further elicited abscopal effects, achieving control of untreated distant tumors. Our findings elucidate the molecular basis of immune escape after radiation and provide an alternative strategy to overcome radioresistance. In summary, this study establishes CD24 as a radiation-responsive innate immune checkpoint regulated by the ANAPC5 / GPAA1 axis. We discovered that the "don't eat me" signal CD24 mediates paradoxical immune evasion after irradiation and propose targeting CD24 in combination with radiotherapy as a novel therapeutic strategy validated in preclinical models. Furthermore, our results suggest that the GPI anchor represents a previously unrecognized vulnerability in irradiated tumors. These findings provide a translational framework for addressing radioresistance through innate immune checkpoint modulation.

[0073] It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. Such modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims appended hereto.

Claims

1. Application of a preparation for detecting CD24 expression levels in the preparation of a TIME product for detecting immune activity in tumor radiotherapy; wherein, The expression level of CD24 is negatively correlated with the immune activity of TIME; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further is any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

2. Application of preparations for detecting CD24 expression levels in the preparation of products for predicting overall survival rate after tumor radiotherapy or immunotherapy; The lower the CD24 expression, the higher the overall survival rate after tumor radiotherapy or immunotherapy; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

3. Use of a preparation that inhibits CD24 expression in the preparation of a product that enhances the abscopal effect of tumor radiotherapy; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and the preparation that inhibits CD24 expression is siRNA or shRNA, further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

4. Use of a preparation that inhibits CD24 expression in combination with radiotherapy for the treatment of tumors; or, use of a preparation that inhibits CD24 expression in the preparation of a product that enhances radiotherapy sensitivity; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and the preparation that inhibits CD24 expression is siRNA or shRNA, further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

5. Use of a preparation that inhibits GPAA1 expression in the preparation of a product for treating tumors; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and the preparation that inhibits CD24 expression is siRNA or shRNA, further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

6. Use of a preparation that inhibits GPAA1 expression in the preparation of a product for enhancing the abscopal effect of tumor radiotherapy; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and the preparation that inhibits CD24 expression is siRNA or shRNA, further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

7. Use of a preparation that inhibits GPAA1 expression in combination with radiotherapy for the treatment of tumors; or, use of a preparation that inhibits GPAA1 expression in the preparation of a product that enhances radiotherapy sensitivity; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and the preparation that inhibits CD24 expression is siRNA or shRNA, further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

8. Application of a preparation that inhibits GPAA1 expression in the preparation of a tumor cell model with low CD24 expression; wherein, The tumor cell model with low CD24 expression is an in vitro irradiated tumor cell model; preferably, the tumor cells are KPC cells with GPAA1 knocked out, and the agent for inhibiting GPAA1 expression is siRNA or shRNA.

9. Application of preparations for detecting CD24 or GPAA1 expression levels in the preparation of products for analyzing the prognostic effect of tumor radiotherapy; wherein, The expression level of CD24 or GPAA1 is negatively correlated with the prognostic effect of tumor radiotherapy; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

10. Preparations for detecting CD24 or GPAA1 expression levels in the preparation and analysis of M1 macrophages, CD8 + T cells, CD8 + Use of a product for any level of infiltration of effector memory T cells; wherein, The CD24 expression level is negatively correlated with the infiltration level; preferably, the tumor is any one of liver cancer, lung cancer, and pancreatic cancer, and further is any one of non-small cell lung cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, ovarian cancer, and prostate cancer.

Images