Journal: Cancer Research

Article Title: Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Mediates Antitumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2616

Figure Lengend Snippet: Radiation enhances tumor cell “do not eat me” signal CD24. A, UMAP plots of pancreatic adenocarcinoma with 10 clusters, NCBI Sequence Read Archive: GSE281288 . CD24 expression overlaid onto UMAP. B, Immunofluorescence images of CD24 (red) on control or irradiated H460 cells. Scale bars, 25 and 5 µm. C and D, Flow cytometry histograms and mean fluorescence intensity (MFI) of CD24 surface expression on control or irradiated H460 ( C ) and PANC-1 ( D ) cells ( n = 3). E, Tumor cell CD24 mean fluorescence intensity from control or irradiated KPC tumor models ( n = 6). F, Flow cytometry histograms and mean fluorescence intensity of CD24 surface expression on H460 cells 48 hours after 2–18 Gy of radiation ( n = 3). IR, 8 Gy irradiation. All data are presented as mean ± SEM and normalized to the control group. A two-tailed Student t test was performed for C–E . One-way ANOVA with a Tukey multiple comparisons test was performed for F . NS, not significant.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with antibodies against CD24 (Abways, cat. #DY1303, RRID: AB_3073211), GPAA1 (Proteintech, cat. #10104-1-AP, RRID: AB_2263708), and ANAPC5 (Proteintech, cat. #67348-1-Ig, RRID: AB_2882606) primary antibodies at 4°C overnight, washed with TBST, and then incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Techniques: Sequencing, Expressing, Immunofluorescence, Control, Irradiation, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Cancer Research

Article Title: Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Mediates Antitumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2616

Figure Lengend Snippet: CD24 inhibition combined with radiation promotes macrophages phagocytosis and activation. A and B, Flow cytometry histograms of CD24 surface expression on H460 ( A ) and PANC-1 ( B ) cells transfected with CD24 -targeting siRNA (si CD24 ), negative-control siRNA (siNC), or isotype control. C and D, Flow cytometry histograms and quantitation of H460 ( C ) and PANC-1 ( D ) cell phagocytosis by THP-1 macrophages, with the indicated treatments ( n = 3). E, Immunofluorescence images and quantitation of H460 cell phagocytosis by THP-1 macrophages (red), with the indicated treatments (green). Arrows, phagocytic events ( n = 6). Scale bars, 50 μm. F, qRT-PCR analysis of Siglec- 10 in THP-1 macrophages transfected with si Siglec- 10 or siNC ( n = 3). G, Flow cytometry histograms and quantitation of irradiated H460 cell phagocytosis by THP-1 macrophages, with the indicated treatments ( n = 3). H – J, Flow cytometry histograms and mean fluorescence intensity (MFI) of CD80 ( H ), CD86 ( I ), and PD-L1 ( J ) on BMDMs cocultured with tumor cells, with the indicated treatments ( n = 3). IR, 8 Gy irradiation. All data are presented as mean ± SEM. One-way ANOVA with a Tukey multiple comparisons test was performed for C–E and G–J . A two-tailed Student t test was performed for F . NS, not significant.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with antibodies against CD24 (Abways, cat. #DY1303, RRID: AB_3073211), GPAA1 (Proteintech, cat. #10104-1-AP, RRID: AB_2263708), and ANAPC5 (Proteintech, cat. #67348-1-Ig, RRID: AB_2882606) primary antibodies at 4°C overnight, washed with TBST, and then incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Techniques: Inhibition, Activation Assay, Flow Cytometry, Expressing, Transfection, Negative Control, Control, Quantitation Assay, Immunofluorescence, Quantitative RT-PCR, Irradiation, Fluorescence, Two Tailed Test

Journal: Cancer Research

Article Title: Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Mediates Antitumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2616

Figure Lengend Snippet: Radiation enhances CD24 membrane trafficking via GPI anchoring. A and B, Western blot analysis of CD24 in control or irradiated H460 ( A ) and PANC-1 ( B ) whole-cell lysate. C, Schematic representation of GPI anchoring. D, Western blot analysis of CD24 in control or irradiated H460 cell membrane and cytosolic lysate. E, Proteomic analysis heatmap of differential proteins in control or irradiated H1299. F and G, Western blot analysis of GPAA1 in control or irradiated H460 ( F ) and PANC-1 ( G ) whole-cell lysate. H, Western blot analysis of GPAA1 and CD24 in H460 cells transfected with si GPAA1 or siNC. I, Flow cytometry histograms of CD24 surface expression on H460 cells, with the indicated treatment or isotype control ( n = 3). MFI, mean fluorescence intensity. J, Flow cytometry histograms and quantitation of H460 cell phagocytosis by THP-1 macrophages, with the indicated treatments ( n = 3). IR, 8 Gy irradiation. All data are presented as mean ± SEM and compared using one-way ANOVA with a Tukey multiple comparisons test. NS, not significant.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with antibodies against CD24 (Abways, cat. #DY1303, RRID: AB_3073211), GPAA1 (Proteintech, cat. #10104-1-AP, RRID: AB_2263708), and ANAPC5 (Proteintech, cat. #67348-1-Ig, RRID: AB_2882606) primary antibodies at 4°C overnight, washed with TBST, and then incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Techniques: Membrane, Western Blot, Control, Irradiation, Transfection, Flow Cytometry, Expressing, Fluorescence, Quantitation Assay

Journal: Cancer Research

Article Title: Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Mediates Antitumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2616

Figure Lengend Snippet: Radiation disrupts APC/C-mediated ubiquitination of GPAA1 at K111. A, Schematic illustration of IP-MS approach. B, GO enrichment analysis (Biological Process) of GPAA1-interacting proteins predicted in IP-MS. C and D, Western blot analysis of cycloheximide (CHX) chase assay in H460 cells treated with MG132 ( C ) or radiation ( D ). E, Western blot analysis followed by IP evaluated the ubiquitination of GPAA1 in indicated treatment groups. F, Western blot analysis of GPAA1 in H460 cells treated with apcin. G, Flow cytometry histograms of CD24 surface expression on H460 cells treated with apcin or isotype control ( n = 3). MFI, mean fluorescence intensity. H, Schematic illustration of screening GPAA1-binding APC/C subunits. I, IP analysis of the GPAA1–ANAPC5 interaction. J, Western blot analysis followed by IP evaluated GPAA1 ubiquitination in H460 cells transfected with si ANAPC5 or siNC. K, Western blot analysis of ANAPC5 and GPAA1 in H460 cells transfected with si ANAPC5 or siNC. L, Flow cytometry histograms of CD24 surface expression on H460 cells transfected with si ANAPC5 , siNC, or isotype control ( n = 3). M, Western blot analysis followed by IP evaluated GPAA1 ubiquitination in 293T cells transfected with different mutants. IR, 8 Gy irradiation. All data are presented as mean ± SEM. One-way ANOVA with a Tukey multiple comparisons test was performed for G . A two-tailed Student t test was performed for L . WT, wild type.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with antibodies against CD24 (Abways, cat. #DY1303, RRID: AB_3073211), GPAA1 (Proteintech, cat. #10104-1-AP, RRID: AB_2263708), and ANAPC5 (Proteintech, cat. #67348-1-Ig, RRID: AB_2882606) primary antibodies at 4°C overnight, washed with TBST, and then incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Techniques: Ubiquitin Proteomics, Protein-Protein interactions, Western Blot, Flow Cytometry, Expressing, Control, Fluorescence, Binding Assay, Transfection, Irradiation, Two Tailed Test

Journal: Cancer Research

Article Title: Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Mediates Antitumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2616

Figure Lengend Snippet: Targeting CD24 sensitizes tumor to radiotherapy and elicits abscopal effect in vivo . A, Schematic illustration of radiation treatment plan in animal models. B and C, Tumor growth curves ( B ) and Kaplan–Meier survival plot ( C ) of Hepa1-6 subcutaneous tumor models, with the indicated treatment ( n = 8). D and E, Tumor growth curves ( D ) and tumor weight ( E ) of KPC subcutaneous tumor models, with the indicated treatment ( n = 6). F and G, Tumor growth curves ( F ) and Kaplan–Meier survival plot ( G ) of KPC subcutaneous tumor models, with the indicated treatment ( n = 8). H, Schematic illustration of abscopal effect evaluation in animal models. I–K, Tumor growth curves of in situ ( I ) and abscopal tumors ( J ) and Kaplan–Meier survival plot ( K ) of KPC subcutaneous tumor models depicted in H ( n = 6). L, Schematic illustration of antibody treatment plan in animal models. M and N, Tumor growth curves ( M ) and Kaplan–Meier survival plot ( N ) of KPC subcutaneous tumor models depicted in L ( n = 7). IR, 8 Gy × 3 irradiation. All data are presented as mean ± SEM. Two-way ANOVA with a Tukey multiple comparisons test was performed for B , D , F , I , J , and M . One-way ANOVA with a Tukey multiple comparisons test was performed for E . A log-rank test was performed for C , G , K , and N . NS, not significant. A, H, and L, Created in BioRender. Kong, L. (2026) https://BioRender.com/rwdzfwn .

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with antibodies against CD24 (Abways, cat. #DY1303, RRID: AB_3073211), GPAA1 (Proteintech, cat. #10104-1-AP, RRID: AB_2263708), and ANAPC5 (Proteintech, cat. #67348-1-Ig, RRID: AB_2882606) primary antibodies at 4°C overnight, washed with TBST, and then incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Techniques: In Vivo, In Situ, Irradiation

Journal: Cancer Research

Article Title: Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Mediates Antitumor Immune Evasion

doi: 10.1158/0008-5472.CAN-25-2616

Figure Lengend Snippet: Low CD24 expression correlates with increased immune cell infiltration and prolonged survival across cancer types. A–E, Immunofluorescence images ( A ) and correlation analysis of CD24 average fluorescence intensity (AFI) with CD8 ( B ), CD68 ( C ), CD86 ( D ), and PD-L1 ( E ) expression from an independent high-grade serous ovarian cancer (HGSOC) array ( n = 44). Scale bars, 200 and 50 μm. F–H, Correlation analysis of CD24 expression levels with M1 macrophage ( F ), CD8 + T-cell ( G ), and CD8 + effector memory T-cell ( H ) infiltration levels in LUSC. Plotted by TIMER 2.0. TPM, transcripts per million. I, Overall survival period of patients with prostate adenocarcinoma (PRAD) receiving radiotherapy ( n = 77) stratified by CD24 expression levels based on the TCGA database. J and K, Overall survival of patients with hepatocellular carcinoma (LIHC; n = 370) stratified by CD24 ( J ) or GPAA1 ( K ) expression levels based on pan-cancer database of Kaplan–Meier plotter. L and M, Overall survival of patients with non–small cell lung cancer (NSCLC; n = 1,044) stratified by CD24 ( L ) or GPAA1 ( M ) expression levels based on SurveExpress lung meta-base. A Pearson correlation test was performed for B–E . R-squared, coefficient of determination. A log-rank test was performed for I–M . Hazard ratio (HR) and its 95% confidence interval were computed via a Cox proportional hazards regression model.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with antibodies against CD24 (Abways, cat. #DY1303, RRID: AB_3073211), GPAA1 (Proteintech, cat. #10104-1-AP, RRID: AB_2263708), and ANAPC5 (Proteintech, cat. #67348-1-Ig, RRID: AB_2882606) primary antibodies at 4°C overnight, washed with TBST, and then incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Techniques: Expressing, Immunofluorescence, Fluorescence