gpr34 antagonist  (MedChemExpress)

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Single-cell RNA sequencing reveals the cellular composition of pancreatic cancer after chemotherapy combined with Surufatinib treatment. a Schematic diagram of the process of this study. First, we performed single-cell RNA (scRNA) sequencing of surgical specimens obtained from patients who were treated with gemcitabine, nab-paclitaxel, and surufatinib in the clinical trial. Patients were divided into responder group and non-responder group according to clinicopathological evaluation. Comparing the differences in macrophage subsets between the two groups, it was found that GPR34 + damage-associated macrophages (DAMs) were absolutely dominant in the treatment-resistant patients. Subsequently, GPR34 + DAMs function and underlying mechanisms were validated in retrospective and prospective pancreatic cancer patient cohorts and mouse models. Finally, targeting GPR34 + DAMs was demonstrated to enhance the efficacy of surufatinib in a preclinical model. Schematic diagram was created with BioRender.com. PDAC: pancreatic ductal adenocarcinoma, DAMs: damage associated macrophages, scRNA: single cell RNA. b Representative contrast-enhanced CT images of responder group (R) and non-responder group (NR) before and after treatment. Patients underwent contrast-enhanced CT and tumor assessment every 8 weeks, and two independent experienced radiologists determined the location and contour of the target lesion. The length of the tumor was measured and counted by pancreatic tumor specialists. The baseline level before treatment as well as the last evaluation before surgery were compared respectively. c Waterfall plot showing efficacy evaluation results from clinical trial patients, with upper dashed line indicating the cutoff for disease progression according to the RECIST (Response Evaluation Criteria in Solid Tumors) V1.1 standard, lower dashed line indicating the cutoff for PR. The final evaluation results are marked above and below each column of the waterfall plot. d UMAP plot displaying the cellular subtypes of all cells ( n = 84380) in the surgical specimens after treatment. e Scale bar chart showing the cell type composition of all patient samples, and aggregated samples from non-responders and responders. f The box plot compared the proportion differences of each cell type between non-responders and responders. A beta regression analysis was used to examine the correlation between the proportions of various cell components and treatment efficacy. g UMAP plot showing macrophage subtypes clustering in all patient samples. mac macrophage, cl cluster. h Bar chart showing the composition of macrophage cluster in all patient samples and aggregated samples from non-responders and responders. i The box plot compared the proportion differences of each macrophage subcluster between non-responders and responders. A beta regression analysis was used to examine the correlation between the proportions of various subclusters and the treatment efficacy. j Heatmap displaying the expression of key functional genes across macrophage subclusters. The characteristic genes of each subpopulation were ranked by log 2 FC, and the top 50 genes with adjusted P -value < 0.05 for each subcluster were selected for visualization and gene set enrichment analysis (GSEA). The main enrichment pathways were selected for display. Each column represents a cell barcode, sorted by cell type, and each row represents a gene. k Bar plot showing the GSEA analysis results for the Mac_cl1 cluster. The analysis was based on the Hallmarks, GOBP and KEGG data sets, and all results were screened based on the scores of the data sets mentioned above. NS no significance, * P < 0.05

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: Single Cell, RNA Sequencing, Sequencing, Biomarker Discovery, Expressing, Functional Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Cohort and in vitro studies reveal the role of GPR34 in macrophages. a Volcano plot showing differentially expressed genes between responder group and non-responder group from the macrophage subclusters of scRNA sequencing data. The horizontal dashed line represents the P -value cutoff ( P < 10⁻⁵⁰), and the vertical dashed line represents the log 2 FC cutoff (-1 or 1). FC fold change, sig significance, R Responder, NR Non-responder. b , c UMAP plot showing the expression of GPR34 in all cells ( b ) and macrophages ( c ). Mac: Macrophage, cl cluster. d Representative mIF staining images (100x) of surgical specimens from responders and non-responders in the clinical trial ( n = 26). Navy: GPR34, Magenta: CD68, Red: CD8, Green: Tim-3, Yellow: CK19, Blue: DAPI. White arrowheads: Tim-3 + CD8 + exhausted T cells (Tex); White arrows: GPR34 + CD68 + macrophages. White scale bar = 100 μm. e Bar plot comparing the proportion of CD68 + macrophages (top) and the proportion of GPR34 + cells among CD68 + macrophages (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. * P < 0.05. f Bar plot comparing the proportion of CD8 + T cells (top) and the proportion of Tim-3 + cells among CD8 + T cells (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. g Representative mIF staining images (200x) of surgical specimens from clinical trial patients ( n = 26). Navy: GPR34, Magenta: CD68, Red: MPO, Green: CD3, Dark yellow: CD20, Yellow: CK19, Blue: DAPI. White scale bar = 50 μm. h Representative mIF staining images (200x) of clinical trial patient surgical specimens ( n = 26). Navy: GPR34, Magenta: CD68, Red: α-SMA, Green: CD31, Dark yellow: CD117, Yellow: CD56, Blue: DAPI. White scale bar = 50 μm. i Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26). One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. j Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26) examined by flow cytometry. One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. k , l Kaplan-Meier curves for overall survival (OS) and Recurrence-free survival (RFS) in the prospective cohort ( k ) and retrospective cohort ( l ), comparing prognosis between groups with high versus low infiltration of GPR34 + macrophages. The cutoff for the proportion of GPR34 + cells among CD68 + cells were dichotomized using a 20% . Log-rank test was used for comparison. m Flow cytometry analysis of GPR34 + cells in tumor tissue versus adjacent non-tumorous tissue from prospective cohort patients ( n = 42). Scatter plot shows the paired infiltration proportion of GPR34 + cells in tumor and normal tissue from the same patient . Two-tailed paired t -test was used. n Flow cytometry analysis of GPR34 + cells in tumor tissue from prospective cohort patients ( n = 42). Bar plots show the infiltration proportions of CD45 + cells, CD8 + T cells, Tim-3 + PD-1 + T cells, MRC1 + macrophages, and MHC-I + macrophages in the low GPR34 ( ≤ 20%) versus high GPR34 ( > 20%) groups. Two-tailed unpaired t-test. Data are presented by mean ± SD. o BMDMs from C57BL/6 mice were cultured until day 5, stimulated with KPC cell TCM for 12 h, followed by treatment with Surufatinib (4 nM) or CSF-1R inhibitor (PLX3397, 20 nM) for 24 h, then analyzed by flow cytometry ( n = 3). Bar plot compares the gMFI of GPR34 among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: In Vitro, Sequencing, Expressing, Staining, Two Tailed Test, Flow Cytometry, Comparison, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage-specific GPR34 knockout improves chemotherapy outcomes in mice. a Orthotopic pancreatic injection of KPC-GFP-LUC cells into Gpr34 flox/flox and Gpr34 Δ Lyz2 mice. After tumor formation, chemotherapy was administered to simulate tumor killing. Tumor bioluminescence intensity was dynamically monitored by bioluminescence imaging. Representative bioluminescence images show tumor growth in each group ( n = 5). b Time-course curve of bioluminescence intensity in orthotopic tumor-bearing mice (n = 5). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SEM. c Bar plot of tumor weight on day 21 post-implantation in orthotopic tumor-bearing mice ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS: no significance, *: P < 0.05. d Flow cytometry analysis of the proportions of CTL, Tex, M1 macrophages, and M2 macrophages in tumor tissues of orthotopic tumor-bearing mice (n = 5). Bar plots show the infiltration differences of immune cells among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. e KPC mice were irradiated with a total dose of 10 Gy, followed by tail vein injection of bone marrow cells from Gpr34 +/+ or Gpr34 −/− mice. After successful transplantation confirmed by flow cytometry. Tumor growth was monitored by B-ultrasound during chemotherapy simulating injury signals ( n = 8–10). Representative B-ultrasound images compare tumor size among groups. Black dashed lines outline tumor boundaries. Tumor volume was calculated as 0.5 * long diameter * (short diameter) ². S: spleen; K: kidney. Black arrows point to the tumor. f Time-course plot of tumor volume monitored by B-ultrasound. Each solid line represents an individual mouse. Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. g Kaplan-Meier curve of KPC spontaneous tumor-bearing mice ( n = 8–10). Log-rank test was used. h mIF staining of paraffin sections from orthotopic tumors in the bone marrow transplantation KPC mice ( n = 6). Representative fluorescence images (top 100x, bottom 400x) show exhausted T cell infiltration among groups. Red: CD8α, Green: Tim-3, White: CK19, Blue: DAPI. White scale bar = 50 μm. i Bar plot showing the proportion of Tim-3 + CD8 + T cells from mIF staining in KPC spontaneous tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. j Fluorescence imaging of macrophages sorted by flow cytometry from tumors of KPC-GFP-LUC orthotopic injection mice model. Representative images show GFP phagocytosis by macrophages in each group ( n = 6). Green: GFP, Blue: DAPI. White scale bar = 20 μm. k Bar plot showing the gMFI of GFP phagocytosis by flow-sorted macrophages from orthotopic tumors ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. l Bar plot of the gMFI of GFP in macrophages from KPC cell orthotopic tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. m Bar plot showing the proportion of MerTK + or AXL + macrophage from flow cytometry analysis in KPC mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used . Data are presented by mean ± SD. n BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, cultured with TCM for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected the proportion of MerTK + or AXL + macrophage. Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 6). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: Knock-Out, Injection, Imaging, Flow Cytometry, Irradiation, Transplantation Assay, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Validation of GPR34 function in macrophage and CD8 + T cell co-culture system. a Gpr34 flox/flox and Gpr34 Δ Lyz2 mice were treated with anti-CD8α or IgG, followed by orthotopic pancreatic injection of KPC-LUC cells. After tumor formation, chemotherapy was administered to simulate an injury signal. Tumor bioluminescence was dynamically monitored. Representative bioluminescence images show tumor growth in different groups ( n = 6). b Time-course curve of bioluminescence imaging for the KPC-LUC orthotopic model ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, *** P < 0.001. c Bar plot showing tumor weight on day 21 in the KPC-LUC orthotopic model ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance. d , e BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then co-cultured with TCM and KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells for 24 hours . Flow cytometry analyzed the expression of functional molecules in BMDMs ( d ) and CD8 + T cells ( e ). Bar plots show levels in Gpr34 +/+ vs Gpr34 −/− groups ( n = 3). Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. f , g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, pre-stimulated with SIINFEKL, then cultured with TCM for 12 h, followed by co-culture with CD8 + T cells from OT-1 mice for 24 h. Flow cytometry detected T cell-specific killing function ( f ) and BMDM antigen presentation function ( g ). Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. h Violin-box plots of cytokine transcript expression in macrophage clusters from scRNA sequencing data. White dot and solid lines in boxes represent medians and quartiles. Two-tailed Wilcoxon test. i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then stimulated with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. qPCR detected Cxcl16 transcript levels. Bar plot compares Cxcl16 transcripts between groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j ELISA detection of cytokine secretion in supernatant from BMDMs stimulated with apoptotic KPC-GFP cells. Bar plot shows CXCL16 protein secretion levels from Gpr34 +/+ and Gpr34 −/− BMDMs ( n = 10). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. k , l BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells. Flow cytometry detected T cell exhaustion ( k ) and cytotoxicity levels ( l ) ( n = 3). One-way ANOVA with Dunnett’s test compared siRNA groups versus control. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: Biomarker Discovery, Co-Culture Assay, Injection, Imaging, Cell Culture, Isolation, Flow Cytometry, Expressing, Functional Assay, Two Tailed Test, Immunopeptidomics, Sequencing, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: LysoPS-GPR34 modulates macrophage efferocytosis and inflammatory cytokine secretion. a , b BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. These were then co-cultured with CD8 + T cells for 24 hours. Flow cytometry detected GFP efferocytosis ( a ) and p-AKT levels ( b ) in BMDMs. Bar plots show differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. c BMDMs from Gpr34 +/+ and Gpr34 −/− mice were treated with LysoPS and co-cultured with CD8 + T cells. Flow cytometry detected MRC1, MHC-I on macrophages, and Tim-3, GZMB on CD8 + T cells. Bar plot shows gMFI differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d , e BMDMs from C57BL/6 mice were treated with LysoPS and AKT inhibitor, then co-cultured with chemotherapy-induced apoptotic KPC-GFP cells for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected GFP uptake by BMDMs ( d ) and exhaustion/cytotoxicity levels of CD8 + T cells ( e ). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. f UMA P plot showing relative expression of efferocytosis-related genes from scRNA sequencing analysis. g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and either chemotherapy-induced apoptotic KPC-GFP cells or normal KPC-GFP cells for 12 h. After removing cells, RNA was extracted for qPCR. Bar plot compares transcript levels among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. h BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with apoptotic KPC cells of negative control for 12 hours. RNA was extracted for qPCR. Bar plot compares transcript differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. i BMDMs from C57BL/6 mice were cultured until day 5, stimulated with TCM and LysoPS for 12 h. ELISA detected CXCL16 secretion in supernatant. Bar plot shows levels in LysoPS vs. control groups. Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: Cell Culture, Flow Cytometry, Co-Culture Assay, Expressing, Sequencing, Negative Control, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage efferocytosis function influences antigen presentation ability through MHC-I. a BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 hours, then analyzed by flow cytometry for GFP uptake. Bar plot shows gMFI of GFP in BMDMs treated with MerTK inhibitor vs control ( n = 3). One-way ANOVA with Dunnett’s test compared MerTKi groups to control. Data are presented by mean ± SD. b BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP cells for 12 hours, treated with MerTK inhibitor, then co-cultured with CD8 + T cells from OT1 mice for 24 hours. Flow cytometry detected MHC-I, SIINFEKL loading, CD80, CD86 on BMDMs. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. c Flow cytometry detection of Tetramer + , PD-1 + , Tim-3 + , and GZMB + cells after co-culture of BMDMs with OT1 CD8 + T cells. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. d Apoptotic KPC cells induced by chemotherapy and labeled with Caspase3/7 green were co-cultured with BMDMs. Phagolysosome formation was detected using pHrodo red. Representative fluorescence microscopy images (1000x) show differences between MerTK inhibitor and control groups ( n = 6). Green: Caspase3/7, Red: pHrodo, Blue: DAPI. White scale bar= 20 μm. e Bar plots show total pHrodo fluorescence intensity (left) and the number of Caspase3/7 + pHrodo + vesicles per cell (right) in BMDMs after incubation with apoptotic cells ( n = 6). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. f Flow cytometry analysis of pHrodo gMFI in BMDMs after incubation with apoptotic cells. Bar plot shows pHrodo gMFI levels between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. g Violin-box plots of lysosome-associated gene transcript expression in macrophage subpopulations from scRNA sequencing data. Solid lines represent medians and quartiles. One-way ANOVA with Kruskal-Wallis H test compared groups (Mac_cl1 as reference). h , i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours. After removing apoptotic cells, RNA was extracted for qPCR. Bar plots show transcript differences between Gpr34 +/+ and Gpr34 −/− BMDMs ( h ) efferocytosis receptors, ( i ) lysosome-related/transcription factors, ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-incubated with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours and analyzed by flow cytometry. Bar plot shows differences in MHC-I protein levels between knockdown and control groups ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. k BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with lysosomal inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Bar plot shows pHrodo gMFI in macrophages from flow cytometry, comparing lysosomal inhibitor group vs control. One-way ANOVA with Dunnett’s test was used . Data are presented by mean ± SD. l , m Flow cytometry detection of macrophage antigen presentation function ( l ) and CD8 + T cell specific killing capacity ( m ) in the BMDM-OT1 CD8 + T cell co-culture system. Bar plots show differences between lysosomal inhibitor and control groups. One-way ANOVA with Dunnett ’ s test was used. Data are presented by mean ± SD. n BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM, MerTK inhibitor/Lysosome inhibitor and chemotherapy-induced apoptotic KPC cells for 12 hours. Bar plot shows differences in MHC-I protein levels between different groups detected by flow cytometry ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. o –q BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, transiently transfected with Cxcl16 siRNA, co-incubated with TCM, LysoPS and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with MerTK inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Flow cytometry detected CD8 + T cell specific killing function (o ), cytotoxic function ( p ), and exhaustion levels ( q ). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Control, Incubation, Co-Culture Assay, Labeling, Fluorescence, Microscopy, Expressing, Sequencing, Two Tailed Test, Transfection, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: GPR34 Antagonist Sensitizes Chemotherapy and Surufatinib. a C57BL/6 mice underwent orthotopic pancreatic injection with KPC-LUC cells. After tumor formation, mice were treated with chemotherapy, Surufatinib, and GPR34 antagonist (GPR34a). Tumor dynamics were monitored by bioluminescence imaging. Representative bioluminescence images show dynamic changes in different treatment groups ( n = 6). b Tumor growth curve from bioluminescence imaging of orthotopic tumor-bearing mice ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD at each time point. c Bar plot showing differences in orthotopic tumor weight after treatment ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d KPC mice underwent B-ultrasound examination twice weekly during treatment. Representative B-ultrasound images show the last examination before experiment endpoint or death ( n = 10). White dashed lines indicate tumor boundaries. S: spleen. K: kidney. e The fold change of the tumor volume at the last examination before death or euthanasia in the KPC spontaneous tumor-bearing model to the baseline level ( n = 10). Two-way ANOVA with Kruskal-Wallis H test was used. Data are presented by mean ± SD. f Kaplan-Meier curves comparing survival of KPC mice receiving different treatments. The experimental endpoint was 102 days after treatment initiation. Log-rank test was used. g Organoids derived from patient tumor tissue were co-cultured with matched PBMCs and treated with different drugs. Apoptosis was labeled with caspase-3/7 probe and imaged ( n = 6). Representative microscopy images show organoid morphology and apoptosis protein levels in different treatment groups. Green: caspase3/7. Black scale bar = 50 μm. h Bar plot showing the gMFI of caspase-3/7 in the organoid-PBMC co-culture model across treatment groups ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily.

Techniques: Injection, Imaging, Derivative Assay, Cell Culture, Labeling, Microscopy, Co-Culture Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Single-cell RNA sequencing reveals the cellular composition of pancreatic cancer after chemotherapy combined with Surufatinib treatment. a Schematic diagram of the process of this study. First, we performed single-cell RNA (scRNA) sequencing of surgical specimens obtained from patients who were treated with gemcitabine, nab-paclitaxel, and surufatinib in the clinical trial. Patients were divided into responder group and non-responder group according to clinicopathological evaluation. Comparing the differences in macrophage subsets between the two groups, it was found that GPR34 + damage-associated macrophages (DAMs) were absolutely dominant in the treatment-resistant patients. Subsequently, GPR34 + DAMs function and underlying mechanisms were validated in retrospective and prospective pancreatic cancer patient cohorts and mouse models. Finally, targeting GPR34 + DAMs was demonstrated to enhance the efficacy of surufatinib in a preclinical model. Schematic diagram was created with BioRender.com. PDAC: pancreatic ductal adenocarcinoma, DAMs: damage associated macrophages, scRNA: single cell RNA. b Representative contrast-enhanced CT images of responder group (R) and non-responder group (NR) before and after treatment. Patients underwent contrast-enhanced CT and tumor assessment every 8 weeks, and two independent experienced radiologists determined the location and contour of the target lesion. The length of the tumor was measured and counted by pancreatic tumor specialists. The baseline level before treatment as well as the last evaluation before surgery were compared respectively. c Waterfall plot showing efficacy evaluation results from clinical trial patients, with upper dashed line indicating the cutoff for disease progression according to the RECIST (Response Evaluation Criteria in Solid Tumors) V1.1 standard, lower dashed line indicating the cutoff for PR. The final evaluation results are marked above and below each column of the waterfall plot. d UMAP plot displaying the cellular subtypes of all cells ( n = 84380) in the surgical specimens after treatment. e Scale bar chart showing the cell type composition of all patient samples, and aggregated samples from non-responders and responders. f The box plot compared the proportion differences of each cell type between non-responders and responders. A beta regression analysis was used to examine the correlation between the proportions of various cell components and treatment efficacy. g UMAP plot showing macrophage subtypes clustering in all patient samples. mac macrophage, cl cluster. h Bar chart showing the composition of macrophage cluster in all patient samples and aggregated samples from non-responders and responders. i The box plot compared the proportion differences of each macrophage subcluster between non-responders and responders. A beta regression analysis was used to examine the correlation between the proportions of various subclusters and the treatment efficacy. j Heatmap displaying the expression of key functional genes across macrophage subclusters. The characteristic genes of each subpopulation were ranked by log 2 FC, and the top 50 genes with adjusted P -value < 0.05 for each subcluster were selected for visualization and gene set enrichment analysis (GSEA). The main enrichment pathways were selected for display. Each column represents a cell barcode, sorted by cell type, and each row represents a gene. k Bar plot showing the GSEA analysis results for the Mac_cl1 cluster. The analysis was based on the Hallmarks, GOBP and KEGG data sets, and all results were screened based on the scores of the data sets mentioned above. NS no significance, * P < 0.05

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: Single Cell, RNA Sequencing, Sequencing, Biomarker Discovery, Expressing, Functional Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Cohort and in vitro studies reveal the role of GPR34 in macrophages. a Volcano plot showing differentially expressed genes between responder group and non-responder group from the macrophage subclusters of scRNA sequencing data. The horizontal dashed line represents the P -value cutoff ( P < 10⁻⁵⁰), and the vertical dashed line represents the log 2 FC cutoff (-1 or 1). FC fold change, sig significance, R Responder, NR Non-responder. b , c UMAP plot showing the expression of GPR34 in all cells ( b ) and macrophages ( c ). Mac: Macrophage, cl cluster. d Representative mIF staining images (100x) of surgical specimens from responders and non-responders in the clinical trial ( n = 26). Navy: GPR34, Magenta: CD68, Red: CD8, Green: Tim-3, Yellow: CK19, Blue: DAPI. White arrowheads: Tim-3 + CD8 + exhausted T cells (Tex); White arrows: GPR34 + CD68 + macrophages. White scale bar = 100 μm. e Bar plot comparing the proportion of CD68 + macrophages (top) and the proportion of GPR34 + cells among CD68 + macrophages (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. * P < 0.05. f Bar plot comparing the proportion of CD8 + T cells (top) and the proportion of Tim-3 + cells among CD8 + T cells (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. g Representative mIF staining images (200x) of surgical specimens from clinical trial patients ( n = 26). Navy: GPR34, Magenta: CD68, Red: MPO, Green: CD3, Dark yellow: CD20, Yellow: CK19, Blue: DAPI. White scale bar = 50 μm. h Representative mIF staining images (200x) of clinical trial patient surgical specimens ( n = 26). Navy: GPR34, Magenta: CD68, Red: α-SMA, Green: CD31, Dark yellow: CD117, Yellow: CD56, Blue: DAPI. White scale bar = 50 μm. i Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26). One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. j Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26) examined by flow cytometry. One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. k , l Kaplan-Meier curves for overall survival (OS) and Recurrence-free survival (RFS) in the prospective cohort ( k ) and retrospective cohort ( l ), comparing prognosis between groups with high versus low infiltration of GPR34 + macrophages. The cutoff for the proportion of GPR34 + cells among CD68 + cells were dichotomized using a 20% . Log-rank test was used for comparison. m Flow cytometry analysis of GPR34 + cells in tumor tissue versus adjacent non-tumorous tissue from prospective cohort patients ( n = 42). Scatter plot shows the paired infiltration proportion of GPR34 + cells in tumor and normal tissue from the same patient . Two-tailed paired t -test was used. n Flow cytometry analysis of GPR34 + cells in tumor tissue from prospective cohort patients ( n = 42). Bar plots show the infiltration proportions of CD45 + cells, CD8 + T cells, Tim-3 + PD-1 + T cells, MRC1 + macrophages, and MHC-I + macrophages in the low GPR34 ( ≤ 20%) versus high GPR34 ( > 20%) groups. Two-tailed unpaired t-test. Data are presented by mean ± SD. o BMDMs from C57BL/6 mice were cultured until day 5, stimulated with KPC cell TCM for 12 h, followed by treatment with Surufatinib (4 nM) or CSF-1R inhibitor (PLX3397, 20 nM) for 24 h, then analyzed by flow cytometry ( n = 3). Bar plot compares the gMFI of GPR34 among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: In Vitro, Sequencing, Expressing, Staining, Two Tailed Test, Flow Cytometry, Comparison, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage-specific GPR34 knockout improves chemotherapy outcomes in mice. a Orthotopic pancreatic injection of KPC-GFP-LUC cells into Gpr34 flox/flox and Gpr34 Δ Lyz2 mice. After tumor formation, chemotherapy was administered to simulate tumor killing. Tumor bioluminescence intensity was dynamically monitored by bioluminescence imaging. Representative bioluminescence images show tumor growth in each group ( n = 5). b Time-course curve of bioluminescence intensity in orthotopic tumor-bearing mice (n = 5). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SEM. c Bar plot of tumor weight on day 21 post-implantation in orthotopic tumor-bearing mice ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS: no significance, *: P < 0.05. d Flow cytometry analysis of the proportions of CTL, Tex, M1 macrophages, and M2 macrophages in tumor tissues of orthotopic tumor-bearing mice (n = 5). Bar plots show the infiltration differences of immune cells among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. e KPC mice were irradiated with a total dose of 10 Gy, followed by tail vein injection of bone marrow cells from Gpr34 +/+ or Gpr34 −/− mice. After successful transplantation confirmed by flow cytometry. Tumor growth was monitored by B-ultrasound during chemotherapy simulating injury signals ( n = 8–10). Representative B-ultrasound images compare tumor size among groups. Black dashed lines outline tumor boundaries. Tumor volume was calculated as 0.5 * long diameter * (short diameter) ². S: spleen; K: kidney. Black arrows point to the tumor. f Time-course plot of tumor volume monitored by B-ultrasound. Each solid line represents an individual mouse. Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. g Kaplan-Meier curve of KPC spontaneous tumor-bearing mice ( n = 8–10). Log-rank test was used. h mIF staining of paraffin sections from orthotopic tumors in the bone marrow transplantation KPC mice ( n = 6). Representative fluorescence images (top 100x, bottom 400x) show exhausted T cell infiltration among groups. Red: CD8α, Green: Tim-3, White: CK19, Blue: DAPI. White scale bar = 50 μm. i Bar plot showing the proportion of Tim-3 + CD8 + T cells from mIF staining in KPC spontaneous tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. j Fluorescence imaging of macrophages sorted by flow cytometry from tumors of KPC-GFP-LUC orthotopic injection mice model. Representative images show GFP phagocytosis by macrophages in each group ( n = 6). Green: GFP, Blue: DAPI. White scale bar = 20 μm. k Bar plot showing the gMFI of GFP phagocytosis by flow-sorted macrophages from orthotopic tumors ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. l Bar plot of the gMFI of GFP in macrophages from KPC cell orthotopic tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. m Bar plot showing the proportion of MerTK + or AXL + macrophage from flow cytometry analysis in KPC mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used . Data are presented by mean ± SD. n BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, cultured with TCM for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected the proportion of MerTK + or AXL + macrophage. Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 6). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: Knock-Out, Injection, Imaging, Flow Cytometry, Irradiation, Transplantation Assay, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Validation of GPR34 function in macrophage and CD8 + T cell co-culture system. a Gpr34 flox/flox and Gpr34 Δ Lyz2 mice were treated with anti-CD8α or IgG, followed by orthotopic pancreatic injection of KPC-LUC cells. After tumor formation, chemotherapy was administered to simulate an injury signal. Tumor bioluminescence was dynamically monitored. Representative bioluminescence images show tumor growth in different groups ( n = 6). b Time-course curve of bioluminescence imaging for the KPC-LUC orthotopic model ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, *** P < 0.001. c Bar plot showing tumor weight on day 21 in the KPC-LUC orthotopic model ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance. d , e BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then co-cultured with TCM and KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells for 24 hours . Flow cytometry analyzed the expression of functional molecules in BMDMs ( d ) and CD8 + T cells ( e ). Bar plots show levels in Gpr34 +/+ vs Gpr34 −/− groups ( n = 3). Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. f , g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, pre-stimulated with SIINFEKL, then cultured with TCM for 12 h, followed by co-culture with CD8 + T cells from OT-1 mice for 24 h. Flow cytometry detected T cell-specific killing function ( f ) and BMDM antigen presentation function ( g ). Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. h Violin-box plots of cytokine transcript expression in macrophage clusters from scRNA sequencing data. White dot and solid lines in boxes represent medians and quartiles. Two-tailed Wilcoxon test. i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then stimulated with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. qPCR detected Cxcl16 transcript levels. Bar plot compares Cxcl16 transcripts between groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j ELISA detection of cytokine secretion in supernatant from BMDMs stimulated with apoptotic KPC-GFP cells. Bar plot shows CXCL16 protein secretion levels from Gpr34 +/+ and Gpr34 −/− BMDMs ( n = 10). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. k , l BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells. Flow cytometry detected T cell exhaustion ( k ) and cytotoxicity levels ( l ) ( n = 3). One-way ANOVA with Dunnett’s test compared siRNA groups versus control. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: Biomarker Discovery, Co-Culture Assay, Injection, Imaging, Cell Culture, Isolation, Flow Cytometry, Expressing, Functional Assay, Two Tailed Test, Immunopeptidomics, Sequencing, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: LysoPS-GPR34 modulates macrophage efferocytosis and inflammatory cytokine secretion. a , b BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. These were then co-cultured with CD8 + T cells for 24 hours. Flow cytometry detected GFP efferocytosis ( a ) and p-AKT levels ( b ) in BMDMs. Bar plots show differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. c BMDMs from Gpr34 +/+ and Gpr34 −/− mice were treated with LysoPS and co-cultured with CD8 + T cells. Flow cytometry detected MRC1, MHC-I on macrophages, and Tim-3, GZMB on CD8 + T cells. Bar plot shows gMFI differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d , e BMDMs from C57BL/6 mice were treated with LysoPS and AKT inhibitor, then co-cultured with chemotherapy-induced apoptotic KPC-GFP cells for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected GFP uptake by BMDMs ( d ) and exhaustion/cytotoxicity levels of CD8 + T cells ( e ). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. f UMA P plot showing relative expression of efferocytosis-related genes from scRNA sequencing analysis. g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and either chemotherapy-induced apoptotic KPC-GFP cells or normal KPC-GFP cells for 12 h. After removing cells, RNA was extracted for qPCR. Bar plot compares transcript levels among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. h BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with apoptotic KPC cells of negative control for 12 hours. RNA was extracted for qPCR. Bar plot compares transcript differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. i BMDMs from C57BL/6 mice were cultured until day 5, stimulated with TCM and LysoPS for 12 h. ELISA detected CXCL16 secretion in supernatant. Bar plot shows levels in LysoPS vs. control groups. Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: Cell Culture, Flow Cytometry, Co-Culture Assay, Expressing, Sequencing, Negative Control, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage efferocytosis function influences antigen presentation ability through MHC-I. a BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 hours, then analyzed by flow cytometry for GFP uptake. Bar plot shows gMFI of GFP in BMDMs treated with MerTK inhibitor vs control ( n = 3). One-way ANOVA with Dunnett’s test compared MerTKi groups to control. Data are presented by mean ± SD. b BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP cells for 12 hours, treated with MerTK inhibitor, then co-cultured with CD8 + T cells from OT1 mice for 24 hours. Flow cytometry detected MHC-I, SIINFEKL loading, CD80, CD86 on BMDMs. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. c Flow cytometry detection of Tetramer + , PD-1 + , Tim-3 + , and GZMB + cells after co-culture of BMDMs with OT1 CD8 + T cells. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. d Apoptotic KPC cells induced by chemotherapy and labeled with Caspase3/7 green were co-cultured with BMDMs. Phagolysosome formation was detected using pHrodo red. Representative fluorescence microscopy images (1000x) show differences between MerTK inhibitor and control groups ( n = 6). Green: Caspase3/7, Red: pHrodo, Blue: DAPI. White scale bar= 20 μm. e Bar plots show total pHrodo fluorescence intensity (left) and the number of Caspase3/7 + pHrodo + vesicles per cell (right) in BMDMs after incubation with apoptotic cells ( n = 6). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. f Flow cytometry analysis of pHrodo gMFI in BMDMs after incubation with apoptotic cells. Bar plot shows pHrodo gMFI levels between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. g Violin-box plots of lysosome-associated gene transcript expression in macrophage subpopulations from scRNA sequencing data. Solid lines represent medians and quartiles. One-way ANOVA with Kruskal-Wallis H test compared groups (Mac_cl1 as reference). h , i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours. After removing apoptotic cells, RNA was extracted for qPCR. Bar plots show transcript differences between Gpr34 +/+ and Gpr34 −/− BMDMs ( h ) efferocytosis receptors, ( i ) lysosome-related/transcription factors, ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-incubated with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours and analyzed by flow cytometry. Bar plot shows differences in MHC-I protein levels between knockdown and control groups ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. k BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with lysosomal inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Bar plot shows pHrodo gMFI in macrophages from flow cytometry, comparing lysosomal inhibitor group vs control. One-way ANOVA with Dunnett’s test was used . Data are presented by mean ± SD. l , m Flow cytometry detection of macrophage antigen presentation function ( l ) and CD8 + T cell specific killing capacity ( m ) in the BMDM-OT1 CD8 + T cell co-culture system. Bar plots show differences between lysosomal inhibitor and control groups. One-way ANOVA with Dunnett ’ s test was used. Data are presented by mean ± SD. n BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM, MerTK inhibitor/Lysosome inhibitor and chemotherapy-induced apoptotic KPC cells for 12 hours. Bar plot shows differences in MHC-I protein levels between different groups detected by flow cytometry ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. o –q BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, transiently transfected with Cxcl16 siRNA, co-incubated with TCM, LysoPS and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with MerTK inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Flow cytometry detected CD8 + T cell specific killing function (o ), cytotoxic function ( p ), and exhaustion levels ( q ). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Control, Incubation, Co-Culture Assay, Labeling, Fluorescence, Microscopy, Expressing, Sequencing, Two Tailed Test, Transfection, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: GPR34 Antagonist Sensitizes Chemotherapy and Surufatinib. a C57BL/6 mice underwent orthotopic pancreatic injection with KPC-LUC cells. After tumor formation, mice were treated with chemotherapy, Surufatinib, and GPR34 antagonist (GPR34a). Tumor dynamics were monitored by bioluminescence imaging. Representative bioluminescence images show dynamic changes in different treatment groups ( n = 6). b Tumor growth curve from bioluminescence imaging of orthotopic tumor-bearing mice ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD at each time point. c Bar plot showing differences in orthotopic tumor weight after treatment ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d KPC mice underwent B-ultrasound examination twice weekly during treatment. Representative B-ultrasound images show the last examination before experiment endpoint or death ( n = 10). White dashed lines indicate tumor boundaries. S: spleen. K: kidney. e The fold change of the tumor volume at the last examination before death or euthanasia in the KPC spontaneous tumor-bearing model to the baseline level ( n = 10). Two-way ANOVA with Kruskal-Wallis H test was used. Data are presented by mean ± SD. f Kaplan-Meier curves comparing survival of KPC mice receiving different treatments. The experimental endpoint was 102 days after treatment initiation. Log-rank test was used. g Organoids derived from patient tumor tissue were co-cultured with matched PBMCs and treated with different drugs. Apoptosis was labeled with caspase-3/7 probe and imaged ( n = 6). Representative microscopy images show organoid morphology and apoptosis protein levels in different treatment groups. Green: caspase3/7. Black scale bar = 50 μm. h Bar plot showing the gMFI of caspase-3/7 in the organoid-PBMC co-culture model across treatment groups ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model. Gpr34 global knockout ( Gpr34 −/− ) mice were generated by crossing Gpr34 flox/flox mice with Dppa3 -Cre mice (C57BL/6Smoc- Dppa3 em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040).

Techniques: Injection, Imaging, Derivative Assay, Cell Culture, Labeling, Microscopy, Co-Culture Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Single-cell RNA sequencing reveals the cellular composition of pancreatic cancer after chemotherapy combined with Surufatinib treatment. a Schematic diagram of the process of this study. First, we performed single-cell RNA (scRNA) sequencing of surgical specimens obtained from patients who were treated with gemcitabine, nab-paclitaxel, and surufatinib in the clinical trial. Patients were divided into responder group and non-responder group according to clinicopathological evaluation. Comparing the differences in macrophage subsets between the two groups, it was found that GPR34 + damage-associated macrophages (DAMs) were absolutely dominant in the treatment-resistant patients. Subsequently, GPR34 + DAMs function and underlying mechanisms were validated in retrospective and prospective pancreatic cancer patient cohorts and mouse models. Finally, targeting GPR34 + DAMs was demonstrated to enhance the efficacy of surufatinib in a preclinical model. Schematic diagram was created with BioRender.com. PDAC: pancreatic ductal adenocarcinoma, DAMs: damage associated macrophages, scRNA: single cell RNA. b Representative contrast-enhanced CT images of responder group (R) and non-responder group (NR) before and after treatment. Patients underwent contrast-enhanced CT and tumor assessment every 8 weeks, and two independent experienced radiologists determined the location and contour of the target lesion. The length of the tumor was measured and counted by pancreatic tumor specialists. The baseline level before treatment as well as the last evaluation before surgery were compared respectively. c Waterfall plot showing efficacy evaluation results from clinical trial patients, with upper dashed line indicating the cutoff for disease progression according to the RECIST (Response Evaluation Criteria in Solid Tumors) V1.1 standard, lower dashed line indicating the cutoff for PR. The final evaluation results are marked above and below each column of the waterfall plot. d UMAP plot displaying the cellular subtypes of all cells ( n = 84380) in the surgical specimens after treatment. e Scale bar chart showing the cell type composition of all patient samples, and aggregated samples from non-responders and responders. f The box plot compared the proportion differences of each cell type between non-responders and responders. A beta regression analysis was used to examine the correlation between the proportions of various cell components and treatment efficacy. g UMAP plot showing macrophage subtypes clustering in all patient samples. mac macrophage, cl cluster. h Bar chart showing the composition of macrophage cluster in all patient samples and aggregated samples from non-responders and responders. i The box plot compared the proportion differences of each macrophage subcluster between non-responders and responders. A beta regression analysis was used to examine the correlation between the proportions of various subclusters and the treatment efficacy. j Heatmap displaying the expression of key functional genes across macrophage subclusters. The characteristic genes of each subpopulation were ranked by log 2 FC, and the top 50 genes with adjusted P -value < 0.05 for each subcluster were selected for visualization and gene set enrichment analysis (GSEA). The main enrichment pathways were selected for display. Each column represents a cell barcode, sorted by cell type, and each row represents a gene. k Bar plot showing the GSEA analysis results for the Mac_cl1 cluster. The analysis was based on the Hallmarks, GOBP and KEGG data sets, and all results were screened based on the scores of the data sets mentioned above. NS no significance, * P < 0.05

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: Single Cell, RNA Sequencing, Sequencing, Biomarker Discovery, Expressing, Functional Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Cohort and in vitro studies reveal the role of GPR34 in macrophages. a Volcano plot showing differentially expressed genes between responder group and non-responder group from the macrophage subclusters of scRNA sequencing data. The horizontal dashed line represents the P -value cutoff ( P < 10⁻⁵⁰), and the vertical dashed line represents the log 2 FC cutoff (-1 or 1). FC fold change, sig significance, R Responder, NR Non-responder. b , c UMAP plot showing the expression of GPR34 in all cells ( b ) and macrophages ( c ). Mac: Macrophage, cl cluster. d Representative mIF staining images (100x) of surgical specimens from responders and non-responders in the clinical trial ( n = 26). Navy: GPR34, Magenta: CD68, Red: CD8, Green: Tim-3, Yellow: CK19, Blue: DAPI. White arrowheads: Tim-3 + CD8 + exhausted T cells (Tex); White arrows: GPR34 + CD68 + macrophages. White scale bar = 100 μm. e Bar plot comparing the proportion of CD68 + macrophages (top) and the proportion of GPR34 + cells among CD68 + macrophages (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. * P < 0.05. f Bar plot comparing the proportion of CD8 + T cells (top) and the proportion of Tim-3 + cells among CD8 + T cells (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. g Representative mIF staining images (200x) of surgical specimens from clinical trial patients ( n = 26). Navy: GPR34, Magenta: CD68, Red: MPO, Green: CD3, Dark yellow: CD20, Yellow: CK19, Blue: DAPI. White scale bar = 50 μm. h Representative mIF staining images (200x) of clinical trial patient surgical specimens ( n = 26). Navy: GPR34, Magenta: CD68, Red: α-SMA, Green: CD31, Dark yellow: CD117, Yellow: CD56, Blue: DAPI. White scale bar = 50 μm. i Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26). One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. j Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26) examined by flow cytometry. One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. k , l Kaplan-Meier curves for overall survival (OS) and Recurrence-free survival (RFS) in the prospective cohort ( k ) and retrospective cohort ( l ), comparing prognosis between groups with high versus low infiltration of GPR34 + macrophages. The cutoff for the proportion of GPR34 + cells among CD68 + cells were dichotomized using a 20% . Log-rank test was used for comparison. m Flow cytometry analysis of GPR34 + cells in tumor tissue versus adjacent non-tumorous tissue from prospective cohort patients ( n = 42). Scatter plot shows the paired infiltration proportion of GPR34 + cells in tumor and normal tissue from the same patient . Two-tailed paired t -test was used. n Flow cytometry analysis of GPR34 + cells in tumor tissue from prospective cohort patients ( n = 42). Bar plots show the infiltration proportions of CD45 + cells, CD8 + T cells, Tim-3 + PD-1 + T cells, MRC1 + macrophages, and MHC-I + macrophages in the low GPR34 ( ≤ 20%) versus high GPR34 ( > 20%) groups. Two-tailed unpaired t-test. Data are presented by mean ± SD. o BMDMs from C57BL/6 mice were cultured until day 5, stimulated with KPC cell TCM for 12 h, followed by treatment with Surufatinib (4 nM) or CSF-1R inhibitor (PLX3397, 20 nM) for 24 h, then analyzed by flow cytometry ( n = 3). Bar plot compares the gMFI of GPR34 among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: In Vitro, Sequencing, Expressing, Staining, Two Tailed Test, Flow Cytometry, Comparison, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage-specific GPR34 knockout improves chemotherapy outcomes in mice. a Orthotopic pancreatic injection of KPC-GFP-LUC cells into Gpr34 flox/flox and Gpr34 Δ Lyz2 mice. After tumor formation, chemotherapy was administered to simulate tumor killing. Tumor bioluminescence intensity was dynamically monitored by bioluminescence imaging. Representative bioluminescence images show tumor growth in each group ( n = 5). b Time-course curve of bioluminescence intensity in orthotopic tumor-bearing mice (n = 5). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SEM. c Bar plot of tumor weight on day 21 post-implantation in orthotopic tumor-bearing mice ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS: no significance, *: P < 0.05. d Flow cytometry analysis of the proportions of CTL, Tex, M1 macrophages, and M2 macrophages in tumor tissues of orthotopic tumor-bearing mice (n = 5). Bar plots show the infiltration differences of immune cells among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. e KPC mice were irradiated with a total dose of 10 Gy, followed by tail vein injection of bone marrow cells from Gpr34 +/+ or Gpr34 −/− mice. After successful transplantation confirmed by flow cytometry. Tumor growth was monitored by B-ultrasound during chemotherapy simulating injury signals ( n = 8–10). Representative B-ultrasound images compare tumor size among groups. Black dashed lines outline tumor boundaries. Tumor volume was calculated as 0.5 * long diameter * (short diameter) ². S: spleen; K: kidney. Black arrows point to the tumor. f Time-course plot of tumor volume monitored by B-ultrasound. Each solid line represents an individual mouse. Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. g Kaplan-Meier curve of KPC spontaneous tumor-bearing mice ( n = 8–10). Log-rank test was used. h mIF staining of paraffin sections from orthotopic tumors in the bone marrow transplantation KPC mice ( n = 6). Representative fluorescence images (top 100x, bottom 400x) show exhausted T cell infiltration among groups. Red: CD8α, Green: Tim-3, White: CK19, Blue: DAPI. White scale bar = 50 μm. i Bar plot showing the proportion of Tim-3 + CD8 + T cells from mIF staining in KPC spontaneous tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. j Fluorescence imaging of macrophages sorted by flow cytometry from tumors of KPC-GFP-LUC orthotopic injection mice model. Representative images show GFP phagocytosis by macrophages in each group ( n = 6). Green: GFP, Blue: DAPI. White scale bar = 20 μm. k Bar plot showing the gMFI of GFP phagocytosis by flow-sorted macrophages from orthotopic tumors ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. l Bar plot of the gMFI of GFP in macrophages from KPC cell orthotopic tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. m Bar plot showing the proportion of MerTK + or AXL + macrophage from flow cytometry analysis in KPC mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used . Data are presented by mean ± SD. n BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, cultured with TCM for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected the proportion of MerTK + or AXL + macrophage. Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 6). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: Knock-Out, Injection, Imaging, Flow Cytometry, Irradiation, Transplantation Assay, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Validation of GPR34 function in macrophage and CD8 + T cell co-culture system. a Gpr34 flox/flox and Gpr34 Δ Lyz2 mice were treated with anti-CD8α or IgG, followed by orthotopic pancreatic injection of KPC-LUC cells. After tumor formation, chemotherapy was administered to simulate an injury signal. Tumor bioluminescence was dynamically monitored. Representative bioluminescence images show tumor growth in different groups ( n = 6). b Time-course curve of bioluminescence imaging for the KPC-LUC orthotopic model ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, *** P < 0.001. c Bar plot showing tumor weight on day 21 in the KPC-LUC orthotopic model ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance. d , e BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then co-cultured with TCM and KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells for 24 hours . Flow cytometry analyzed the expression of functional molecules in BMDMs ( d ) and CD8 + T cells ( e ). Bar plots show levels in Gpr34 +/+ vs Gpr34 −/− groups ( n = 3). Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. f , g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, pre-stimulated with SIINFEKL, then cultured with TCM for 12 h, followed by co-culture with CD8 + T cells from OT-1 mice for 24 h. Flow cytometry detected T cell-specific killing function ( f ) and BMDM antigen presentation function ( g ). Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. h Violin-box plots of cytokine transcript expression in macrophage clusters from scRNA sequencing data. White dot and solid lines in boxes represent medians and quartiles. Two-tailed Wilcoxon test. i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then stimulated with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. qPCR detected Cxcl16 transcript levels. Bar plot compares Cxcl16 transcripts between groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j ELISA detection of cytokine secretion in supernatant from BMDMs stimulated with apoptotic KPC-GFP cells. Bar plot shows CXCL16 protein secretion levels from Gpr34 +/+ and Gpr34 −/− BMDMs ( n = 10). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. k , l BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells. Flow cytometry detected T cell exhaustion ( k ) and cytotoxicity levels ( l ) ( n = 3). One-way ANOVA with Dunnett’s test compared siRNA groups versus control. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: Biomarker Discovery, Co-Culture Assay, Injection, Imaging, Cell Culture, Isolation, Flow Cytometry, Expressing, Functional Assay, Two Tailed Test, Immunopeptidomics, Sequencing, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: LysoPS-GPR34 modulates macrophage efferocytosis and inflammatory cytokine secretion. a , b BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. These were then co-cultured with CD8 + T cells for 24 hours. Flow cytometry detected GFP efferocytosis ( a ) and p-AKT levels ( b ) in BMDMs. Bar plots show differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. c BMDMs from Gpr34 +/+ and Gpr34 −/− mice were treated with LysoPS and co-cultured with CD8 + T cells. Flow cytometry detected MRC1, MHC-I on macrophages, and Tim-3, GZMB on CD8 + T cells. Bar plot shows gMFI differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d , e BMDMs from C57BL/6 mice were treated with LysoPS and AKT inhibitor, then co-cultured with chemotherapy-induced apoptotic KPC-GFP cells for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected GFP uptake by BMDMs ( d ) and exhaustion/cytotoxicity levels of CD8 + T cells ( e ). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. f UMA P plot showing relative expression of efferocytosis-related genes from scRNA sequencing analysis. g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and either chemotherapy-induced apoptotic KPC-GFP cells or normal KPC-GFP cells for 12 h. After removing cells, RNA was extracted for qPCR. Bar plot compares transcript levels among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. h BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with apoptotic KPC cells of negative control for 12 hours. RNA was extracted for qPCR. Bar plot compares transcript differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. i BMDMs from C57BL/6 mice were cultured until day 5, stimulated with TCM and LysoPS for 12 h. ELISA detected CXCL16 secretion in supernatant. Bar plot shows levels in LysoPS vs. control groups. Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: Cell Culture, Flow Cytometry, Co-Culture Assay, Expressing, Sequencing, Negative Control, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage efferocytosis function influences antigen presentation ability through MHC-I. a BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 hours, then analyzed by flow cytometry for GFP uptake. Bar plot shows gMFI of GFP in BMDMs treated with MerTK inhibitor vs control ( n = 3). One-way ANOVA with Dunnett’s test compared MerTKi groups to control. Data are presented by mean ± SD. b BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP cells for 12 hours, treated with MerTK inhibitor, then co-cultured with CD8 + T cells from OT1 mice for 24 hours. Flow cytometry detected MHC-I, SIINFEKL loading, CD80, CD86 on BMDMs. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. c Flow cytometry detection of Tetramer + , PD-1 + , Tim-3 + , and GZMB + cells after co-culture of BMDMs with OT1 CD8 + T cells. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. d Apoptotic KPC cells induced by chemotherapy and labeled with Caspase3/7 green were co-cultured with BMDMs. Phagolysosome formation was detected using pHrodo red. Representative fluorescence microscopy images (1000x) show differences between MerTK inhibitor and control groups ( n = 6). Green: Caspase3/7, Red: pHrodo, Blue: DAPI. White scale bar= 20 μm. e Bar plots show total pHrodo fluorescence intensity (left) and the number of Caspase3/7 + pHrodo + vesicles per cell (right) in BMDMs after incubation with apoptotic cells ( n = 6). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. f Flow cytometry analysis of pHrodo gMFI in BMDMs after incubation with apoptotic cells. Bar plot shows pHrodo gMFI levels between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. g Violin-box plots of lysosome-associated gene transcript expression in macrophage subpopulations from scRNA sequencing data. Solid lines represent medians and quartiles. One-way ANOVA with Kruskal-Wallis H test compared groups (Mac_cl1 as reference). h , i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours. After removing apoptotic cells, RNA was extracted for qPCR. Bar plots show transcript differences between Gpr34 +/+ and Gpr34 −/− BMDMs ( h ) efferocytosis receptors, ( i ) lysosome-related/transcription factors, ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-incubated with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours and analyzed by flow cytometry. Bar plot shows differences in MHC-I protein levels between knockdown and control groups ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. k BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with lysosomal inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Bar plot shows pHrodo gMFI in macrophages from flow cytometry, comparing lysosomal inhibitor group vs control. One-way ANOVA with Dunnett’s test was used . Data are presented by mean ± SD. l , m Flow cytometry detection of macrophage antigen presentation function ( l ) and CD8 + T cell specific killing capacity ( m ) in the BMDM-OT1 CD8 + T cell co-culture system. Bar plots show differences between lysosomal inhibitor and control groups. One-way ANOVA with Dunnett ’ s test was used. Data are presented by mean ± SD. n BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM, MerTK inhibitor/Lysosome inhibitor and chemotherapy-induced apoptotic KPC cells for 12 hours. Bar plot shows differences in MHC-I protein levels between different groups detected by flow cytometry ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. o –q BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, transiently transfected with Cxcl16 siRNA, co-incubated with TCM, LysoPS and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with MerTK inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Flow cytometry detected CD8 + T cell specific killing function (o ), cytotoxic function ( p ), and exhaustion levels ( q ). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Control, Incubation, Co-Culture Assay, Labeling, Fluorescence, Microscopy, Expressing, Sequencing, Two Tailed Test, Transfection, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: GPR34 Antagonist Sensitizes Chemotherapy and Surufatinib. a C57BL/6 mice underwent orthotopic pancreatic injection with KPC-LUC cells. After tumor formation, mice were treated with chemotherapy, Surufatinib, and GPR34 antagonist (GPR34a). Tumor dynamics were monitored by bioluminescence imaging. Representative bioluminescence images show dynamic changes in different treatment groups ( n = 6). b Tumor growth curve from bioluminescence imaging of orthotopic tumor-bearing mice ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD at each time point. c Bar plot showing differences in orthotopic tumor weight after treatment ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d KPC mice underwent B-ultrasound examination twice weekly during treatment. Representative B-ultrasound images show the last examination before experiment endpoint or death ( n = 10). White dashed lines indicate tumor boundaries. S: spleen. K: kidney. e The fold change of the tumor volume at the last examination before death or euthanasia in the KPC spontaneous tumor-bearing model to the baseline level ( n = 10). Two-way ANOVA with Kruskal-Wallis H test was used. Data are presented by mean ± SD. f Kaplan-Meier curves comparing survival of KPC mice receiving different treatments. The experimental endpoint was 102 days after treatment initiation. Log-rank test was used. g Organoids derived from patient tumor tissue were co-cultured with matched PBMCs and treated with different drugs. Apoptosis was labeled with caspase-3/7 probe and imaged ( n = 6). Representative microscopy images show organoid morphology and apoptosis protein levels in different treatment groups. Green: caspase3/7. Black scale bar = 50 μm. h Bar plot showing the gMFI of caspase-3/7 in the organoid-PBMC co-culture model across treatment groups ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Macrophage Gpr34 conditional knockout mouse model ( Gpr34 Δ Lyz2 ) was constructed by Gpr34 flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2 -CreERT mice (B6.129P2- Lyz2 tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing.

Techniques: Injection, Imaging, Derivative Assay, Cell Culture, Labeling, Microscopy, Co-Culture Assay

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: ABHD16A–LysoPS enhances ILC3 activation via the GPR34–STAT3–AKT signaling pathway. A, MNK3 cells were treated with or without LysoPS (10 μmol/L); the levels of IL22, pAKT, AKT, pSTAT3, and STAT3 were assessed by Western blotting. B, Western blotting was performed to check IL22, pAKT, AKT, pSTAT3, and STAT3 levels in MNK3 cells cocultured with MFC cells, Abhd16a -knockdown MFC cells, or Abhd16a -knockdown MFC cells with simultaneous supplementation of LysoPS (10 μmol/L). C, Western blotting was conducted to assess IL22, pAKT, AKT, pSTAT3, and STAT3 levels in MNK3, Gpr34 -knockdown MNK3, and perifosine-pretreated MNK3 cells, all under LysoPS supplementation (10 μmol/L). D, ELISA analysis of IL22 production in supernatants from MNK3 cells under the same treatments as in B . E, ELISA analysis of IL22 production in the supernatant from MNK3 cells under the same treatments as in C . F, ELISA analysis of IL22 production by ILC3s and ILC3s treated with LysoPS in the presence or absence of perifosine. G and H, LysoPS (2.5 mg/kg) was administered to Abhd16a -knockdown gastric cancer mice and control mice in the presence or absence of a GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg) or AKT inhibitor (perifosine, 20 mg/kg). The representative living images on days 3, 10, and 17 ( G ) and tumor volume at the end of the experiment ( H ) are shown. Scale bar, 1.000e+4 – 1.000e + 5 p/s/cm 2 /sr; n = 5 per group. I–K, The proportions of ILC3s ( I ), pSTAT3 + ILC3s ( J ), and IL22 + ILC3s ( K ) in total CD45 + cells derived from orthotopic gastric cancer tissues. Mice treatments were the same as G and H . **, P < 0.01; ns, nonsignificant.

Article Snippet: For the animal experiment, LysoPS (2.5 mg/kg, Sigma, 858144P), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), AKT inhibitor (perifosine, 20 mg/kg, MedChemExpress, HY-50909), XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845), IL22 Monoclonal Antibody (200 μg/mouse, eBioscience, 16-7222-82, RRID: AB_2016695), rmIL22 (500 ng/mouse, ABclonal, RP02942), and CD90.2 (150 μg/mouse, BioXCell, BE0066, RRID: AB_1107682) for ILC3 depletion were intraperitoneally injected once every 3 days.

Techniques: Activation Assay, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Combination therapy enhances the anti–PD-L1 immunotherapeutic effect in gastric cancer. A and B, After tumor formation, the orthotopic gastric cancer mice ( n = 5 per group) were treated with anti–PD-L1 (100 μg per mouse), GPR34 inhibitor (20 mg/kg), or XBP1s inhibitor (30 mg/kg) every 3 days or ACh inhibitor (2.5 mg/kg) daily. Combinations of anti–PD-L1 with each inhibitor followed the every 3-day dosing schedule for a total duration of 2 weeks via i.p. injection. Living images were used to monitor tumor progression at 5-day intervals from the time of drug administration ( A ); IHC and mIF were performed to detect PD-L1 and XBP1s levels and proportions of CD4 + (green) and CD8 + (red) T cells in gastric cancer tissues at the end of treatments ( B ). Scale bars, 1.000e+5 –∼ 5.000e + 5 p/s/cm 2 /sr for living images; 200 μm for IHC; 50 μm for immunofluorescence. C and D, Representative images ( C ) and tumor volume ( D ) of subcutaneous tumors. The administration protocol for the mice was consistent with the description provided in A and B . **, P < 0.01; ***, P < 0.001.

Article Snippet: For the animal experiment, LysoPS (2.5 mg/kg, Sigma, 858144P), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), AKT inhibitor (perifosine, 20 mg/kg, MedChemExpress, HY-50909), XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845), IL22 Monoclonal Antibody (200 μg/mouse, eBioscience, 16-7222-82, RRID: AB_2016695), rmIL22 (500 ng/mouse, ABclonal, RP02942), and CD90.2 (150 μg/mouse, BioXCell, BE0066, RRID: AB_1107682) for ILC3 depletion were intraperitoneally injected once every 3 days.

Techniques: Injection, Immunofluorescence