Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl CD4 Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Knock-Out, Generated, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 fl/fl CD4 Cre mice exhibit improved anti-tumor mmune responses. (A-C) Naïve CD4 + T cells were isolated from CD4 Cre or GPCR68 fl/fl CD4 Cre mice and activated using anti-CD3 and anti-CD28 using the culture media under physiologic neutral pH (7.4) or varying pH 6.0, 6.5, or 7.8. Flow cytometry plots showing the expression of IFN-γ and IL-2 in CD4 + T cells from CD4 Cre and GPCR68 fl/fl CD4 Cre mice. Each panel represents the frequency of IFN-γ + and IL-2 + cells. (B) Bar graph summarizing the percentage of IFN-γ + CD4 + T cells at each pH level for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (C) Bar graph showing the percentage of IL-2 + CD4 + T cells at each pH for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (D) Experimental timeline depicting tumor induction and treatment protocol in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (E) Tumor growth curves in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (F) Tumor weight in CD4 Cre versus GPCR68 fl/fl CD4 Cre mice at the time of harvesting on day 21. (G) Representative images of excised tumors at day 21. (H) Flow cytometric analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (I) Flow cytometric analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Flow Cytometry, Expressing, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Anti-tumor effects of borate bioactive glass (BOLT) in B16 tumor. (A) Schematic illustration depicting the induction of B16 melanoma tumors, followed by treatment with BOLT at various time points, and tumor harvesting for subsequent analysis. (B) Tumor growth curves showing tumor volume in Control and BOLT-treated B16 melanoma tumors in mice. (C) Tumor weight at the time of harvesting in the BOLT-treated group compared to the Control. (D) Representative images of excised tumors from Control and BOLT-treated mice. (E) In vivo imaging of tumor-bearing mice in both the Control and BOLT-treated groups. (F) Flow cytometry analysis showing IFN-γ production in CD4 + and CD8 + T cells following BOLT treatment compared to Control. (G) Flow cytometry analysis demonstrated TNF-α production in CD4 + and CD8 + T cells in the BOLT-treated group, with a significant increase observed in CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data represent the mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Control, In Vivo Imaging, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Combinational treatment of BOLT and anti-CTLA-4 blockade enhances anti-tumor immune response in B16 melanoma. (A) C57BL/6 mice were subcutaneously injected with 1 × 10 5 B16 melanoma cells on day 0 to induce tumors. On day 7, mice were randomly divided into groups and treated with either BOLT alone (intratumoral injection administered on alternate days starting from day 7), anti-CTLA-4 (intraperitoneal injection administered on days 9, 11, 13, and 15), or a combination of both treatments. PBS was used as a vehicle Control, while IgG was used as anti-CTLA-4 Control. Tumor growth was monitored throughout the treatment period, and tumors were harvested for analysis on day 21. (B-C) Tumor growth curves and area under the curve (AUC) analysis for WT mice treated with BOLT, with or without anti-CTLA-4 antibody, following subcutaneous injection of B16 melanoma cells. Tumor growth was monitored, and analysis was conducted on day 21. (D) Representative images of excised tumors at day 21, showed reduced tumor size in combination-treated mice. (E, F) Flow cytometry analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (G, H) Flow cytometry analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Injection, Control, Flow Cytometry

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: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

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: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

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: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

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: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

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: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

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