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: For antigen-specific assays, BMDMs were pulsed with 1 μg/mL SIINFEKL peptide (MCE, HY-P1489) for 2 hours.

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: 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: For antigen-specific assays, BMDMs were pulsed with 1 μg/mL SIINFEKL peptide (MCE, HY-P1489) for 2 hours.

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

Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) Zeta potential and quantification of OVA protein adsorption on Aze-Chol NP@OVA nanovaccines. ( B ) STORM image of Aze-Chol NP@OVA. Green (rhodamine B), Aze-Chol NP; red (Cy5), OVA. ( C ) Representative confocal fluorescence images of DC2.4 cells incubated with Cy5-OVA or Aze-Chol NP@Cy5-OVA (Aze-Chol NP at 20 μg/ml). Red, Cy5-OVA; blue, nuclei. Scale bars, 10 μm. ( D and E ) Flow cytometry analysis of OVA uptake by BMDCs incubated with free OVA or Aze-Chol NP@OVA (Aze-Chol NP at 20 μg/ml) for 24 hours. (D) Representative histograms; (E) MFI quantification. FITC, fluorescein isothiocyanate. ( F ) Heatmap showing MFI of CD80, CD86, CD40, and MHCII expression on BMDCs after incubation with OVA, Aze-Chol NP, or Aze-Chol NP@OVA for 24 hours. ( G and H ) Biodistribution kinetics of Aze-Chol NP@Cy5-OVA (Aze-Chol NP at 20 mg/kg) in ILNs of C57BL/6 mice following subcutaneous administration. (G) Ex vivo fluorescence imaging and (H) semiquantitative analysis at indicated time points postinjection with free Cy5-OVA or Aze-Chol NP@Cy5-OVA. ( I and J ) Flow cytometry analysis of Cy5-OVA uptake by (I) resident CD8α + CD11c + MHCII + cDC1s and (J) migratory CD103 + CD11c + MHCII + cDC1s in ILNs following subcutaneous injection with free Cy5-OVA or Aze-Chol NP@Cy5-OVA. ( K ) SIINFEKL-MHCI complex levels on BMDCs after incubation with OVA (10 μg/ml) or Aze-Chol NP@OVA (Aze-Chol NP at 20 μg/ml) for 24 hours. ( L and M ) OT-I CD8 + T cell proliferation and activation after 72-hour coculture with OVA- or Aze-Chol NP@OVA–treated BMDCs. Left shows representative flow cytometry plots; right shows quantification of (L) proliferation (by CFSE dilution) and (M) activation (by CD69 expression). ( N ) In vivo evaluation of OVA-specific CTL responses. Representative flow cytometry plots and quantification of OVA peptide–specific target cell lysis percentages are shown. ** P < 0.01 and **** P < 0.0001.

Article Snippet: OVA peptide (SIINFEKL) and E7 peptide (AGQAEPDRAHYNIVTFCCKCDS) were synthesized by Sangon Biotech (Shanghai, China), which were purified by reversed-phase high-performance liquid chromatography to a purity exceeding 95%.

Techniques: Zeta Potential Analyzer, Adsorption, Fluorescence, Incubation, Flow Cytometry, Expressing, Ex Vivo, Imaging, Injection, Activation Assay, In Vivo, Lysis

Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) Schematic showing the immunization schedule of C57BL/6 mice subcutaneously (sc) injected with PBS (G1; Group 1), OVA alone (G2; Group 2), CpG + OVA (G3; Group 3), or Aze-Chol NP@OVA nanovaccine (G4; Group 4; Aze-Chol NP at 20 mg/kg) once weekly for 3 weeks. ( B ) Flow cytometry quantification of cDCs presenting SIINFEKL peptide on H-2K b in dLNs of immunized mice. ( C ) Representative dot plots (left) and quantification (right) of OVA-specific (SIINFEKL-MHCI tetramer-positive) CD8 + T cells in peripheral blood after immunization. ( D and E ) Representative dot plots (D) and quantification (E) of IFN-γ–positive CD8 + T cells in peripheral blood postimmunization. ( F ) Serum IFN-γ concentrations quantified by ELISA at day 21 postimmunization. ( G ) Experimental timeline for assessing antigen-specific immune responses after immunization with OVA, CpG + OVA, or Aze-Chol NP@OVA. ( H ) Representative ELISPOT images of IFN-γ–secreting splenocytes stimulated ex vivo with SIINFEKL peptide for 48 hours. ( I and J ) Quantification of (I) IFN-γ spot-forming units (SFU) and (J) sum of spot volume (SSV) from splenocytes stimulated as described in (G). ( K ) Levels of cytokines (IFN-γ, TNF-α, IL-2, and granzyme B) associated with T cell activation and effector function, measured by ELISA after ex vivo stimulation of splenocytes with SIINFEKL peptide for 72 hours. ( L ) Percentages of specific tumor cell lysis measured by Hoechst 33342 staining following 72-hour coculturing of splenocytes from immunized mice with OVA-expressing B16F10 cells. ( M ) Schemes showing the tumor challenge experiment design. D 0, D 5, D 10 and D15 indicate Day 0, Day 5, Day 10 and Day 15, respectively. ( N ) Average (left) and individual (right) tumor growth curves of C57BL/6 mice bearing B16F10-OVA tumors, treated as indicated. ( O ) Survival curves of tumor-bearing mice after treatment with PBS, OVA, CpG + OVA, or Aze-Chol NP@OVA. ( P ) Representative immunofluorescence staining showing CD8 + T cell infiltration (red) and nuclei (blue) in tumor sections from indicated treatment groups. Scale bars, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: OVA peptide (SIINFEKL) and E7 peptide (AGQAEPDRAHYNIVTFCCKCDS) were synthesized by Sangon Biotech (Shanghai, China), which were purified by reversed-phase high-performance liquid chromatography to a purity exceeding 95%.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Ex Vivo, Activation Assay, Lysis, Staining, Expressing, Immunofluorescence

Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) Therapeutic regimen of Aze-Chol NP-OVAp vaccine in the B16F10-OVA melanoma model. ( B ) (Left) Average and (right) individual tumor growth curves of C57BL/6 mice treated with OVA peptide, CpG + OVA peptide, and Aze-Chol NP-OVAp (Aze-Chol NP at 20 mg/kg). ( C ) Frequency of OVA antigen–specific effector memory CD8 + T cells in various tissues, analyzed by flow cytometry. ( D ) Effector function of tumor-infiltrating CD8 + T cells. Percentages of TNF-α, IFN-γ, or granzyme B–secreted CD8 + T cells within tumor after OVA peptide restimulation are shown. ( E ) Proportion of multifunctional CD8 + T cells capable of simultaneously secreting one, two, and three effector cytokines (TNF-α, IFN-γ, and/or granzyme B) in tumor was displayed as a 10 by 10 dot plot. ( F ) Schematic of therapeutic regimen and tumor rechallenge study of Aze-Chol NP-E7p in combination with αPD-L1 antibody in TC-1 cervical cancer model. ip, intraperitoneal. ( G ) Average TC-1 tumor growth curves of C57BL/6 mice treated with αPD-L1 antibody, Aze-Chol NP-E7p, and Aze-Chol NP-E7p in combination with αPD-L1 antibody (Aze-Chol NP at 20 mg/kg). ( H and I ) (H) Survival curves of C57BL/6 mice following indicated treatments were obtained using the Kaplan-Meier method, (I) along with pie charts representing the percentage of cured mice quantified by tumor-free survival (>60 days). ( J and K ) Long-term immune effect induced by Aze-Chol NP-E7p against TC-1 rechallenge. (J) Average tumor growth curves and (K) survival curves of cured mice from indicated groups after TC-1 tumor rechallenge on day 45. Age-matched naive C57BL/6 mice served as controls. ( L and M ) Representative flow cytometry plots and frequencies of central memory (CD44 + CD62L + ) CD8 + T cells (top) and CD4 + T cells (down) in the (L) spleen and (M) bone marrow. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: OVA peptide (SIINFEKL) and E7 peptide (AGQAEPDRAHYNIVTFCCKCDS) were synthesized by Sangon Biotech (Shanghai, China), which were purified by reversed-phase high-performance liquid chromatography to a purity exceeding 95%.

Techniques: Flow Cytometry