Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A promotes Fl-BMDC maturation via ADAM10. a Predicted accurate model of the interaction of HPFs with mouse ADAM10 (pLDDT = 72.7). The predicted local distance difference test score (pLDDT) was used to evaluate per-residue confidence, with values ≥ 70 indicating reliable backbone modeling. b Representative fluorescence images showing interactions of PA0833 or HPF with ADAM10 on the surface of Fl-BMDCs after 6 h of treatment with PA0833, HPF, or HPF with GI254023X ( n = 3 per group). The antigens used were PA0833 or HPF. The arrowheads indicate cells that captured the antigens. c Representative flow cytometry histogram (left) and quantification (right) of MHCII + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. d Representative flow cytometry histogram (left) and quantification (right) of CD40 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. e Representative flow cytometry histogram (left) and quantification (right) of CD80 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. f Representative flow cytometry histogram (left) and quantification (right) of CD86 + cell frequencies gated on CD11c + cells from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. g Serum levels of TNF-α or IL-6 secreted from Fl-BMDCs after 7.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 6 per group). The data were pooled from two independent experiments. Each data point indicates a biological replicate in ( c – g ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( c – g ). Fl-BMDCs Flt3L-induced bone marrow dendritic cells, pLDDT predicted local-distance difference test

Article Snippet: Recombinant mouse GM-CSF (Sino Biological, Cat# 51048-MNAH), recombinant mouse IL-4 (Sino Biological, Cat# 51084-MNAE), and recombinant human Flt3L (Sino Biological, Cat# 10315-HNAE) were utilized for the cultivation of bone marrow-derived dendritic cells (BMDCs).

Techniques: Residue, Fluorescence, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Oncolytic adenovirus delivery of neoantigens sensitizes low-mutation tumors to anti-PD-1 therapy and prevents metastasis

doi: 10.1038/s41392-025-02511-5

Figure Lengend Snippet: Construction and in vivo validation of NeoViron. a ‒ d C57BL/6 mice inoculated with mICCN-4 or Hep53.4 cells were treated with PBS, vector or AdSVP-Flt3L. For the mICCN-4 model, treatment was initiated on day 8, with intratumoral injections of 1 × 10⁸ pfu OAV administered every other day for a total of five doses. For Hep53.4 tumors, treatment commenced on Day 6 with intratumoral injections of 2 × 10⁸ pfu OAV every other day for five doses. a , b Left, tumor images at the end point. Middle, tumor growth curves ( n = 6 mice per group). c , d CD103+ cDC1s were examined by flow cytometry ( c ) or immunofluorescence staining (MHC II+ CD103+ cells) ( d ). e Design of NeoViron, coexpressing neoantigens and Flt3L. f – h C57BL/6 mice inoculated with mICCN-4 were treated with PBS or 1 × 10⁸ pfu OAV (vector, AdSVP-NAg mICC , AdSVP-Flt3L or NeoViron) from Day 8 every other day for a total of five doses. f Left, experimental schematic. Middle, tumor images at the end point. Right, tumor growth curves ( n = 6 mice per group). g Percentages of PD1+ GZMB+ CD8+ T cells and PD1+ GZMB- CD8+ T cells infiltrating the tumor tissues of each group. h Representative immunofluorescence images of GZMB+ CD8+ T cells in tumor tissues. The data are shown as the means ± SDs ( a – g ) and are representative of two ( a – h ) independent experiments. Significance was calculated via one-way ANOVA ( a – c , g ) or two-way ANOVA ( a , b , f ). * P < 0.05, ** P < 0.01, *** P < 0.001. ns not significant

Article Snippet: One hundred milligrams of tumor tissue were finely sliced and incubated at 37 °C in 200 μL of PBS for 2 h. The supernatants were then collected and analyzed via a mouse Flt3L ELISA Kit (Proteintech, USA).

Techniques: In Vivo, Biomarker Discovery, Plasmid Preparation, Flow Cytometry, Immunofluorescence, Staining, Immunopeptidomics

Journal: Signal Transduction and Targeted Therapy

Article Title: Oncolytic adenovirus delivery of neoantigens sensitizes low-mutation tumors to anti-PD-1 therapy and prevents metastasis

doi: 10.1038/s41392-025-02511-5

Figure Lengend Snippet: NeoViron-induced CD8+ Trm cells prevent tumor metastasis. a ‒ g C57BL/6 mice inoculated with mICCN-4 or Hep53.4 cells were treated with PBS, vector or NeoViron/AdSVP-NAg Hep + AdSVP-Flt3L 5 times, followed by tumor excision on day 16. One week later, liver/lung metastasis models were established by injecting tumor cells via the spleen/tail vein. a , d Experimental schematic. b , e Tumor area of liver metastases and counts of lung metastases analyzed by ImageJ. c , f HE-stained images of liver and lung metastases at the end of the experiment. g Multiplex immunofluorescence images showing CD8+ Trm cells in liver and lung metastases. The data are shown as the means ± SDs ( b , e ) and are representative of two independent experiments ( a – h ). Significance was calculated via the Kruskal‒Wallis test ( b , e ). * P < 0.05, *** P < 0.001. ns not significant

Article Snippet: One hundred milligrams of tumor tissue were finely sliced and incubated at 37 °C in 200 μL of PBS for 2 h. The supernatants were then collected and analyzed via a mouse Flt3L ELISA Kit (Proteintech, USA).

Techniques: Plasmid Preparation, Staining, Multiplex Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

doi: 10.1101/2025.06.14.659663

Figure Lengend Snippet: a t-SNE clustering of cell types in the tumors of CRC patients . b Heatmap depicting the correlation of cell types in the tumors of CRC patients. c Heatmap depicting the correlation of immune cell clusters from scRNA-seq data of NCG HIS and NCG- Gfi1 -/- HIS mice. d Schematic of MC & MM or BD & SC neutrophils sorted from BM of NCG- Gfi1 -/- HIS mice were cultured with G-CSF or Flt3L. e After culture for 4 days, proportions of CD11b + CD14 + cells among human WBCs were determined by flow cytometry ( n = 3). f After culture for 4 days, proportions of SSC hi CD66b + cells among human WBCs were determined by flow cytometry ( n = 3). g Schematic of neutrophils at various stages sorted from BM and PB of non-tumor donors were cultured with Flt3L. h Proportions of CD11b + CD14 + cells in each group were determined by flow cytometry ( n = 5). i Schematic of sorted monocytes, MC & MM, and CD11b + CD14 + population transdifferentiated from MC & MM for T cell suppression, giemsa staining and transcriptome detection. j Suppression index of human T cell proliferation co-cultured with monocytes, MC & MM, and CD11b + CD14 + population transdifferentiated from MC & MM ( n = 5). k Giemsa staining of monocytes, MC & MM, and monocytes transdifferentiated from MC & MM by Flt3L. l Heatmap of PMN-MDSC, trans-differentiation, monocytes/macrophages and DC-related gene markers in monocytes, MC & MM, and CD11b + CD14 + population transdifferentiated from MC & MM ( n = 4). All data are mean ± SD and were analyzed by two-tailed, unpaired Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Reagents and cytokines for cell culture and tissue digestion were Flt3L (Sinobiological, Cat#:10315-H07B), G-CSF (Sinobiological, Cat#:10007-H01H), DMEM (Biological Industries, Cat#: 06-1055-57-1ACS), RPMI 1640 (Biological Industries, Cat#: 01-100-1ACS), fetal bovine serum (FBS) (Gibco, Cat#: 10099141), penicillin/streptomycin (P/S) (Gibco, Cat#: 15140122), Dulbecco’s phosphate buffered saline (Biological Industries) (Bioind, Cat#: 02-023-1ACS), collagenase mix (SIGMA, Cat#: 9001-12-1), DNase I (SIGMA, Cat#: DN25), Ficoll (GE Healthcare, Cat#: 17544203), Red Blood Cell Lysing Buffer Hybri-Max (SIGMA, Cat#: R7757-100ML), Atezolizumab (anti PD-L1, MedChemExpress, Cat#: HY-P9904) and Lysing Solution 10× Concentrate (BD, Cat#: 349202).

Techniques: Cell Culture, Flow Cytometry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

doi: 10.1101/2025.06.14.659663

Figure Lengend Snippet: a t-SNE clustering of cell types in the tumors of PDAC patients . b Heatmap depicting the correlation of cell types in the tumors of PDAC patients. c Flow cytometry Gating strategy for analysis of CD11b + CD14 + expression on neutrophils in different stages of human BM and PB ( n = 5). d The developmental stage distribution of human CD54 - neutrophils in the BM ( n = 5). e After culture with Flt3L for 4 days, proportions of CD11b + CD14 + cells in each group were determined by flow cytometry ( n = 5). All data are mean ± SD and were analyzed by two-tailed, unpaired Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Reagents and cytokines for cell culture and tissue digestion were Flt3L (Sinobiological, Cat#:10315-H07B), G-CSF (Sinobiological, Cat#:10007-H01H), DMEM (Biological Industries, Cat#: 06-1055-57-1ACS), RPMI 1640 (Biological Industries, Cat#: 01-100-1ACS), fetal bovine serum (FBS) (Gibco, Cat#: 10099141), penicillin/streptomycin (P/S) (Gibco, Cat#: 15140122), Dulbecco’s phosphate buffered saline (Biological Industries) (Bioind, Cat#: 02-023-1ACS), collagenase mix (SIGMA, Cat#: 9001-12-1), DNase I (SIGMA, Cat#: DN25), Ficoll (GE Healthcare, Cat#: 17544203), Red Blood Cell Lysing Buffer Hybri-Max (SIGMA, Cat#: R7757-100ML), Atezolizumab (anti PD-L1, MedChemExpress, Cat#: HY-P9904) and Lysing Solution 10× Concentrate (BD, Cat#: 349202).

Techniques: Flow Cytometry, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

doi: 10.1101/2025.06.14.659663

Figure Lengend Snippet: a Schematic of NCG- Gfi1 -/- HIS mice were hydrodynamically i.v. injection of pLIVE-G-CSF or Flt3L plasmids. b representative flow cytometry plots of BM cells 7 days after the injection were shown. c As depicted in the schematic, A375 xenograft NCG- Gfi1 -/- HIS model was established, and pLIVE-Flt3L plasmid was injected i.v. hydrodynamically. d Tumor growth of NCG- Gfi1 -/- HIS mice with PBS or Flt3L treatment (PBS, n = 3; Flt3L, n = 3). e Tumor weight of NCG-Gfi1 -/- HIS mice with PBS or Flt3L treatment (PBS, n = 3; Flt3L, n = 3) f Proportions of neutrophils (CD11b + CD66b + ) in the BM, SP, PB and tumors of NCG- Gfi1 -/- HIS mice with or without Flt3L treatment ( n = 3). g Proportions of human neutrophils at different developmental stages in the BM of NCG- Gfi1 -/- HIS mice with or without Flt3L treatment ( n = 3). h As depicted in the schematic, A375 xenograft NCG- Gfi1 -/- HIS model was established, and pLIVE-Flt3L plasmid was injected i.v. hydrodynamically while the anti-PD-L1 antibodies were injected i.p. (150 µg/dose, once every three days). i Tumor growth of NCG- Gfi1 -/- HIS mice with PBS, Flt3L and/or anti-PD-L1 treatment ( n = 4). j Tumor weight of NCG-Gfi1 -/- HIS mice with PBS, Flt3L and/or anti-PD-L1 treatment ( n = 4). k Proportion of different immune cell populations in the tumor of NCG- Gfi1 -/- HIS from each group ( n = 4). l The proportion of human T cells (CD3 + ), monocytes (CD11b + CD14 + ) and neutrophils (SSC hi CD66b + ) in the tumor of NCG- Gfi1 -/- HIS from each group ( n = 4). All data are mean ± SD and were analyzed by two-tailed, unpaired Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Reagents and cytokines for cell culture and tissue digestion were Flt3L (Sinobiological, Cat#:10315-H07B), G-CSF (Sinobiological, Cat#:10007-H01H), DMEM (Biological Industries, Cat#: 06-1055-57-1ACS), RPMI 1640 (Biological Industries, Cat#: 01-100-1ACS), fetal bovine serum (FBS) (Gibco, Cat#: 10099141), penicillin/streptomycin (P/S) (Gibco, Cat#: 15140122), Dulbecco’s phosphate buffered saline (Biological Industries) (Bioind, Cat#: 02-023-1ACS), collagenase mix (SIGMA, Cat#: 9001-12-1), DNase I (SIGMA, Cat#: DN25), Ficoll (GE Healthcare, Cat#: 17544203), Red Blood Cell Lysing Buffer Hybri-Max (SIGMA, Cat#: R7757-100ML), Atezolizumab (anti PD-L1, MedChemExpress, Cat#: HY-P9904) and Lysing Solution 10× Concentrate (BD, Cat#: 349202).

Techniques: Injection, Flow Cytometry, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

doi: 10.1101/2025.06.14.659663

Figure Lengend Snippet: a A375 xenograft NCG HIS model was established, and Flt3L plasmid was injected i.v. hydrodynamically. Tumor growth of NCG HIS mice with PBS or Flt3L treatment (PBS, n = 3; Flt3L, n = 3). b Tumor weight of NCG HIS mice with PBS or Flt3L treatment (PBS, n = 3; Flt3L, n = 3). c Proportions of neutrophils (CD11b + CD66b + ) in the BM, SP, PB and tumors of NCG HIS mice with or without Flt3L treatment ( n = 3). d Proportions of human neutrophils at different developmental stages in the BM of NCG HIS mice with or without Flt3L treatment ( n = 3). All data are mean ± SD and were analyzed by two-tailed, unpaired Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Reagents and cytokines for cell culture and tissue digestion were Flt3L (Sinobiological, Cat#:10315-H07B), G-CSF (Sinobiological, Cat#:10007-H01H), DMEM (Biological Industries, Cat#: 06-1055-57-1ACS), RPMI 1640 (Biological Industries, Cat#: 01-100-1ACS), fetal bovine serum (FBS) (Gibco, Cat#: 10099141), penicillin/streptomycin (P/S) (Gibco, Cat#: 15140122), Dulbecco’s phosphate buffered saline (Biological Industries) (Bioind, Cat#: 02-023-1ACS), collagenase mix (SIGMA, Cat#: 9001-12-1), DNase I (SIGMA, Cat#: DN25), Ficoll (GE Healthcare, Cat#: 17544203), Red Blood Cell Lysing Buffer Hybri-Max (SIGMA, Cat#: R7757-100ML), Atezolizumab (anti PD-L1, MedChemExpress, Cat#: HY-P9904) and Lysing Solution 10× Concentrate (BD, Cat#: 349202).

Techniques: Plasmid Preparation, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

doi: 10.1101/2025.06.14.659663

Figure Lengend Snippet: a Proportions of human monocytes (CD11b + CD14 + ) and neutrophils (SSC hi CD66b + ) and neutrophils at different developmental stages in the BM of CDX NCG- Gfi1 -/- HIS mice from each treatment group ( n = 4). b Proportions of human monocytes (CD11b + CD14 + ) and neutrophils (SSC hi CD66b + ) and neutrophils at different developmental stages in the spleen of CDX NCG- Gfi1 -/- HIS mice from each treatment group ( n = 4). c Proportions of human monocytes (CD11b + CD14 + ) and neutrophils (SSC hi CD66b + ) and neutrophils at different developmental stages in the PB of CDX NCG- Gfi1 -/- HIS mice from each treatment group ( n = 4). d Proportions of human neutrophils at different developmental stages in PB of NCG- Gfi1 -/- HIS mice with anti-PDL1 or Flt3L treatment ( n = 4). All data are mean ± SD and were analyzed by two-tailed, unpaired Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Reagents and cytokines for cell culture and tissue digestion were Flt3L (Sinobiological, Cat#:10315-H07B), G-CSF (Sinobiological, Cat#:10007-H01H), DMEM (Biological Industries, Cat#: 06-1055-57-1ACS), RPMI 1640 (Biological Industries, Cat#: 01-100-1ACS), fetal bovine serum (FBS) (Gibco, Cat#: 10099141), penicillin/streptomycin (P/S) (Gibco, Cat#: 15140122), Dulbecco’s phosphate buffered saline (Biological Industries) (Bioind, Cat#: 02-023-1ACS), collagenase mix (SIGMA, Cat#: 9001-12-1), DNase I (SIGMA, Cat#: DN25), Ficoll (GE Healthcare, Cat#: 17544203), Red Blood Cell Lysing Buffer Hybri-Max (SIGMA, Cat#: R7757-100ML), Atezolizumab (anti PD-L1, MedChemExpress, Cat#: HY-P9904) and Lysing Solution 10× Concentrate (BD, Cat#: 349202).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

doi: 10.1101/2025.06.14.659663

Figure Lengend Snippet: Human MC & MM-stage neutrophils, predominant in BM and constituting most TINs within the TIME across cancer types, exhibit immunosuppressive and tumor-promoting properties. Tumor-driven expansion of these cells occurs in BM and periphery of cancer patients and NCG- Gfil -/- HIS mice, where CD63 and Galectin-3 distinguish them from other neutrophils, while their transdifferentiation into monocytic cells via Flt3L retards tumor growth.

Article Snippet: Reagents and cytokines for cell culture and tissue digestion were Flt3L (Sinobiological, Cat#:10315-H07B), G-CSF (Sinobiological, Cat#:10007-H01H), DMEM (Biological Industries, Cat#: 06-1055-57-1ACS), RPMI 1640 (Biological Industries, Cat#: 01-100-1ACS), fetal bovine serum (FBS) (Gibco, Cat#: 10099141), penicillin/streptomycin (P/S) (Gibco, Cat#: 15140122), Dulbecco’s phosphate buffered saline (Biological Industries) (Bioind, Cat#: 02-023-1ACS), collagenase mix (SIGMA, Cat#: 9001-12-1), DNase I (SIGMA, Cat#: DN25), Ficoll (GE Healthcare, Cat#: 17544203), Red Blood Cell Lysing Buffer Hybri-Max (SIGMA, Cat#: R7757-100ML), Atezolizumab (anti PD-L1, MedChemExpress, Cat#: HY-P9904) and Lysing Solution 10× Concentrate (BD, Cat#: 349202).

Techniques: