Journal: Cancer Communications

Article Title: Targeting TREM2 + and TIMD4 + Macrophages Reprograms the Tumor Microenvironment and Enhances PD-1 Blockade Efficacy in Intrahepatic Cholangiocarcinoma

doi: 10.34133/cancomm.0056

Figure Lengend Snippet: TREM2 + macrophages promote POSTN + CAF formation at the tumor boundary to establish an immune barrier restricting CD8 + T cell infiltration in ICC. (A and B) DEGs analysis (A) and GO enrichment (B) of up-regulated genes in fibroblasts from Trem2 −/− versus WT tumors (Kras-p19-induced ICC mice). (C and D) UMAP plot of human fibroblast cell subtypes (C) and POSTN expression levels (D). (E) Spatial transcriptomics shows expression level and distribution of genes in ICC samples. The dotted line demarcates the boundary between the tumor and peritumoral tissue. (F to H) mIF staining showing spatial distribution (F and G) and quantitative analysis (H) of CD8 + T cells in the presence or absence of an immune barrier ( **** P < 0.0001; Mann–Whitney U test). (I) IHC was performed to evaluate the expression levels of Ck19, Postn, and Cd8a, and SR was performed to evaluate collagen abundance, between the anti-Fap treatment group and the control group. For detailed procedures regarding the preparation of the anti-Fap therapeutic agent, please refer to Materials and Methods. (J) α-SMA and POSTN expression detected by mIF staining in LX-2 cocultured with Trem2 + macrophages or Trem2 − macrophages or treated with TGF-β ( * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant; one-way ANOVA with Dunnett’s test). (K and L) Survival (Kaplan–Meier) analysis of POSTN (K) and TREM2-POSTN coexpression (L) in the FuDan-ICC cohort. Abbreviations: ICC, intrahepatic cholangiocarcinoma; CAF, cancer-associated fibroblast; DEGs, differentially expressed genes; UMAP, Uniform Manifold Approximation and Projection; GO, Gene Ontology; WT, wild type; apCAF, antigen-presenting cancer-associated fibroblast; HSC, hepatic stellate cell; iCAF, inflammatory cancer-associated fibroblast; myCAF, myofibroblastic cancer-associated fibroblast; Prolif_Fibro, proliferating fibroblast; IHC, immunohistochemistry; PanCK, pan-cytokeratin; FAP, fibroblast activation protein; OS, overall survival.

Article Snippet: For polarization, naïve CD4 + T cells (1 × 10 6 cells/ml) were cultured with TGF-β (5 ng/ml; catalog no. HY- P70648 , Medchemexpress), IL-2 (100 U/ml), and reduced anti-CD3/CD28 activation beads (0.5 μg/ml) for 3 to 5 days.

Techniques: Analysis, Expressing, Spatial Transcriptomics, Tissue, Staining, MANN-WHITNEY, Control, Immunohistochemistry, Activation Assay

Journal: Cancer Communications

Article Title: Targeting TREM2 + and TIMD4 + Macrophages Reprograms the Tumor Microenvironment and Enhances PD-1 Blockade Efficacy in Intrahepatic Cholangiocarcinoma

doi: 10.34133/cancomm.0056

Figure Lengend Snippet: TREM2 + macrophages promote immunosuppressive microenvironment in ICC by regulating CD8 + T cell exhaustion and Treg development. (A) Schematic diagram of the experimental workflow for macrophage and T cell coculture assays. Macrophage subsets were isolated from ICC tumors, and naive Cd4 + T cells or Cd8 + T cells were isolated from the spleens of healthy mice for subsequent coculture experiments. (B) Flow cytometry analysis of Foxp3 expression in naive Cd4 + T cells cocultured with sorted Trem2 − macrophages or Trem2 + macrophages for 2 days in the presence of IL-2 and TGF-β. Cells were gated sequentially on single cells, live cells, and Cd4 + T cells. Representative flow cytometry plots and quantification of Foxp3 + cells among Cd4 + T cells are shown ( * P < 0.05, **** P < 0.0001; one-way ANOVA by Dunnett’s test). (C) Flow cytometry analysis of Foxp3 expression in naive Cd4 + T cells cocultured with sorted Trem2 − macrophages or Trem2 + macrophages for 9 days in the presence of IL-2 but without exogenous TGF-β. Cells were gated sequentially on single cells, live cells, and Cd4 + T cells. Representative flow cytometry plots and quantification of Foxp3 + cells among Cd4 + T cells are shown ( * P < 0.05, **** P < 0.0001, ns, not significant; one-way ANOVA by Dunnett’s test). (D) Batf expression in nTreg cells cocultured with macrophages ( *** P < 0.001; ns, not significant; one-way ANOVA by Dunnett’s test). (E) Spearman correlation analysis between Treg signatures and immune-related genes in the FuDan-ICC cohort (Spearman test). (F) Correlation of TNFSF13B with FOXP3 expression in the Xena pan-cancer cohort (Spearman test). (G) Batf expression in nTreg cells in indicated groups. Flow cytometry analysis of Batf expression in Treg cells treated with recombinant TNFSF13B in the presence or absence of a TNFSF13B inhibitor. Representative histogram plots and quantification of Batf + cells among Foxp3 + T cells are shown ( *** P < 0.001, **** P < 0.0001; one-way ANOVA by Dunnett’s test). (H) Pd-1 expression in Cd8 + T cells stimulated by macrophage subtypes ( * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns, not significant; one-way ANOVA by Dunnett’s test). (I) In vitro cytotoxicity assay assessing the killing activity of OT-1 Cd8 + T cells against OVA-expressing ICC tumor cells after coculture with macrophage subsets. Tumor cells were visualized by crystal violet staining. Representative images of residual OVA-ICC tumor cells are shown for the control, Cd8 + T cell alone, Trem2 + macrophage–Cd8 + T cell coculture, and Timd4 + macrophage–Cd8 + T cell coculture groups. (J) Heatmap of immunosuppressive molecule expression in macrophage subcell types (scaled value). (K) Spatial transcriptomics of TIMD4 expression and localization in tumor tissue of 2 ICC patients. (L and M) Spearman correlation of TIMD4 with immunosuppressive markers [PDCD1 (L), VSIG4 (M)] in tumor and adjacent tissues (EHBH-ICC cohort). (N) Multiplex immunofluorescence staining of ICC tissue showed the spatial distribution of PanCK, CD8A, and PD-1 signals in tumor and adjacent tissues (PanCK marks epithelial tumor cells, CD8A marks CD8 + T cells, and PD-1 marks PD-1 + cells; the white dashed line indicates the tumor-adjacent tissue boundary; Magnified images of the boxed adjacent-tissue region and single-channel images are shown). (O) Ligand–receptor interaction analysis between macrophage subsets and exhausted CD8 + T cells in adjacent tissues based on scRNA-seq data. The chord diagram shows predicted immune checkpoint ligand–receptor pairs. (P) Bulk RNA-seq analysis of Cd8 + T cells cocultured with sorted Trem2 + macrophages or Timd4 + macrophages. Heatmap shows scaled mRNA expression levels of selected inhibitory receptor- and exhaustion-related genes compared with control Cd8 + T cells. (Q) GO enrichment of up-regulated DEGs in Cd8 + T cells cocultured with Trem2 + macrophages. (R) Expression levels of fatty acid metabolism-related genes and Csf1 in Cd8 + T cells under coculture with macrophages (the mRNA expression levels were log2-transformed; ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant; one-way ANOVA by Dunnett’s test). Abbreviations: ICC, intrahepatic cholangiocarcinoma; WT, wild type; TREM2, triggering receptor expressed on myeloid cells 2; TIMD4, T cell immunoglobulin and mucin domain-containing protein 4; TGF-β, transforming growth factor-β; IL-2, interleukin-2; FOXP3, forkhead box P3; BATF, basic leucine zipper ATF-like transcription factor; Treg, regulatory T cell; nTreg, natural regulatory T cell; TNFSF13B, tumor necrosis factor ligand superfamily member 13B; PD-1, programmed cell death protein 1; OVA, ovalbumin; mIF, multiplex immunofluorescence; PanCK, pan-cytokeratin; RNA-seq, RNA sequencing; DEG, differentially expressed gene; GO, Gene Ontology; TPM, transcripts per million; TME, tumor microenvironment.

Article Snippet: For polarization, naïve CD4 + T cells (1 × 10 6 cells/ml) were cultured with TGF-β (5 ng/ml; catalog no. HY- P70648 , Medchemexpress), IL-2 (100 U/ml), and reduced anti-CD3/CD28 activation beads (0.5 μg/ml) for 3 to 5 days.

Techniques: Isolation, Flow Cytometry, Analysis, Expressing, Recombinant, In Vitro, Cytotoxicity Assay, Activity Assay, Staining, Control, Spatial Transcriptomics, Tissue, Multiplex Assay, Immunofluorescence, RNA Sequencing, Transformation Assay

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet: Simultaneous application of IL-4 and Arg promotes efferocytosis and improves the pro-regenerative microenvironment (A) Schematic illustration of the in vitro efferocytosis assessment. (B and C) Macrophage efferocytosis assessment via flow cytometry, showing representative scatterplots of macrophages engulfing apoptotic cells (B) and quantification of the efferocytosis rate after 24 h (C). (D) Morphological evaluation of macrophage efferocytosis, showing representative cell tracking images of macrophages (labeled with CMFDA) engulfing apoptotic cells (labeled with CM-Dil) after 24 h of co-culture. (E) Quantification of the efferocytosis rate after 6, 24, and 48 h determined by flow cytometry under Control and IL-4 + Arg conditions. (F) Experimental design for detecting microenvironmental changes following efferocytosis. (G) ROS quantification following efferocytosis. (H) Enzyme-linked immunosorbent assay (ELISA) quantification of TGF-β1 and IL-10 in the culture supernatants. Error bars represent SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test (C, G, and H) or two-way ANOVA followed by Sidak’s multiple comparisons test (E). Sample sizes: n = 3 biological replicates (B–E); n = 5 biological replicates, each performed with 3 technical replicates (G) and n = 3 biological replicates, each performed with 3 technical replicates (H). Scale bars, 100 μm (D) and 20 μm (inset).

Article Snippet: Mouse TGF-β1 ELISA Kit , ABclonal , Cat# RK00057.

Techniques: In Vitro, Flow Cytometry, Cell Tracking Assay, Labeling, Co-culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet: Transcriptomic changes underlying synergistic regulation of macrophages by IL-4 and Arg (A) RT-qPCR analysis of M2-related marker gene expression ( Tgfb1 , Il10 , Arg1 , and Cd206 ) in macrophages. (B) Schematic illustration of the bone marrow-derived macrophages (BMDMs) isolation, total RNA extraction, and transcriptomic sequencing workflow. (C) Functional annotation of upregulated genes using Gene Ontology (GO) enrichment analysis. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis emphasizing significant signaling pathways. (E) RT-qPCR analysis of Tgfb1 and Il10 mRNA expression levels of macrophage after treatment with the IL-4 + Arg combination in the presence or absence of nor-NOHA. (F) ELISA quantification of secreted TGF-β1 and IL-10 protein concentrations in the culture supernatants under nor-NOHA inhibition. Error bars represent SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test (A, E, and F). Sample sizes: n = 3 biological replicates (C and D); n = 3 (A and F) and 4 (E) biological replicates, each performed with 3 technical replicates.

Article Snippet: Mouse TGF-β1 ELISA Kit , ABclonal , Cat# RK00057.

Techniques: Quantitative RT-PCR, Analysis, Marker, Gene Expression, Derivative Assay, Isolation, RNA Extraction, Sequencing, Functional Assay, Protein-Protein interactions, Expressing, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet:

Article Snippet: Mouse TGF-β1 ELISA Kit , ABclonal , Cat# RK00057.

Techniques: Cell-Signaling, Recombinant, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Software