Journal: Cell reports

Article Title: Mapping the genetic landscape establishing a tumor immune microenvironment favorable for anti-PD-1 response

doi: 10.1016/j.celrep.2025.115698

Figure Lengend Snippet: (A) Overview of single dose paradigm where tumors are harvested 48 h after a single dose of aPD1 or isotype control in order to capture the initial effects of treatment. (B) UMAP plot depicting an overview of heterogeneity in single-cell transcriptomes derived from responder and non-responder strain tumors and highlighting the T/NK cell cluster. (C) Subclustering of the T/NK cell cluster reveals transcriptionally distinct cell subsets showing enrichment of subclusters 3 and 13 in responding tumors. (D) Fraction of exhausted CTLs (cluster 3) as portion of all cells in responder and non-responder strain groups, Wilcoxon test, *** p < 0.001, * p < 0.05, respectively. (E) Fraction of IFN-γ + CTLs (cluster 13) as portion of all cells in responder and non-responder strain groups, Wilcoxon test, ** p < 0.01; n.s., not significant, respectively. (F) Expression of an 18-gene signature reflecting IFN-γ + stimulation applied to all cells in each cluster (left) or macrophage subcluster (right), with dot size proportional to the relative number of cells of each type, dot color relative to the median IFN-γ + stimulation score, and bars depicting the difference in summed pathway score between responder and non-responder cells within each (sub)cluster. (G) Fraction of IFN-γ-stimulated macrophage clusters (clusters 2, 5, and 26) as a portion of all cells in responder and non-responder strain groups, Wilcoxon test, **** p < 0.0001, * p < 0.05, respectively. (H) Visium spatial transcriptomics images showing expression of CD45 ( Ptprc ) (top row) and Cxcl9 (bottom row) in spatially resolved spots of tumors harvested from responder (CC75F1) and non-responder (CC80F1) mouse tumors. Hematoxylin and eosin stains showed no evidence of necrosis in these tumor sections. (I) Percentage of Ptprc + spatial transcriptomics spots co-expressing either macrophage lineage markers ( Adgre1, Cd68, Itgam , and Cxcl9 ) or dendritic cell markers (Batf3 and/or Zbtb46) along with CTL markers ( Thy1, Cd8a , and Gzmb ) across all tissue regions in responder vs. non-responder strain tumors. **** p < 0.0001, permutation test (see ). See also .

Article Snippet: Anti-Human/Mouse CD11b APC-Cy7 (clone M1/70) , Tonbo Biosciences , CAT#25-0112; RRID:AB_2621625.

Techniques: Control, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a Representative flow cytometry plots gated on CD45 + living cells isolated from colonic lamina propria of WT and Pellino1-mKO male mice in normal, acute 1.5% DSS, and AOM/DSS groups. Migratory macrophages (CD11b + CX3CR1 int ), resident macrophages (CD11b + CX3CR1 hi ). b (Left) Percentage of CD11b + CX3CR1 hi macrophages, (Right) CD11b + CX3CR1 int macrophage infiltration into the colonic lamina propria in WT and Pellino1-mKO male mice in normal, acute 1.5% DSS, and AOM/DSS groups (H 2 O, n = 4; DSS, n = 5; AOM/DSS, n = 5). These percentages for both CD11b + CX3CR1 hi and CD11b + CX3CR1 int were determined by gating among CD45 + cells. c (Left) Representative images of the wound healing assay. Wound healing assays were performed to examine the migration of BMDMs of WT and Pellino1-mKO under stimulation of 100 ng/mL LPS and 20 ng/mL CCL5 for 24 h. Initial wounded areas were marked with a dashed line. Scale bar = 100 μm. (Right) Quantification of migration areas using ImageJ ( n = 4). d (Left) Representative images of the migration assay. BMDMs from WT and Pellino1-mKO mice were induced to migrate with stimulation from 100 ng/mL LPS, 20 ng/mL CCL5, and 100 ng/mL TGF β for 24 h. Scale bar = 50 μm. (Right) Quantification of migrated cells within the field of view area using ImageJ ( n = 7). e (Left) CFSE-labeled MC38 tumor cells were co-cultured with BMDMs from WT and Pellino1-mKO mice for 6 h in the presence of 100 ng/mL LPS. (Right) Phagocytic index was calculated using the following formula: phagocytic index (%) = (number of F4/80 + CSFE + cells)/(number of F4/80 + cells) × 100 ( n = 5). f Expression levels of mRNA associated with macrophage migration in colon tissues of WT and Pellino1-mKO male mice of normal (WT, n = 5; Pellino-mKO, n = 4), acute 1.5% DSS ( n = 6), and AOM/DSS (WT, n = 5; Pellino-mKO, n = 6) groups were assessed by qRT-PCR and normalized to GAPDH expression. Data were represented as mean ± SD in ( b – f ). All statistical comparisons were made using two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: The following fluorochrome-labeled mAbs and staining reagents were used according to the manufacturer’s protocol: PE- or PE-Cy7-anti-B220 (clone RA3-6B2, eBioscience), PerCP-Cy5.5- or PE-Cy7-anti-CD3 (clone 145-2C11, eBioscience), FITC- or PE-Cy7-anti-CD4 (clone RM4-5, eBioscience), PE- or APC-anti-CD8 (clone 53-6.7, eBioscience), APC- or PE-Cy7-anti-CD11b (clone M1/70, eBioscience), PE-Cy7-anti-CD11c (clone N418, eBioscience), PerCP-Cy5.5-anti-CD19 (clone 1D3, eBioscience), PerCP-Cy5.5-anti-CD45 (clone 30-F11, eBioscience), PE-Cy7-anti-CX3CR1 (clone SA011F11, BioLegend), FITC-anti-Ly6G (clone 1AB, eBioscience), APC-anti-Ly6C (clone HK1.4, eBioscience), FITC-anti-MHC2 (clone M5/114.15,2, eBioscience), PerCP-Cy5.5-anti-GR-1 (clone RB6-8C5, eBioscience), PE-anti-F4/80 (clone BM8, eBioscience), FITC-anti-CD80 (clone 16-10A1, eBioscience), PE-Cy7-anti-CD86 (clone GL1, eBioscience), APC-anti-CD206 (clone MR6F3, eBioscience), FITC-anti-CD282 (TLR2) (clone 6C2, eBioscience), PE-anti-CD284 (TLR4) (clone HTA125, eBioscience), PE-Cy7-anti-CD369 (Dectin-1) (clone Bg1fpj, eBioscience), and APC-anti-CD36 (clone HM36, eBioscience).

Techniques: Flow Cytometry, Isolation, Wound Healing Assay, Migration, Labeling, Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test