Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) Representative images and quantification of CCR7 + DCs (panCK − HLA-DR + LAMP3 + , yellow) near BVs (CD31 + PDPN − , magenta), or LVs (CD31 + PDPN + , cyan) in human tumors (HNSCC, NSCLC, and EC). Scale bar represents 20 μm. Whole-tumor sections were analyzed for EC and NSCLC. Numbers of fields of view (FOVs) analyzed per HNSCC sample are as follows: HNSCC1–04 n = 7; HNSCC1–06 n = 16; HNSCC1–07 n = 11; HNSCC2–01 n = 126; HNSCC2–06 n = 455; HNSCC2–09 n = 180; HNSCC2–11 n = 122; HNSCC2–12 n = 79; HNSCC2–15 n = 205; HNSCC2–26 n = 293; HNSCC2–35 n = 175. One bar = one patient . (B) Representative images and quantification of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) or LVs (CD31 + LYVE-1 + ; cyan) in mouse tumors (MC38, B16F10, and D4M3.A-OVA). Scale bar represents 10 μm. Whole-tumor sections were analyzed. One bar = one mouse. (C) Frequencies of BV-, LV- and non-vessel-associated CCR7 + DCs in mouse MC38 tumors 3 days post anti-CD40 or anti-PD-1 treatment. Whole-tumor sections were analyzed. One bar = one mouse. (D) (Left) Representative images of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) in MC38 tumors inoculated in Ccr7 ko/wt and Ccr7 ko/ko mice, 3 days post anti-PD-1 treatment. (Right) Distribution of the area of CCR7 + DC surfaces in clusters relative to their distance to closest BVs and plotted as percentage of total CCR7 + DC cluster area. CCR7 + DC surfaces from clusters associated with LVs and those not in clusters were excluded from the analysis. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = average value of all clusters in each genotype ( Ccr7 ko/ko n = 5 mice, 56 clusters; Ccr7 wt /ko n = 6 mice, 28 clusters; and Ccr7 wt /wt n = 3 mice, 19 clusters). Two-way ANOVA with multiple comparisons, mean with SEM; **** p < 0.0001 for comparison at 10 and 20 μm from closest BVs. (E) (Left) Representative images of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) and Ccl19 ( Ccl19 -eYFP + Tomato + ; white) in Ccl19 -ieYFP reporter mice (left image) or CCL21 (white, right image) in MC38 tumors. (Right) Frequencies of perivascular CCR7 + DC clusters associated with Ccl19 -covered BVs or within CCL21 + areas of the tumors among total perivascular CCR7 + DC clusters. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = one mouse. Unpaired t test, mean with SEM; *** p < 0.001. (F) (Left) Representative images of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) in MC38 tumors inoculated in Ccl19 wt/wt and Ccl19 ko/ko mice, 2 days post anti-PD-1treatment. (Right) Quantification of BV- or LV-associated CCR7 + DC clusters in MC38 tumors from Ccl19 wt/wt and Ccl19 ko/ko mice. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = one mouse, whiskers represent min to max. Unpaired t test; * p < 0.05. (G) Heatmap depicts log 2 -transformed averaged expression of Ccl19 in indicated immune and non-immune populations in the TME of multiple mouse tumor models (breast, , lung [and GSE201247 ], and pancreatic , ). (H) (Left) Synthetic images of CCR7 + DCs (yellow), blood endothelial cells (BECs; magenta), lymphatic endothelial cells (LECs; cyan), and CCL19 + fibroblasts (green) in one representative NSCLC patient analyzed by spatial transcriptomics. (Right) Box plots depict the enrichment scores of CCL19 + fibroblasts within the neighborhood of BV-associated CCR7 + DCs, in four human NSCLC. Data are shown for both permuted (median enrichment scores from 1,000 permutations) and observed datasets. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = one sample. Paired t test, whiskers represent mean to max; * p < 0.05. (I) Heatmap depicts log 2 -transformed averaged expression of CCL19 in indicated immune and non-immune populations in the TME of multiple human cancer types (HNSCC, n = 40, n = 18 patients; CRC, n = 23, n = 64 patients; ESCC, n = 58 patients ; NSCLC, n = 32, n = 7 patients; BRCA, n = 29 patients ; and PRCA, n = 18 patients ). A cross indicates that the cellular population was not detected. See also – .

Article Snippet: Unconjugated rabbit anti-human LAMP3 (polyclonal) , Atlas Antibodies , Cat#HPA051467;RRID: AB_2681475.

Techniques: Comparison, Transformation Assay, Expressing, Spatial Transcriptomics

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) Heatmaps depict the enrichment of immune and non-immune cell types in the immediate neighborhood of CCR7 + DCs in NSCLC spatial transcriptomic data ( n = 4). (B) (Left) Representative FOV displaying CCR7 + DCs (HLA-DR + LAMP3 + ; yellow) located near BVs (CD31 + PDPN − ; magenta) and Tregs (CD4 + FOXP3 + ; white) in one HNSCC sample using high-plex whole-tissue imaging. Scale bar represents 20 μm. (Right) Box plots display the frequencies of BV-associated, LV-associated, and non-vessel-associated CCR7 + DCs close (<5 μm) to Tregs among all tumor CCR7 + DCs with nearby Tregs. Wilcoxon test, whiskers represent min to max; * p < 0.05. (C) Correlations between CCR7 + DCs and Tregs within CD45 + cells, as determined by scRNA-seq in multiple human cancer types. Spearman rank correlation; significant correlations are shown with a fitted red line. (D) (Left) Scheme outlining the analyses of CCR7 + DCs and Tregs in NSCLC samples. Patients with numerous (>5) CCR7 + DC clusters ( n = 12) were selected for downstream analyses. (Right) Frequency of CCR7 + DCs (CD11c + LAMP3 + ) with at least one nearby (<50 μm) Treg (CD4 + FOXP3 + ) in each individual patient. Numbers of FOVs analyzed per sample are as follows: NR01, n = 126; NR06, n = 455; NR09, n = 180; NR12, n = 79; NR26, n = 293; R11, n = 122; R15, n = 205; R35, n = 175; R37, n = 459; R45, n = 276. (E) (Left) Scheme outlining the analysis of tumor biopsies from HNSCC patients before immunotherapy (pre-IO). Patients were divided into non-responders (NR, n = 5) and responders (R, n = 5) based on the assessment of clinical response at 6 months. (Right) CCR7 + DC shortest distance to Tregs, T CONV , and CD8 + T cells in NR versus R tumors. Data are shown for all CCR7 + DCs compiled (NR tumors, n = 1,457 cells; R tumors, n = 1,324 cells). Unpaired t test, whiskers represent min to max; **** p < 0.0001. Numbers of FOVs analyzed per sample as in (D). (F) (Left) Scheme outlining the analyses of CCR7 + DC-CD8 + T cell niches. (Right) Frequencies of CCR7 + DC-CD8 + T cell niches with or without Tregs in their proximity (<100 μm). Two-way ANOVA with multiple comparisons, whiskers represent min to max; * p < 0.05. Numbers of FOVs analyzed per sample as in (D). (G) Representative FOV displaying CCR7 + DCs (FSCN1 + cells; FSCN1 in yellow) located near BVs (CD31 + LYVE-1 − cells; CD31 in magenta) and Tregs (FOXP3 + cells; FOXP3 in white) in untreated MC38 tumors. Scale bar represents 50 μm. (H) Correlations between the numbers of CCR7 + DCs and Tregs per mg of tumor tissue, as determined by fluorescence-activated cell sorting (FACS) analyses of MC38 and D4M3. A tumors. Spearman rank correlation; significant correlations are shown with a fitted red line. (I) Box plots show the frequencies of tumor CCR7 + DCs close (<5 μm) to Tregs that are associated to BVs or LVs in MC38 tumors ( n = 7). Whole-tumor sections were analyzed. Paired t test, whiskers represent min to max; **** p < 0.0001. See also and .

Article Snippet: Unconjugated rabbit anti-human LAMP3 (polyclonal) , Atlas Antibodies , Cat#HPA051467;RRID: AB_2681475.

Techniques: Imaging, Fluorescence, FACS

Journal: bioRxiv

Article Title: Cross-species analysis identifies genotype-driven vulnerabilities in lung adenocarcinoma

doi: 10.1101/2025.08.24.671070

Figure Lengend Snippet: A) UMAP visualization and stacked plots of human epithelial cells from patients with LUAD grouped by three major oncogenic genotypes. B) UMAP visualization and stacked plots of mouse epithelial cells from LUAD GEMMs grouped by three key oncogenic genotypes. C) Heatmap of the top differentially expressed genes between epithelial cells from patients with LUAD harboring the three oncogenic mutations. D) Heatmap analysis showing the top differentially expressed genes between the epithelial cells of LUAD GEMMs harboring the three oncogenic mutations. E) Venn diagrams reveal common human and mouse STK11 -specific genes in epithelial cells. F) Dotplots of the STK11 -specific expression of the nine overlapping genes in epithelial cells. G) Box plot comparison of PDE4D mRNA expression among different LUAD genotypes from the TCGA dataset. H) Violin plots comparing PDE4D mRNA expression among different LUAD genotypes in human cell lines from the CCLE database. I) Venn diagrams reveal common human and mouse EGFR -specific differentially-expressed genes in epithelial cells. J) Bubble plots verify EGFR -specific expression of twenty-three overlapping genes in epithelial cells. K) Box plot comparison of OGDH mRNA expression among different LUAD genotypes from the TCGA dataset. L) Violin plots comparing OGDH mRNA expression among different LUAD genotypes in human cell lines from the CCLE database. M) Kaplan-Meier survival curve utilizing the TCGA lung adenocarcinoma mRNA expression dataset for the OGDH gene. N) Kaplan-Meier survival curve using the TCGA EGFR -mutated lung adenocarcinoma mRNA expression dataset for the OGDH gene.

Article Snippet: Rabbit polyclonal anti-human unconjugated oxoglutarate dehydrogenase (OGDH)(Atlas Antibodies, Cat #HPA020347), rabbit polyclonal anti-human unconjugated cAMP-specific 3’,5’-cyclic phosphodiesterase 4D (PDE4D) (Abcam, Cat #ab14613), and rabbit polyclonal anti-human unconjugated macrophage receptor with collagenous structure (MARCO) (Atlas Antibodies, Cat #HPA063793) were optimized using a Ventana Medical Systems Discovery Ultra platform (Ventana Medical Systems, Oro Valley, AZ).

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Cross-species analysis identifies genotype-driven vulnerabilities in lung adenocarcinoma

doi: 10.1101/2025.08.24.671070

Figure Lengend Snippet: A) Representative H&E and OGDH staining in patients with LUAD. Scale bars, 5 mm. B) Representative H&E and OGDH staining of LUAD GEMMs. Scale bars, 100 μm. C) Immunoblots verifying CRISPR-mediated OGDH ablation in human LUAD cell lines. sgNT served as a non-targeting gRNA control. Actin served as a control to verify equal protein loading.

Article Snippet: Rabbit polyclonal anti-human unconjugated oxoglutarate dehydrogenase (OGDH)(Atlas Antibodies, Cat #HPA020347), rabbit polyclonal anti-human unconjugated cAMP-specific 3’,5’-cyclic phosphodiesterase 4D (PDE4D) (Abcam, Cat #ab14613), and rabbit polyclonal anti-human unconjugated macrophage receptor with collagenous structure (MARCO) (Atlas Antibodies, Cat #HPA063793) were optimized using a Ventana Medical Systems Discovery Ultra platform (Ventana Medical Systems, Oro Valley, AZ).

Techniques: Staining, Western Blot, CRISPR, Control

Journal: bioRxiv

Article Title: Cross-species analysis identifies genotype-driven vulnerabilities in lung adenocarcinoma

doi: 10.1101/2025.08.24.671070

Figure Lengend Snippet: A) Comparison of average per-patient PDE4D mRNA expression among different human LUAD genotypes in scRNA-seq data from epithelial cells. B) Representative immunohistochemical staining of PDE4D in human LUAD specimens stratified by oncogenic genotype, accompanied by violin plots quantifying PDE4D expression across genotypes. Scale bars, 300 μm. C) Violin plots comparing the average per mouse PDE4D mRNA expression in the epithelial cells of the three LUAD GEMMs used in this study. D) Representative images of PDE4D IHC of the three LUAD GEMMs and violin plots comparing the PDE4D IHC quantification. Scale bars,100 μm. E) In vitro 2D assays comparing cell proliferation in H1944 and H23 LUAD cell lines upon CRISPR-mediated ablation of PDE4D (sgNT served as a non-targeting gRNA control). F) Representative images and corresponding quantitative analysis of tumor mass from subcutaneous H1944 xenografts following genetic ablation of PDE4D. G) Dose-response curves illustrate the sensitization of H1944 cells to MRTX849 upon PDE4D ablation seven days post-treatment and MRTX IC50s comparison upon PDE4D ablation. H) Comparison of the average per-patient OGDH mRNA expression among different human LUAD genotypes in the scRNA-seq data of epithelial cells. I) Representative immunohistochemical staining of OGDH in human LUAD specimens stratified by oncogenic genotype, accompanied by violin plots quantifying OGDH expression across genotypes. Scale bars, 300 μm. J) Violin plots comparing the average OGDH mRNA expression per mouse in epithelial cells of the three LUAD GEMMs used in this study. K) Representative images of OGDH IHC in LUAD GEMMs from different genetic backgrounds and violin plots comparing the OGDH IHC quantification. Scale bars, 100 μm. L) In vitro 2D assays comparing cell proliferation in various human LUAD cell lines (H1975, PC9, H441, H358, A549, H2212) upon OGDH CRISPR-mediated ablation (sgNT serves as a non-targeting gRNA control). M) Representative images and corresponding quantitative analysis of tumor mass from subcutaneous H1975 xenografts following genetic ablation of OGDH . N) Representative images of Ki67 IHC staining in H1975 tumors and violin plots comparing Ki67 staining in H1975 tumors. Scale bars are 100 μm. O) Dose-response curves illustrate the sensitization of H1975 cells to Osimertinib upon OGDH ablation 72 h post-treatment, after OGDH ablation and Osimertinib IC50s compared to OGDH ablation. P) Representative images and corresponding quantitative analysis of tumor mass from subcutaneous PC9 xenografts following genetic ablation of OGDH . Q) Representative images of Ki67 IHC staining in PC9 tumors and violin plots comparing Ki67 staining in PC9 tumors after OGDH ablation.

Article Snippet: Rabbit polyclonal anti-human unconjugated oxoglutarate dehydrogenase (OGDH)(Atlas Antibodies, Cat #HPA020347), rabbit polyclonal anti-human unconjugated cAMP-specific 3’,5’-cyclic phosphodiesterase 4D (PDE4D) (Abcam, Cat #ab14613), and rabbit polyclonal anti-human unconjugated macrophage receptor with collagenous structure (MARCO) (Atlas Antibodies, Cat #HPA063793) were optimized using a Ventana Medical Systems Discovery Ultra platform (Ventana Medical Systems, Oro Valley, AZ).

Techniques: Comparison, Expressing, Immunohistochemical staining, Staining, In Vitro, CRISPR, Control, Immunohistochemistry