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