Fig 2: ICAM-1 is overexpressed in head and neck cancer stromaA. Immunofluorescence of ICAM-1 (green) and vimentin (red) in representative cancer biopsies from two distinct patients with head and neck carcinoma. B. ICAM-1 immunohistological staining in human head and neck (n=50) carcinomas. Left panel shows low ICAM-1 detection and right panel shows high ICAM-1 detection. Scale bar, 100 µm. C. Plot of mean quick score quantification for ICAM-1 specific decoration in tumor stroma from 48 head and neck carcinoma samples relative to the mean quick score for presence of invasive carcinoma cell nodules.

Fig 3: Monocyte/platelet EVs activate HUVEC in vitro. [EVs were collected from isolated monocyte/platelet aggregates, isolated platelets or THP1 cells incubated with vehicle (V) or TNF-a (50 ng/ml) for 60 min, in presence or absence of Iloprost (2 µM; PGI2) for 60 min. HUVEC were incubated with the different EV sets (10 × 106/ml) overnight. Cells were stained for flow cytomentry analysis and supernatants collected and analysed for cytokine release. (a) Confocal images of the uptake by HUVEC stained with Phalloidin (red) after 24 h of BODIPY labelled EV isolated from TNF-a stimulated cells (white arrows, left panel) or from unstimulated cells (white arrow, right panel). (b) IL-6 levels by ELISA. (c-d) Quantification of ICAM-1 and VCAM-1 expression (MFI units) in HUVEC treated with different subsets of monocyte/platelet derived EVs, platelet-derived EVs, THP1-derived EVs. (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA post Bonferroni test, mean ± SEM of n = 3–6 cell preparation incubated with distinct EV preparations from different donor cells)]

Fig 4: ICAM-1 controls proinvasive ECM remodelingA. Percentage of gel contraction by hDF six days after RNAi transfection and subsequent LIF stimulation (left panel, n=2 in triplicates). Schematic representation of the experimental conditions (right panel). B. Percentage of gel contraction by CAF 6 days after RNAi transfection (left panel, n=2 in triplicates). Schematic representation of the experimental conditions (right panel). C. Immunoblot of ICAM-1 in hDF following LIF or TGFß1 stimulation, in the presence (48h) or absence of the LIF blocking antibody. Immunoblot of tubulin was the internal control. D. Immunoblot of ICAM-1 in three hDF #1, #2 and #3) and in three CAF (Head and Neck, Breast and Lung). Immunoblot as control. E. Percentage of gel contraction by CAF transfected with control RNAi (siLuc) or RNAi targeting ICAM-1 (siICAM-1#1, #2, #3 and #4) (n=3 in triplicates, mean + s.d, ***p<0.001). F. Representative images of H&E coloration of paraffin-embedded sections of SCC12 3D-cultures in response to CAF transfected with control (siLuc) or targeting ICAM-1 (siICAM-1#1, #2, #3 and #4) siRNA (n=3, I.I., invasion index, mean ± s.d., ***P<0.001). Scale bar 100µm. G. Percentage of gel contraction by LIF-stimulated or not (Veh.) HDF subsequently transfected with control (siLuc) or ICAM-1-targeting ICAM-1 (siICAM-1#1 and #2) RNAi (n=3 in triplicates, mean + s.d, ***p<0.001). H. Representative images of H&E staining of paraffin-embedded sections of SCC12 in response to control (veh) or LIF-activated hDF subsequently transfected with control (siLuc) or ICAM-1-targeting siICAM-1#1 and #2) RNAi ((n=3, I.I., invasion index, mean ± s.d., ***P<0.001). Scale bar 100µm.

Fig 5: Protein validation of transcriptional markers reveals nonbinary latency specificity. (A) FACS analysis of CCR6 in day 7 latency IIb proliferating B cells and latency III LCLs stratified by ICAM-1 expression. (B) Quantification of mean fluorescence intensity of CCR6 expression data from panel A. (C) Percentage of CCR6-positive cells represented in panel A. (D) FACS analysis of CCR7 in day 7 latency IIb proliferating B cells and latency III LCLs stratified by ICAM-1 expression. (E) Quantification of mean fluorescence intensity of CCR7 expression presented in panel D. (F) Percentage of CCR7-positive cells represented in panel D. Each bar represents the average of results from 3 independent donors *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s pairwise t test). All error bars denote SEM.