Journal: Advanced Healthcare Materials

Article Title: Bioengineering a Patient‐Derived Vascularized Lung Tumor‐on‐Chip Model to Decipher Immunomodulation by the Endothelium

doi: 10.1002/adhm.202403446

Figure Lengend Snippet: Patient‐derived lung endothelial cells isolation and transcriptomic characterization. A) Overview of isolation of endothelial cells (ECs) from juxtatumoral tissue of surgical NSCLC samples. B) Patients’ clinical data. C) Hierarchical clustering of bulk RNA‐Seq data of commercial (HMVEC‐L) and patient‐derived (patient #11) lung ECs, in standard 2D monolayer culture or 3D microvessel model ( n = 1 sample per condition). D) Geneset enrichment scores (ssGSEA), computed from bulk RNA‐Seq data of commercial and patient‐derived (patient #11) lung ECs, in standard 2D monolayer culture or 3D microvessel model, are shown as heatmap ( n = 1 sample per condition). Rows indicate datasets. Columns indicate signatures. As positive dataset control, the pseudo‐bulk data from an in vivo lung endothelial single‐cell RNA‐Seq atlas were used. [ ⁹ ] The first 13 columns correspond to published lung endothelial signatures (four in vitro signatures, nine in vivo signatures) as described in. [ ⁹ ] As non‐endothelial negative control signatures, fibroblast signatures were used: one normal fibroblast signature and seven cancer‐associated fibroblast (CAF) signatures (CAF‐S1 subtypes).

Article Snippet: Approximately 250 consecutive serial sections (each 2 μm thick) were recorded for each microvessel, from which 3D projection images were generated using IMARIS (v9.0.2, Bitplane, USA) or FIJI software.

Techniques: Derivative Assay, Isolation, RNA Sequencing, Control, In Vivo, In Vitro, Negative Control

Journal: Advanced Healthcare Materials

Article Title: Bioengineering a Patient‐Derived Vascularized Lung Tumor‐on‐Chip Model to Decipher Immunomodulation by the Endothelium

doi: 10.1002/adhm.202403446

Figure Lengend Snippet: Phenotypic and functional characterization of engineered patient‐derived lung microvessels. A) Confocal imaging of engineered vessels. Representative images of VE‐cadherin (red), ZO‐1 (green), and Hoechst 33342 (blue) staining on a vessel generated with patient‐derived ECs (patient #4) after 24 h culture (left panel). Representative 3D projection (HMVEC‐L cells, right panel, see also Movie , Supporting Information). Scale bar = 50 µm. B) Microvessel permeability. Endothelial barrier apparent permeability (Papp) was determined by FITC‐dextran (70 kDa) permeability assay. Commercial lung endothelial HMVEC‐L cells and acellular collagen served as positive and negative controls, respectively. The mean ± standard error of the mean (SEM) are shown. Each point represents an independent microvessel, n = 4–6 per condition. Data distribution was assessed using the Shapiro–Wilk test and did not meet the assumption of normality; consequently, statistical analysis was conducted using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. C) Activation by TNF‐α. mRNA levels of ICAM‐1, VCAM‐1, and E‐selectin in lung microvessels were analyzed by mRNA under resting conditions and after activation with TNF‐α (100 ng mL −1 for 4 h). Commercial lung endothelial HMVEC‐L cells served as positive control. The housekeeping β2‐microglobuline (B2M) gene was used to normalize RNA contents. Relative expression = 2 −∆∆Ct . Mean 2 −∆∆Ct is presented ± SEM, n = 4 independent microvessels per condition. Data distribution was assessed using the Shapiro–Wilk test and did not meet the assumption of normality; consequently, statistical analysis was conducted using Mann–Whitney test. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. D) Leucocyte adhesion assays. Adhesion assays were carried out by perfusing fluorescently labelled Jurkat T lymphocytes on resting or TNF‐α treated lung microvessels. Scale bars = 50 µm. E) Quantifications of adhesion assays. Number of firmly adherent T‐lymphocytes was quantified per area. The mean ± SEM are shown, n = 3–4 independent microvessels per condition. Commercial lung endothelial HMVEC‐L cells served as positive control. Data distribution was assessed using the Shapiro–Wilk test and met the assumption of normality; consequently, statistical analysis was conducted using one‐way ANOVA Tukey's multiple comparisons test. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Approximately 250 consecutive serial sections (each 2 μm thick) were recorded for each microvessel, from which 3D projection images were generated using IMARIS (v9.0.2, Bitplane, USA) or FIJI software.

Techniques: Functional Assay, Derivative Assay, Imaging, Staining, Generated, Permeability, Activation Assay, Positive Control, Expressing, MANN-WHITNEY