Fig. 2

Protein expression patterns and heterogeneity of CDH2 and Piezo1 expression in ADC- and SCC-derived ECs. a Cluster map comparing the protein expression patterns of NEC-A, PEC-A, TEC-A, and TEC-S. Functional category gene enrichment tests were performed using heatmap.2 and the gregmisc package in the R statistical environment (http://cran.r-project.org/web/packages/gplots). Red indicates higher expression levels, and blue indicates lower expression levels in the two cell types. b The first and second principle components, PC1 and PC2, from the principle component analysis (PCA). The complete proteomic data sets for the four EC groups were subjected to PCA analysis to capture the maximum variance among ECs, using the SIMCA-P + V12.0.1 software package. The principal components (proteins from the original four groups) were marked with the corresponding name and group color. c Migration assay. The migration of NEC (pool of NEC-A and NEC-S), TEC-S, and TEC-A cells (1.8 × 10⁴) was detected using transwell chambers (6.5 mm, 8.0-μm), after induction by M131 medium supplemented with 20% or 3% MVGS or tumor cell CM at 37 °C for 12 h. To study EC-induced VSMC migration, cocultivation was established by plating VSMCs (2.4 × 10⁴) onto the filter inserts and transferring to the EC monolayer (5.0 × 10⁶), then incubating in M131 medium supplemented with 0.2% fetal bovine serum at 37 °C for 16 h. The cells were stained with 20% Giemsa solution. The number of migrated cells was counted in five randomly selected fields under a microscope (200×). d Heterogeneity of CDH2 and Piezo1 expression in ADC- and SCC-derived ECs. Expression of Piezo1 or CDH2 in HUVECs after being cultured in CM-1, CM-2 (SPC-A-1 or L-78 cell-conditioned medium, respectively), Coculture 1 (cocultured with the ADC cell line SPC-A-1), or Coculture 2 (cocultured with the SCC cell line L-78). e Consecutive sections were prepared to detect CD105, CDH2, and Piezo1 expression in the endothelial cells of tumor samples from 68 patients (36 with SCC and 32 with ADC), and CD105 expression was detected as a positive control. The fields shown in dotted lines were enlarged two-fold in the right column, and microvessels are identified using black arrows, based on positive staining for CD105. Expression of Piezo1 or CDH2 in ADC- and SCC-derived ECs, and CDH2 in ECs from tumor tissues (T) and paired paratumor (P) and normal (N) tissues of 32 patients with ADC were statistically analyzed. The intensity of staining (IS) was scored as 0: negative, 1: weak, 2: moderate and 3: strong. The percentage of positive (PP) cells was scored as 0 (PP ≤ 5%), 1 (6% ≤ PP ≤ 25%), 2 (26% ≤ PP ≤ 50%), 3 (51% ≤ PP ≤ 75%), and 4 (PP ≥ 75%). The staining in endothelial cells was scored by multiplying the PP by the IS (immunoreactive score = PP × IS): 0 (score: 0–2, negative), 1+ (score: 3–4, moderately positive), 2++ (score: 5–6, strongly positive), and 3+++ (score: 7–8, very strongly positive)