EC protein expression profiling based on iTRAQ analysis
Primary ECs that were positive for expression of EC markers (CD31, CD34, CD144, CD105, and VWF) but negative for expression of a vascular smooth muscle cell (VSMC) marker (α-SMA) and monocyte markers (CD11b and CD45b), with high purity and viability (CD105 expression > 98%, AcLDL uptake > 90%), were prepared (Fig. 1; Additional file 1). A total of 1820 proteins were identified. Gene set enrichment analysis of the differentially expressed proteins among ADC-related ECs shown in Additional file 2: Figure S1, including the 81 proteins unique to TECs from ADC (TEC-A) (compared to paratumor-derived ECs from ADC (PEC-A) and normal tissue-derived ECs (NEC-A)), which were mainly involved in transcription activity, protein prepare, hydrogen peroxide catabolic processes, NF-κB transcription factor activity.
The proteomic profiles were similar in TECs by heatmap analysis (Fig. 2a). A few principal components accounted for 63.3% (PC1 and PC2) of the total data variation, and the locations of the TECs were very close together. Then, the TEC proteomic profiles were analyzed in pairs (Fig. 2b). The results show the proteins (including CDH2, Piezo1, EPS8, NAP1L1, FAM98B, HSPBP1, RPS15 and so on) responsible for segregation into two groups. Only 31 proteins (see Additional file 3: Table S1) were differentially expressed in both groups, among which CDH2 and Piezo1 were the two most significantly upregulated proteins in the TEC-A group. The expression levels of 15 significantly altered proteins were verified by western blotting (Additional file 4: Figure S2A). Similar changes were observed in PEC-A, TEC-A and tumor cell–EC cocultivation samples. Therefore, all types of isolated ECs provide a good basis for studying tumor angiogenesis and heterogeneity.
Heterogeneity of lung cancer-derived ECs
EC migration and positive regulation of smooth muscle cells are two important biological processes relevant to angiogenesis. The migration of TECs induced by either M131 medium containing 20% or 3% MVGS or tumor cell-conditioned medium (CM) was significantly faster than that of NECs (pool of NEC-A and NEC-S), but TEC-A migration was even faster (P < 0.01) than that of TEC-S in the CM group. Moreover, during EC- VSMC cocultivation, although all types of ECs enhanced the migration of VSMCs, TEC-A did so most markedly (Fig. 2c).
The expression levels of Piezo1 and CDH2 were upregulated in primary human umbilical vein endothelial cells (HUVECs) cocultured with the lung ADC cell line SPC-A-1 but not the SCC cell line L-78 or CM from SPC-A-1 or L-78 cells (Fig. 2d). Positive immunostaining of Piezo1 and CDH2 (score > 3) was significantly more frequent in TEC-A than in TEC-S samples (P = 0.01 and P < 0.0001, respectively; Fig. 2e). In SCC, the expression of CDH2 was much higher in TECs than in NECs or PECs. Furthermore, the prognostic values revealed that high tumor CDH2 expression was related to a shorter survival time in LC patients (P = 0.017); this correlation was more significant in ADC (n = 720, P = 0.008) but not significant in SCC (n = 524, P = 0.220). However, no similar correlation between Piezo1 expression and survival time was observed (Additional file 4: Figure S2B). These data indicated that CDH2 likely plays more important functional roles in ADC development than in SCC development.
Effect of CDH2 on angiogenesis
Loss- or gain-of-function analysis revealed that CDH2 expression significantly promoted EC proliferation, motility and capillary-like tube formation, whereas silencing endogenous CDH2 had the opposite effect (Additional file 5: Figure S3). A markedly increased density of neovessels (VWF-positive) was observed in Matrigel plugs containing CDH2-overexpressing ECs (Additional file 6: Figure S4A). Preclinical and clinical data have shown that a peptidic CDH2 antagonist (exherin, ADH-1) causes rapid tumor vascular disruption and apoptosis [9]. To date, limited research has been conducted on CDH2-expressing ECs. When treated with 0.4 mg/mL ADH-1 for 24 h, the CDH2 overexpression group (mainly located in the cell membrane) was significantly inhibited, but this effect was not obvious in the control group. ADH-1 induced apoptosis in a dose- and CDH2-dependent manner in ECs (Additional file 6: Figure S4B&C).
Mechanism of CDH2-mediated angiogenesis
With CDH2 overexpression, epithelial cell markers (E-cadherin, EpCAM, and P-cadherin), neural and basal cell adhesion molecules (NCAM-1, NrCAM, and BCAM), leukocyte rolling and attachment molecules (L-, E-, and P-selectin), and strictly EC-specific adhesion molecules (VE-cadherin) were all downregulated. Moreover, proteins known to positively regulate leukocyte migration (RANTES, ENA-78, IL-6, MCP-2, and IP-1α) were also significantly downregulated. Three proteins involved in the VEGFR signaling pathway (VEGFA, VEGFD, and VEGFR3) were significantly upregulated. Furthermore, the phosphorylation levels of two major MAPK signaling pathways, namely, the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways, were significantly increased, and CDH2 was confirmed to induce HIF-1α, VEGF, and VEGFR3 accumulation (Additional file 7: Figure S5). Therefore, the MAPK/ERK and MAPK/JNK signaling pathways are likely to play crucial roles in CDH2-induced HIF-1α/VEGF-mediated angiogenesis.
Clinical value of CDH2 in ADC
The adjacent normal tissues were nearly negative for CDH2 expression, but tumor cells in the lung tissues exhibited very strong positive staining (Fig. 3a, clinical data were listed in Additional file 8: Table S2). The expression of CDH2 in cancer cells from SCC and ADC did not differ significantly (scored by PP × IS), but its localization in cancer cells was heterogeneous. Therefore, expression was further rescored by the intensity of staining (IS, score: 0~3) in the membrane, cytoplasm and nucleus. CDH2 expression in the cytoplasm or nucleus was significantly higher in cancer cells form ADC than in those from SCC (P < 0.05). Heterogeneity of CDH2 cleavage resulted in different regulatory behaviors related to cancer cell–cell adhesion and cell migration/ proliferation [10].
Positive CDH2 staining (score > 3) was seen in 22 and 66% of TECs from SCC and ADC tumors, respectively (Fig. 3b). High CDH2 expression in TECs was significantly positively related to ADC (P < 0.001) and tumor stage (P < 0.05) in LC patients, and positively related to tumor stage and visceral pleural metastasis in the ADC subtype (P < 0.05, Fig. 3b and c; Additional file 9: Table S3). Significantly poorer prognosis and overall survival were associated with patients with high TEC CDH2 expression and LC (P = 0.023) or ADC (n = 86, P = 0.025) but not SCC (n = 53, P = 0.623) (Fig. 3d). These data suggest that CDH2 may serve as a prognostic indicator of relative risk for patients with ADC.