Cancer cells increase endothelial cell tube formation and survival by activating the PI3K/Akt signalling pathway

Human umbilical vein endothelial cells (HUVECs, pooled donors) were obtained from Lonza (Walkersville, MA) and maintained in Endothelial Basel Medium-2 (EBM-2) with supplements (Lonza). Replicated cultures were obtained by trypsinization and were used at passages < 6. The human lung adenocarcinoma cell line CL1-5 was established previously [22]. The cells were grown in RPMI 1640 medium (Thermo Fisher Scientific, Rockford, IL) supplemented with 10% FBS. All cell lines were kept at 37 °C in a humidified atmosphere containing 5% CO ₂ . The Transwell Permeable Supports (Corning, Tewksbury, MA) with a 0.4 μm polycarbonate membrane were used in the co-culture model system to separate HUVECs and CL1-5 cells into the different compartments. One hour prior to co-culture, 5 × 10⁴ HUVECs in 500 μl EBM-2 were grown in 24-well plate with or without Matrigel coating (BD Biosciences, San Jose, CA). An equivalent number of CL1-5 cells in 200 μl EBM-2 were seeded into the top chamber of a transwell insert, which was then placed directly on top of the 24-well plate containing the HUVECs. When performing tube formation and TUNEL assay, HUVECs were seeded on Matrigel in 24-well plate. After incubation of overnight (mRNA and protein expression) or the indicated time (tube formation and TUNEL assay), HUVECs were harvested for further analysis as described below. For the motility assay, the 8 μm polycarbonate membrane insert was used in the co-culture system where HUVECs were in the upper and CL1-5 cells were in the lower compartment as described in the migration assay section.

For F-actin staining, cells were plated on 12-mm-diameter coverslips for 24 h, washed twice with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 min at room temperature. After two additional washes with PBS, cells were permeabilized with 0.1% Triton X-100 for 2–5 min and washed again with PBS. Rhodamine conjugates of Phalloidin (Thermo Fisher Scientific) were used to stain F-actin. The cells were stained for 40 min at room temperature. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The fluorescence images of F-actin and nuclei were visualized by confocal microscopy (ZEISS, Germany).

The Transwell Permeable Support (Corning) with an 8-μm polycarbonate membrane insert was used in the cell migration model where HUVECs were allowed to migrate. Approximately 2 × 10⁴ HUVECs in 200 μL EBM-2 medium were loaded into each 24-well insert in triplicate with or without CL1-5 cells in the lower chamber at 37 °C in a 5% CO₂ incubator. After approximately 20 h, the migrated cells were fixed with methanol, stained with Giemsa solution (Sigma, St. Louis, MO) and counted at 200x magnification under a light microscope.

Matrigel Basement Membrane Matrix (BD Biosciences) was diluted with EBM-2 medium and coated in 24-well plates at 37 °C for 1 h. Then, 5 × 10⁴ HUVECs were seeded alone or co-cultured with an equivalent number of CL1-5 cells in the EBM-2 medium on Matrigel. Co-cultured CL1-5 cells were seeded in transwells and incubated in the same well with HUVECs. The tube formation ability of HUVECs was measured at 1, 2, 6, 12 and 24 h with or without CL1-5 cells. In inhibitor experiments, HUVECs were treated with the PI3K inhibitor LY294002 (5 μM) and the COX-2 inhibitor celecoxib (10 μM) (Sigma) for 12 h and co-cultured with CL1-5 cells. After incubation, the number of tubes and nodes of the tubular structures was quantified.

Total RNA was extracted from HUVECs, which were co-cultured with or without CL1-5 cells. First-strand cDNA for real-time quantitative PCR (QPCR) analysis was obtained from 5 μg of total RNA using a random primer and SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Reactions were detected by the SYBR Green approach (Thermo Fisher Scientific). Ten nanograms of cDNAs served as templates to detect gene expression. Experiments were performed three times in triplicate. Details of the specific primers designed for QPCR to determine relative levels of gene expression are shown in Table 1.

All experiments were performed as previously described [23]. After transfer to nitrocellulose membranes, the following primary antibodies were used: α-actinin (Merck Millipore, Billerica, MA), β-catenin (SANTA CRUZ BIOTECHNOLOGY, Dallas, Texas), Akt (Cell Signaling Technology, Beverly, MA), phospho-Akt (Ser473) (Cell Signaling Technology), PI3K (SANTA CRUZ BIOTECHNOLOGY), phospho-PI3K p85 (Tyr458)/p55 (Tyr199) (Cell Signaling Technology), PARP (Cell Signaling Technology) and Caspase 3 (Cell Signaling Technology). Chemiluminescent signals were detected by the Fujifilm LAS-3000 system (Fujifilm, Tokyo, Japan), and β-actin and α-tubulin (Sigma) (Merck Millipore) were used as the loading control. To determine the Rac-1 activity, the Active Rac1 Pull-Down and Detection Kit was used, according to the manufacturer’s protocol (Thermo Fisher Scientific).

The mRNA profiles of HUVECs co-cultured with or without CL1-5 cells were analysed using the Affymetrix Human Genome U133 Plus 2.0 GeneChip according to the manufacturer’s protocols (Santa Clara, CA) by the National Taiwan University Microarray Core Facility for Genomic Medicine. The raw data were analysed by GeneChip Operating software (GCOS). Pathway analyses of the differentially expressed genes were performed using the DAVID programme [24].

The data were presented as the means ± standard deviations, and the significance of differences was analysed using Student’s t-test. All experiments were performed in triplicate. To evaluate the prognostic ability of the selected candidate genes, we examined their association with clinical data using a published microarray dataset GSE30219 [25]. The intensity values of the probes from expression profile of GSE30219 were first rescaled using a quantile normalization method, and then log-transformed to a base-2 scale. The prognostic test was performed as previously described [15]. Briefly, the differentially expressed genes were employed to test its association with overall survival and disease-free survival by univariate Cox proportional hazard regression analysis. For those genes with a significant Cox regression coefficient, a risk score method was used to calculate the signature and construct the risk score function. The risk score function was a linear combination of gene expression weighted by the regression coefficient from Cox regression. The median risk score was used as the cut-off point for patient classification. Kaplan-Meier method was used to estimate survival curve and difference between curves was evaluated by log-rank test. Multivariate Cox proportional hazards regression analysis was employed to evaluate independent prognostic factors, and age, gender and stage were used as covariates. All statistical tests were two-tailed and P < 0.05 was considered statistically significant.

To clarify the interaction between endothelial cells and cancer cells, we established a co-culture system using the Corning 0.4 μm polycarbonate membrane. In this model, HUVECs were seeded in the lower chamber and CL1-5 lung cancer cells were seeded in the upper compartment. This system was labelled as HUVEC/CL1-5 in the figures, and HUVECs without cancer cells were labelled as HUVEC.

We used the phenotypic changes of HUVECs to evaluate the effects of lung cancer cells on angiogenesis in the co-culture system. The morphology of HUVECs alone revealed a fusiform cell shape and formed a tight cluster. In contrast, after co-culture with CL1-5 cells, the HUVECs elongated, spread and lost contact with each other, as shown in Fig. 1a.

Angiogenesis is a multi-step process that includes proliferation, migration and tube formation of endothelial cells. Endothelial cells migrate along chemoattractants, which are secreted in the microenvironment. Here, we used the transwell migration assay to create the chemical gradient by putting cancer cells in the lower chamber. The data also showed that the migration capacity of HUVECs increased significantly (2.2-fold) when co-cultured with CL1-5 cells compared with HUVECs alone (P < 0.05) (Fig. 1b).

To evaluate the ability to undergo angiogenesis, the tube formation experiment is the key method used in vitro. When HUVECs were seeded on Matrigel, they gradually formed capillary-like tubular structures, and the capillary-like tubes connected to each other created a mesh-like structure on the gel. The capillary-like tubular structure was formed more densely, reached a peak at 2 h and finally disappeared in a time-dependent manner. Quantitative analysis of capillary-like tubular structures showed that the number of tubes and nodes significantly increased when HUVECs were co-cultured with CL1-5 cells at each time point (P < 0.05) (Fig. 1c). It appeared that the angiogenic ability of HUVECs increased after co-culture with cancer cells. On the other hand, the viability of endothelial cells is also critical in tumour angiogenesis [26]. As shown in Fig. 1c, the tubular structures decreased and the cell death increased as time went by. To further investigate whether the viability of HUVECs was altered by cancer cells, TUNEL assay was performed at 6, 12, 24 and 30 h after seeding HUVECs on Matrigel-coated plate. HUVECs were recovered from Matrigel by Cell Recovery Solution and estimated the apoptotic percentage. The percentages of apoptotic HUVECs were low at 6 h and showed no difference between co-culture and non-co-culture groups. However, the apoptotic percentage of HUVECs alone was increased 1.5 ~ 2-fold higher than that of HUVECs co-culture with CL1-5 cells and in a time dependent manner after 12, 24 and 30 h of tube formation (P < 0.05) (Fig. 1d). This indicated that co-culture with cancer cells would decrease the apoptotic percentage of endothelial cells.

As shown in Fig. 1, many phenotypes of HUVECs changed when co-cultured with cancer cells. To identify the specific molecular mechanism, we used an Affymetrix microarray to identify the gene expression profile. We found that ~7000 genes were up-regulated and down-regulated over 2.5-fold in HUVECs co-cultured with CL1-5 cells compared to HUVECs alone. The differentially expressed genes were subjected to pathway analysis using DAVID. The top 10 significant pathways are listed in Additional file 1: Table S1. These functional pathways provided us with useful hints to confirm the molecular changes of HUVECs altered by cancer cells.

According to pathway organization, we identified that PIK3CA, PIK3R3, AKT3, ITGAV, ITGB3 and RAC1 appeared in the top 2^nd, 4^th and 5^th pathways (i.e., PI3K-Akt signalling pathway, Jak-STAT signalling pathway and RAP1 signalling pathway, respectively) and were correlated with the observed phenotypes. Although the changes in the expression of PIK3R1 and CTNNB1 were under 2.5-fold, these genes were also selected for further analysis due to their role in these three pathways. Moreover, based on a reference search, we further identified some genes enriched in the lower ranking of DAVID that predicted functional annotation or some genes with under a 2.5-fold change but related to the phenotype change of HUVECs, including ACTN1, PTGS2 (COX-2), CXCL8, VCAM1 and ICAM1. These candidate genes have been shown to be involved in survival, migration and angiogenesis. The expression of these genes was validated by QPCR, as shown in Fig. 2. In survival-related genes (Fig. 2a), all of the investigated genes were up-regulated in HUVECs with cancer cell co-culture. Among these, the mRNA levels of PIK3R1 and CTNNB1 were increased more than 2-fold, and those of PIK3R3, PIK3CA and AKT3 were increased 1.6- to 1.9-fold after co-culture with cancer cells. Nevertheless, the expression of these genes exhibited the same trend as the microarray data. In addition, all of the migration- and angiogenesis-related genes in HUVECs with CL1-5 co-culture were expressed 2-fold more than the genes in HUVECs alone (Fig. 2b, c). The comparison of the expression level between microarray and QPCR was summarized in Additional file 1: Table S2. The protein level and activity (phosphorylation status) were also confirmed by Western blot and showed a significant increase after co-culture with cancer cells (Fig. 3a, b).

Based on our results, the migration ability of HUVECs was elevated after co-culture with cancer cells (Fig. 1b). In cell migration, Rho GTPases are important regulators of cell cytoskeleton dynamics [27]. Here, we showed that Rac-1 was bound by GTP after co-culture with CL1-5 cells (Fig. 3c). It revealed that Rac-1 was activated in HUVECs after co-culture with CL1-5, and this might contribute to the increasing motility of HUVECs after interaction with lung cancer cells.

Lamellipodia and filopodia are crucial in cell motility [28‐30]. During cell movement, protrusions are stimulated at the leading edge of cells. We used rhodamine phalloidin to stain and label cytoskeleton F-actin in HUVECs and observed fluorescence images by confocal microscopy. The results showed that the cell margin of HUVECs alone group was smoother than that of the co-culture group. The cytoskeletal distribution of F-actin in HUVECs was rearranged after co-culture with CL1-5 cells. Filopodia stuck out of the lamellipodia (Fig. 3d). In addition, the number of filopodia increased in HUVECs after co-culture with CL1-5 cells (P < 0.05) (Fig. 3e).

In previous data, PI3K and COX-2 expression were significantly up-regulated in HUVECs after co-culture with CL1-5 cells (Figs. 2 and 3a). These proteins were reported to be involved in cell survival, migration and angiogenesis. Therefore, we chose inhibitors of PI3K (LY294002) and COX-2 (celecoxib) to clarify their effects on the capillary-like tubular formation capacity and cell viability of HUVECs after co-culture with CL1-5 cells. After being treated with 5 μM LY294002 and 10 μM celecoxib in HUVECs of the co-culture system, the capillary-like tubular structure on Matrigel was reduced significantly at 12 h compared with that of HUVEC/CL1-5 without inhibitor treatment. The number of tubes and nodes were also decreased in HUVEC/CL1-5 with treatment with LY294002 and celecoxib compared to the non-treated control (P < 0.05) (Fig. 4a). Furthermore, the apoptotic percentage increased from 18% to 78% compared with the non-treated control after LY294002 treatment by TUNEL staining assay (P < 0.05) (Fig. 4b). This apoptosis process involved Caspase 3 and PARP cleavage (Fig. 4c). These data revealed that PI3K and COX-2 were important in endothelial cell angiogenesis and survival. Manipulating PI3K and COX-2 could reverse the phenotypic changes of HUVECs after co-culture with lung cancer cells.

The data mentioned above indicated that many genes in endothelial cells were up-regulated after co-culture with cancer cells and correlated with angiogenesis, migration and anti-apoptosis phenotypes. Generally, the tumour specimen may contain not only endothelial cells but also surrounding tissue when the tumour section was dissected. Thus, we sought to determine whether these up-regulated gene sets could be employed to predict patient outcome. Based on microarray screening and QPCR validation (Fig. 2, Additional file 1: Table S2), 13 genes were used to construct the prognostic gene signatures. In these genes, two predicted the opposite clinical outcome compared to our results (ITGB3 and PIK3R1) and thus were not included in signature calculation. Using the remaining 11 genes that were differentially expressed after co-culture with cancer cells, we identified the gene signatures to predict the overall survival and disease-free survival of NSCLC patients. The 11-gene signature (12 probes from 11 genes) was identified based on the patient cohorts that had been published previously [25]. Detailed information on the probes, genes and risk score formula for each gene signature is described in Additional file 1: Table S3. Patients with a high expression gene signature exhibited shorter median overall survival and disease-free survival than patients with a low expression gene signature (P = 0.0035 and P = 0.0026, respectively; log rank test; Fig. 5a, b). Furthermore, we selected the hub genes or more upstream genes to narrow down the gene number, which may be more applicable in clinical testing. The 5-gene signature (6 probes from 5 genes) could more significantly predict overall survival and disease-free survival (P = 0.0009 and P = 0.0016, respectively; log rank test; Fig. 5c, d). A multivariate Cox proportional hazards regression with covariates of gender, age and stage was used to evaluate the prognostic independence of gene signatures in the published cohort (n = 293 in overall survival prediction; n = 278 in disease-free survival prediction). Except for the 11-gene set in disease-free survival, which was at the borderline but still had a trend (P = 0.0521), the gene signatures were significant after considering the effects of covariates (Table 2). The results indicated that the gene signatures from cancer cell-stimulated endothelial cells were correlated with extended overall survival and disease-free survival in this published cohort.