Tumor-endothelium reciprocal interactions in co-culture: changes of genetics and phenotypic characteristics of endothelial and laryngeal cells

Cell culture and study designs

HUVEC cell line was supplied by Dr Timucin Avsar, and HEp-2 cell line was supplied by Dr Hasan Acar. HEp2 cells were transfected with a green fluorescent protein (GFP) to aid in the discrimination of the cells from HUVECs. GFP-HEp-2 cell line was obtained in the project of Hasan Acar et al. (TÜBİTAK-112S498). Both cell types were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, USA) with 10 % fetal bovine serum (FBS, Gibco, USA) and 1 % penicillin/streptomycin (Gibco, USA) at 37 °C in a 5 % CO2 incubator. Stable GFP-expressing HEp-2 cells were expanded in growth media. For direct co-culture, HUVECs were grown to 80 % confluence for 24 h, and then GFP-HEp- two cells were added at a 4:1 ratio. Two types of indirect co-culture were performed. For the first type, which was used for the invasion assay, cells were cultured in 24-well Transwell chambers (Corning, USA) with or without Matrigel. The second type of indirect co-culture, used for proliferation, migration, and gene expression analyses, involved the use of conditioned medium (CM).

Preparation of the CM

HEp-2 and HUVEC cells were seeded into T75 culture flasks and cultured in DMEM for 30 h until reaching approximately 80 % confluence. The medium was then collected and centrifuged at 1,500 rpm for 5 min, and the supernatant was filtered to obtain CM for further studies. Various concentrations of CM, specifically 10 , 25, and 100 %, were prepared and utilized in experiments. For optimization, experiments were conducted with 100 % growth medium (cell control) and different CM concentrations (10 , 25, 50, and 100 %) in proliferation assays. Based on these results, 10 % CM was selected as the optimal concentration for subsequent experiments, including migration and gene expression analyses.

Morphology analysis

To evaluate the effect of co-culture on cell morphology, GFP-HEp-2 and HUVEC cell lines, both in mono-culture and direct co-culture, were examined daily for 120 h using an inverted laser scanning confocal microscope (Nikon A1R1, Japan). GFP was excited with a 488 nm laser line, and emission was detected at 510 nm.

Trypan blue staining

Cell viability was evaluated using a trypan blue exclusion assay. A total of 3×10⁵ cells per well were seeded in a growth medium in 6-well plates. After overnight attachment and growth, control cells in mono-culture were fed with the growth medium, while co-culture cells were fed with 10 and 100 % CMs. Trypan blue staining was performed as previously described [19].

XTT proliferation assay

Cell growth rates in mono-cultures and co-cultures were determined by XTT assay. Cells were seeded into 96-well plates at a density of 5×10³ per well. After overnight attachment and growth, mono-culture cells as control were fed with culture mediums, and the co-culture cells were fed with CMs of 10 and 25 %. Cell viability was assessed at 24, 48, and 72 h. The XTT kit (BI, USA) was used according to the manufacturer’s instructions. Results were obtained by detecting absorbance at a wavelength of 460 nm with the microplate reader (Biotek-Epoch, USA). The cell proliferation rate (%) was calculated using the formula (OD of sample/OD of control)×100. Three replicate wells were used for each analysis.

Real-time analysis of cell proliferation assay (RTCA)

Cell growth of mono-cultures and co-cultures of HEp-2 and HUVEC cells was continuously monitored using the xCELLigence RTCA MP instrument (ACEA Biosciences, USA). Briefly, cells were seeded into wells of E-plate View 16 (ACEA Biosciences, USA) at a density of 10⁴/100 µL per well. After overnight attachment and growth, mono-culture cells as control were fed with growth mediums, and the co-culture cells were fed with CMs 10 and 100 % at the 25th hour, consistent with the XTT assay. RTCA assay was performed according to the previously published method [20]. The impedance was measured every 15 min for at least 120 h at 37 °C in a 5 % CO2 atmosphere. Cell proliferation rate was registered as an increase in the cell index (CI). All experiments were performed in duplicate. CI is a parameter used to monitor cell proliferation changes in real time. It is measured through electrical impedance-based systems like xCELLigence, reflecting cell density and attachment strength [21].

Wound healing and invasion assays

Cells in the growth medium were seeded in 24-well plates and were adhered to overnight (>90 % confluence). To suppress the proliferation of the cells, they were pretreated with mitomycin C (5 μg/mL) (Sigma, Germany) for 2 h. A wound-healing assay was subsequently performed to evaluate cell migration capacity. Photographs of the same area of the wound were taken at certain times (3, 6, 9, 12, 24, 48, 72 h) using a light inverted microscope (Leica, Germany). The migration distances were measured using Image J software (LOCI, USA) based on the images at time “0” as the reference point. To evaluate invasive capacity, a 24-well Transwell chamber invasion assay (Corning, USA) coated with Matrigel (Corning, USA) was used. Three fields (×100) were randomly selected for counting. The experiment is illustrated in Supplementary Material, Figure S1. Different co-culture systems were selected based on the requirements of each experiment: CM was used for migration, proliferation, and gene expression assays, while Transwell chambers were employed for invasion assays to provide a physical barrier that better simulates in vivo extracellular matrix interactions.

Quantitative real-time polymerase chain reaction (qRT-PCR)

For real-time PCR analysis, the HEp-2s and HUVECs were in vitro co-cultured for 24 h (short-term) and 21 days (long-term). Mono-culture HUVECs and mono-culture HEp-2s served as controls. Total RNA was extracted from the co-culture and mono-culture systems using the TRIzol reagent (Invitrogen, USA). For cDNA synthesis, 1 µg of total RNA from each pooled sample was reverse transcribed into cDNA using a 2-step RT-PCR kit (Vivantis, Malaysia) according to the manufacturer’s instruction. cDNA was diluted 1:9 in sterile water. qPCR was performed on a Lightcycler 480 instrument (Roche, Light Cycle 480 II, Germany) using GoTaq^® qPCR Master Mix (Promega, USA). The housekeeping gene GAPDH was used as the internal control for HEp-2s. The other housekeeping gene, B2M, was used as the internal control for HUVECs. The genes (CDH2, CCL21, CXCL8, ITGB, MMP2, MMP9, and VIM) were selected from those reported in the literature to be associated with cancer. The relative fold change in target gene expression was calculated using the comparative cycle threshold (2−ΔΔCT) method. All experiments were conducted in triplicate. Specific primers for PCR amplification were designed using IDT (http://eu.idtdna.com/home/home.aspx). The primers used are listed in Supplementary Material, Table S1.

Statistical analysis

Data were shown as mean + standard deviation (SD) from three independent experiments and analyzed using GraphPad Prism 6.00 (GraphPad Software, USA). Student’s t-test was performed between groups for comparing treated and untreated cell groups. Statistical significance is indicated as p<0.05, p<0.01 and p<0.001.

Co-culture induces morphological changes in HEp-2 and HUVEC cells

To evaluate the direct co-culture effect on HEp-2 and HUVEC cell morphology, we examined the cells using confocal and light microscopes. Control samples of HEp-2, grown separately, exhibited their characteristic rounded and connected morphology (Figure 1A–C). In contrast, after 120 h of co-culture, HEp-2 cells displayed a spindle-shaped morphology, becoming disjointed and scattered. Meanwhile, HUVECs in mono-culture appeared ‘teardrop-like’, but in co-culture, they developed net-like formations similar to a vascular network (Figure 1B–D).

Figure 1:

Confocal microscope images of GFP-HEp-2 and HUVEC cells at 10× and 40×magnification. Left panel mono-cultured GFP-HEp-2 cells in the GFP channel. Right panel co-cultured GFP- HEp-2 cells in GFP and TD merged channel. Co-cultured HEp-2 cells exhibited elongated shuttles (B, D), while monocultured HEp-2 cells maintained a rounded form (A, C). HUVECs formed net-like structures in co-culture (B). GFP, green fluorescence protein; TD, transmitted detector, bright field.

Dose- and time-dependent effects of conditioned medium on cell proliferation

Treatment with 10 and 25 % HUVEC-CM significantly increased HEp-2 proliferation at 24 h by 1.6-fold and 1.5-fold, respectively (p<0.001 for both), but did not affect HEp-2 viability at 48 and 72 h (Figure 2A). Similarly, 10 % HEp2-CM treatment significantly increased HUVEC proliferation at 24 and 48 h by 1.5-fold and 1.3-fold, respectively (p=0.010 and p=0.002), with no significant changes in HUVEC viability at 72 h (Figure 2B). Cell proliferation was evaluated by cell counting, and it was found that 10 and 100 % HUVEC-CM significantly reduced HEp-2 cell numbers by 1.6-fold and 2.6-fold at 24 h (p=0.003 and p<0.001). At 72 h, the reduction was 1.6-fold and 3.2-fold, respectively (p=0.003 and p<0.001) (Figure 2C). These results indicate that HUVEC-conditioned medium inhibits HEp-2 cell proliferation in a dose and time-dependent manner. Additionally, Figure 2D demonstrates a significant reduction in HUVEC cell numbers following 24 h of treatment with 10 and 100 % HEp2-CM, showing reductions of 1.3-fold and 2.5-fold, respectively (p=0.026 and p<0.001). However, at 72 h, the number of HEp-2 cells treated with 100 % HUVEC-CM decreased much more significantly than the control cells, reaching up to a 32.8-fold reduction (p<0.001). These results underscore the substantial inhibitory effect of 100 % HUVEC-CM on HEp-2 cell proliferation over time.

Figure 2:

Effect of indirect co-culture on the cell viability of HEp-2 (A, C) and HUVEC (B, D). After HEp-2 and HUVEC cells treatment with DMEM 10 % FBS (control), HEp2-CM (10 %, 25 %), or HUVEC-CM (10 %, 25 %) for 24, 48, and 72 h, and XTT assay was performed to determine the viability of cells. HEp-2 (A) and HUVEC (B) in CM showed higher proliferation potentials than controls, depending on CM concentration and incubation time. After HEp-2 and HUVEC cells were treated with DMEM 10 % FBS (control), HEp2-CM, or HUVEC-CM for 24 and 72 h, cell proliferation was evaluated by trypan blue staining. The cell counts of HEp-2 (C) and HUVEC (D) were decreased significantly compared to that in the control mediums. *p<0.05, ** p<0.01, *** p<0.001. CM, conditioned medium.

In our study, discrepancies were observed between the results of the XTT assay and the trypan blue exclusion assay. While the XTT assay indicated that the CM increased cell proliferation at early time points, the trypan blue assay showed a reduction in cell numbers.

Real-time monitoring of dynamic cell proliferation and attachment using the xCELLigence system

To assess the effects of CM on the proliferation and attachment of HEp-2 and HUVEC cells, the cells were monitored every 15 min for 120 h using the xCELLigence system. As illustrated in Figure 3A, the CI for each cell type, including controls, reached its peak at approximately 60 h after treatment with CM. Significant differences were observed between the HEp-2 control cells and those treated with 10 % CM, as well as between the HEp-2 control cells and those treated with 100 % CM (p=0.012 and p<0.001, respectively). Consequently, 10 and 100 % HUVEC-CM resulted in a 1.1-fold and 2.3-fold decrease in the HEp-2 CI (Figure 3B). In contrast, HUVEC cells showed minimal response to CM treatment, with cell indices nearly identical between the control and treated groups. No significant differences were observed in the HUVEC control cells and those treated with 10 % CM. However, treatment with 100 % HEp2-CM significantly reduced HUVEC proliferation (p=0.013) (Figure 3C).

Figure 3:

Effects of conditioned medium on HEp-2 and HUVEC cell index: Time-dependent analysis and quantification. Effects of 10 and 100 % CM during 120 h exposure on the viability of HEp-2 and HUVECs were measured based on the cell index by the xCELLigence system. (A) The cell index plot of HEp-2 (red line), HUVEC (light green line), HEp-2-100 % CM (purple line), HEp-2-10 % CM (orange line), HUVEC-100 % CM (blue line), HUVEC-10 % CM (dark green), baseline (black line). The bar diagram represents the cell index of HEp-2 (B) and HUVEC (C) in various percentage concentrations of HEp-2-CM and the HUVEC-CM models. *p<0.05, *** p<0.001. CM, conditioned medium.

Enhanced wound healing capacity of HEp-2 and HUVEC cells induced by CMs

Whether CMs induce cell migration in HEp-2 and HUVEC cells was investigated using a gap closure assay to measure differences in migration capacities. By the 24th h, co-cultured HEp-2 cells had completely closed the gaps, while co-cultured HUVEC cells achieved complete gap closure by the 48th h. The wound-healing rates were significantly higher in both HEp-2 and HUVEC cells treated with CM, showing 2.3-fold and 3-fold increases, respectively, compared to cells grown in serum-free medium (p=0.013 and p=0.030) (Figure 4A and B). The light microscope images of HEp-2 and HUVEC migration were consistent with the results (Figure 4C and D). These findings suggest that both HEp-2-CM and HUVEC-CM significantly promote the migration capacity of HEp-2 and HUVEC cells.

Figure 4:

Effects of conditioned medium and co-culture on migration and invasion of HEp-2 and HUVEC cells. (A) Migration of HEp-2 cells under three different conditions: Serum-free medium, growth medium, and 10 % HUVEC-CM, evaluated over 24 h (B) Migration of HUVEC cells under three different conditions: Serum-free medium, growth medium, and 10 % HEp-2-CM, evaluated over 24 h. the percentage of migration was calculated based on the wound-healing assay. (C) Representative images of HEp-2 cell migration at 0 and 24 h under control (growth medium) and 10 % HUVEC-CM conditions. (D) representative images of HUVEC cell migration at 0 and 24 h under control (growth medium) and 10 % HEp-2-CM conditions. Blue arrows indicate the wound area. The spreading speeds of HEp-2 (A) and HUVEC (B) cells in 10 % CM along the wound edge were faster than those in controls. Comparison of the invasion percentage of HEp-2 (E) and HUVEC (F) cells under control conditions and co-culture conditions. Matrigel invasion assay results show a statistically significant reduction in HEp-2 cell invasion in the co-culture group compared to the control group (p=0.049). Representative images of HEp-2 (G) and HUVEC (H) cell invasion through Matrigel (Matrigel +) and without Matrigel (Matrigel -) under control and co-culture conditions. Matrigel (+) images depict cells that successfully invaded through the Matrigel layer, while Matrigel (−) images show non-specific migration. Similar to HEp-2 cells, Matrigel (+) and Matrigel (−) images demonstrate the invasion and migration ability of HUVEC cells in both conditions. *p<0.05. CM, conditioned medium; ns, not significant.

Decreased invasion capacity of HEp-2 cells induced by co-culture with HUVECs

Fewer HEp-2 cells co-cultured with HUVECs were seen invading across the Matrigel compared to the control cells. This suggests that co-cultured HUVECs reduced the invasion potential of HEp-2 cells by approximately 2-fold (p=0.049) (Figure 4E). Conversely, HUVECs co-cultured with HEp-2 cells did not exhibit a significantly higher invasion capacity compared to HUVECs cultured alone (p=0.287) (Figure 5F). The light microscope images of HEp-2 and HUVEC invasion were consistent with the results (Figure 4G and H).

Figure 5:

Gene expression profiles of HEp-2 and HUVEC cells treated with HUVEC CM or HEp-2 CM. qRT-PCR of CDH2 (A), CLC21 (B), CXCL8 (C), ITGB (D), MMP2 (E), MMP9 (F) and VIM (G) genes in co-cultured HEp-2 and HUVEC cells treated with corresponding CM for 24 h or 21 days. The expression level of CCL21 mRNA (B) was markedly increased (88.4-fold) in HUVEC cells when treated with HEp2-CM at 21 days. *p<0.05, **p<0.01, ***p<0.001. CM, conditioned medium; Co-24 h, co-culture condition after 24 h; Co-21 d, co-culture condition after 21 days.

Gene expression patterns of HEp-2 and HUVEC cells treated with HUVEC CM or HEp- 2 CM

Changes in proliferation, migration, and invasion in HEp-2 and HUVEC cells treated with CM or in co-culture were observed, leading to the investigation of the underlying molecular mechanisms. Accordingly, the mRNA expression levels of genes significantly associated with cancer prognosis were evaluated. The mRNA levels of CDH2, CCL21, CXCL8, ITGB, MMP2, MMP9, and VIM genes were significantly downregulated in HEp-2 cells grown in co-culture, with expression levels reduced by up to 0.05-fold at either 24 h or 21 days (Figure 5). Similarly, the mRNA levels of CXCL8 and ITGB were significantly downregulated in HUVEC cells grown in co-culture at 24 h or 21 days (Figure 5C and D). Meanwhile, a significant increase in the expression level of CCL21 mRNA (88.4-fold) was detected in HUVEC cells at 21 days (p=0.039) (Figure 5B).