Decidual NK cell-derived conditioned medium enhances capillary tube and network organization in an extravillous cytotrophoblast cell line

dNK-CM increased HTR8/SVneo proliferation (a; MTS assay) and migration (b; calcein AM-based Transwell plate migration assay) in a dose-dependent manner. For MTS assays, serum-starved HTR8/SVneo were seeded in serum-free DMEM/F12 in the absence or presence of dNK-CM, and incubated for 72 h, after which time MTS was added. The results were expressed as % of control = OD490 nm [assay group] × 100/OD490 nm [control medium group]. For the migration assay, HTR8/SVneo were loaded to Matrigel-coated Transwell inserts and incubated in either DMEM/F12/5% FBS or dNK-CM/5% FBS in the bottom overnight, followed by 1 h incubation with calcein AM. Migrated and calcein AM-labeled cells were then shaken off from the bottom of the plate by trypsinization. The plates with labeled cells were read and the results expressed as % of control = RFU [assay group] × 100/RFU [control medium group]. The assay was repeated for a total of 4 times. Dotted line and p value: Kruskal–Wallis non-parametric ANOVA. Dunn's multiple post-hoc test: *p < 0.05; **p < 0.01; ***p < 0.001, compared with control. Results normalized to mean control result (100%) for each experiment.

dNK-CM enhanced HTR8/SVneo, and primary CTB, capillary tube and network organization. Serum-starved HTR8/SVneo cells were added to pre-solidified Matrigel. Overnight incubation with either dNK-CM/5% FBS or DMEM/F12/5% FBS (control) revealed differences between control medium-treated and dNK-CM-treated groups in terms of capillary tube and network morphology (a) and total network tube length (b), using image analysis software. These concentration-dependent findings were confirmed using primary first-trimester CTB (c and d). Dotted line and p value: Kruskal–Wallis non-parametric ANOVA. Dunn's multiple post-hoc test: *p < 0.05; **p < 0.01; ***p < 0.001, compared with control (b); Mann–Whitney U test (d). Results normalized to mean control result (100%) for each experiment. Scale bar: 250 μm.

Vascular phenotype and adhesion molecule expressions on HTR8/SVneo cells, by flow cytometry with FITC-conjugated antibodies (upper panels) and by confirmatory immunohistochemistry staining on primary CTB cells (lower panels). Flow cytometry: red: isotype control; green: control medium-treated; blue: dNK-CM-treated. dNK-CM increased ICAM-1 expression by HTR8/SVneo and primary CTB cells compared with control serum Both control medium- and dNK-CM-treated HTR8/Svneo and primary CTB cells expressed VE-cadherin with no difference between groups. Other vascular and adhesion molecules were poorly expressed. Scale bar: 70 μm.

Anti-ICAM-1 antibody blocked dNK-CM-mediated enhancement of HTR8/SVneo migration (a) and capillary tube formation (b and c). Serum-starved HTR8/SVneo cells were loaded into top of the inserts and pre-incubated with anti-ICAM-1 antibody prior to filling bottom of the plate with control medium or dNK-CM. The remainder of the procedures was performed by regular migration assay. The HTR8/SVneo migration was increased by dNK-CM, and the enhanced effect was reduced by anti-ICAM-1 antibody. For the capillary tube formation on Matrigel, serum-starved HTR8/SVneo cells were pre-incubated with anti-ICAM-1 antibody before adding to Matrigel. dNK-CM-induced enhancement of capillary tube formation was partially reversed by anti-ICAM-1 antibody. Dotted line and p value: Kruskal–Wallis non-parametric ANOVA. Dunn's multiple post-hoc test: *p < 0.05; **p < 0.01; ***p < 0.001, compared with control; ^†p < 0.05; ^†††p < 0.001, compared with dNK-CM. Results normalized to mean control result (100%) for each experiment. Scale bar: 250 μm.

dNK-CM activated PI3K/AKT and p38 MAPK pathways in HTR8/SVneo. HTR8SVneo were plated on Matrigel and exposed to serum-free DMEM/F12 (control) and serum-free dNK-CM. After 24 h incubation, cells were harvested for Western Blot analysis. High levels of p44/p42 ERK were observed in both control medium- and dNK-CM-treated HTR8/SVneo. While pAKT and pp38 were low in the control group, higher levels of pAKT and pp38 were observed in HTR8/SVneo exposed to serum-free dNK-CM.

PI3K/AKT and p38 MAPK inhibitors (LY294002 and SB202190, respectively) reduced HTR8/SVneo ICAM-1 expression (a), migration (b), and capillary tube and network formation (c and d). HTR8/SVneo cells were pre-treated with either LY294002 or SB202190, and then processed for capillary tube and network formation on Matrigel. The capillary tube and network formation was enhanced by dNK-CM, and the enhanced effect was reversed by either LY294002 or SB202190 (c and d). HTR8/SVneo cells were harvested for FACS analysis to assess ICAM-1 expression. ICAM-1 expression was enhanced by dNK-CM, and the enhanced effect was reduced either by LY294002 or SB202190 (a). For migration assay, serum-starved HTR8/SVneo cells were loaded into top of the inserts and pre-treated with LY294002 or SB202190 prior to filling bottom of the plate with medium or dNK-CM. dNK-CM was able to increase HTR8/SVneo migration, this effect was inhibited by either LY294002 or SB202190. In (a), red: isotype control; green: control medium-treated; blue: dNK-CM-treated; brown: dNK-CM and inhibitor treated (LY or SB). In (b) and (d), the dotted line and p value: Kruskal–Wallis non-parametric ANOVA. Dunn's multiple post-hoc test: *p < 0.05, compared with control; ^††p < 0.01; ^†††p < 0.001, compared with dNK-CM. Results normalized to mean control result (100%) for each experiment. Scale bar: 250 μm.