Figure 2.

LncSNHG5 in breast CAFs primes the premetastatic niche by inducing angiogenesis and vascular leakiness. (A) Tube formation (upper panel) and spheroid sprouting (bottom panel) of HUVECs were measured after treatment with CM from lncSNHG5-knockdown CAFs and control CAFs (scale bar, 100 µm). (B-C) CM from lncSNHG5-knockdown CAFs or control CAFs was applied to construct HUVEC monolayers, and the invasive GFP⁺ tumor cells across the HUVEC monolayers were assessed by transwell assay (B). The permeability of HUVEC monolayers was tested by rhodamine-dextran (70 kDa) staining (C). (D-E) HUVECs were cultured in CM derived from lncSNHG5-knockdown and control CAFs for 30 h, and the mRNA and protein expression levels of ZO-1 and Occludin in HUVECs were examined using qRT-PCR and WB. (F) MDA-MB-231 cells mixed with NFs/Ctrl, CAFs/shNC or CAFs/shlncSNHG5 were orthotopically injected into nude mice for 2 weeks. The mice were injected with rhodamine-dextran before lung metastasis, and the vascular permeability of mouse lungs is shown (scale bar, 100 µm). (G) The fluorescence levels of rhodamine-dextran in mouse lungs were calculated using ImageJ software and normalized to DAPI levels. (H) The microvessel density in mouse lungs was quantified by counting the microvessel numbers under a fluorescence microscope. (I) Representative images of fluorescent staining of ZO-1 (green), Occludin (red) and CD31 (pink) in lung tissue are shown (scale bar, 100 µm), and the vascular structures were labeled using CD31 (pink). (J) The protein levels of ZO-1 and Occludin in mouse lung tissues before metastasis were assessed by western blotting. The data represent the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).