Basement membrane extract

For cell migration and invasion assays, the cells were seeded out and grown overnight, followed by transfection with miRNA inhibitors. The transfected cells were grown for 48 hours, followed by serum-deprivation for 24 hours. 50,000 cells were then assayed in each well of a 96-well Boyden Chamber (R&D Systems, Minnesota, USA) for migration and invasion according to the manufacturer’s protocol. 20% foetal bovine serum was used as the chemoattractant. Basement Membrane Extract (R&D Systems, Minnesota, USA) was used as the matrix barrier for the invasion assay.

MCF-7 cells were seeded into the wells of a 24-well plate (2 × 10⁴ cells/well) coated with basement membrane extract (R&D systems) and allowed to adhere overnight. Cells were stimulated with EVs (10 µg/ml) for 8 h and every 10 min one picture was captured at two distinct positions per well using the BZ-X800 fluorescence microscope (Keyence). Single cells (10 – 20 per condition) were tracked using the Image J software (version 1.52p).

Cancer cell invasion was analyzed by a modified Boyden chamber assay [24 (link)]. Briefly, MCF-7 cells were seeded in triplicates onto a polycarbonate membrane (10 µm pore diameter, Pieper Filter) coated with basement membrane extract (R&D systems) in the upper wells of the chamber and were stimulated with EVs (1 µg/ml) for 96 h. The number of invasive cells in the lower wells of the chamber was counted and related to an unstimulated control. For inhibition of EV uptake, cells were pre-incubated with Dynasore (12.5 µM) for 2 h prior to EV addition, and cell invasion was quantified after 48 h due to the toxicity of the inhibitor upon longer incubation times.

All the cell culture medium and fetal bovine serum (FBS), Trypsin–EDTA (0.25%), antibiotic solutions (penicillin and streptomycin), and Dispase II were purchased from Gibco (USA). Hyaluronic acid (HA) and Collagen I (Col I) were purchased from Sigma-Aldrich (USA). Basement Membrane Extract (BME), an extract of the murine EHS transplantable tumor line that overproduces the matrix components present in fetal basement membranes, was purchased from R&D (USA). Immortalized human keratinocytes, the HaCaT cell line, and human umbilical vein endothelial cells, HUVECs, were purchased from the Chinese Academy of Medical Science & Peking Union Medical College (China). HaCaT and HUVEC cells were maintained in α-modified Eagle medium (α-MEM) and Dulbecco's Modified Eagele Medium (DMEM), separately, and are supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin.

The tubuloids, derived from human cortical kidney tissue, were obtained from Hubrecht Organoid Technology (HUB), Utrecht, the Netherlands (OSR-2020-30b), and were cultured according to Schutgens et al. [10 (link)] Briefly, the tubuloids were maintained at 37 °C and 5% v/v CO₂ in Basement Membrane Extract (BME) (R&D Systems, Abingdon, UK) and cultured in expansion medium (ADMEM/F12 supplemented with 1% penicillin/streptomycin, HEPES, GlutaMAX, N-acetylcysteine (1 mM; Sigma-Aldrich, Zwijndrecht, the Netherlands) and 1.5% B27 supplement (Gibco, Life Technologies, Paisley, UK), supplemented with 1% Rspo3-conditioned medium (U-Protein Express, Utrecht, The Netherlands), EGF (50 ng ml^–1; Peprotech, London, UK), FGF-10 (100 ng ml^–1, Peprotech, London, UK), Rho-kinase inhibitor Y-27632 (10 µM; Abmole, Brussels, Belgium) and A8301 (5 µM; Tocris Bioscience, Abingdon, UK)). For tubuloids differentiation, the medium was changed to ADMEM/F12 supplemented with 1% penicillin/streptomycin, HEPES and GlutaMAX, defined as differentiation medium and the tubuloids were maintained in culture for 7 days (Additional file 1).