Apotome 1

Immunofluorescence microscopy (IF) (Zeiss Axio Imager Z1 coupled with an Apotome 1 (Carl Zeiss Vision Swiss AG, Feldbach, Switzerland)) and the Philips CM12 transmission electron microscopy (TEM) (FEI, Eindhoven, The Netherlands) were used to assess the expression of BBB characteristics in the hBLECs on DIV7 of the co-culture period (Figure A4). The tight junction (TJ) proteins claudin 5 (Figure A4a) and junctional adhesion molecule A (JAM-A) (Figure A4b) were shown to be expressed continuously in cell-cell contacts. Furthermore, the adherens junction molecule vascular endothelial cadherin (VE-cadherin) (Figure A4c) as well as the TJ-associated scaffolding protein ZO-1 (Figure A4d) were also detected and continuously expressed. Formation of these cell junctions was further corroborated by TEM (Figure A4e). To functionally demonstrate the induction of barrier properties in the hBLEC monolayer, TEER was measured daily during the 7 days of co-culture. A significant daily increase was found reaching a plateau on DIV6 and DIV7 of the co-culture period, resulting in TEER values of around 60 Ω∙cm² (Figure A4f).

Macrophages were infected with GFP labelled P. aeruginosa as described above. After 2.5 hrs of gentamicin treatment, infected J774 cells were washed twice with PBS and incubated with 50 nM Lysotracker red DND-99 (Molecular Probes) in DMEM for 10 min to stain lysosomes. Cells were then washed with PBS and fixed with 4% paraformaldehyde in PBS and mounted on glass slides in Vectashield (Vector Laboratories, Inc) with 4′,6-diamidino-2-phenylindole (DAPI) to stain the nucleus. The slides were examined using an upright fluorescence microscope (Axioimager Z2, Zeiss) equipped with an Apotome 1 for optical sectioning. A 63X Apochromat Objective (NA 1.4) was used, transmitted light was acquired using differential interference contrast (DIC), Fluorescein isothiocyanate (FITC) filter was used to visualize GFP expressing bacteria and Lysotracker red fluorescence was acquired using a texas red filter set.

Organoids were fixed in 4% paraformaldehyde (20 min at room temperature) before embedding (first in a 2% agar solution, and then in paraffin). Organoid sections (5-µm width), as well as sections from matching primary BC tissues, were prepared and stained for ER (#13258, Cell Signaling Technology), PR (#8757, Cell Signaling Technology), HER2 (#2165, Cell Signaling Technology) and Ki67 (#MA5-14520, Thermo Fisher Scientific) with a VENTANA automated staining system (Roche) at CUSL. Image acquisition was performed using an AxioImager Z1 microscope equipped with an Apotome1 (Zeiss).