Cell culture and in vitro models
4T1 mouse triple negative mammary carcinoma cells were kept in Roswell Park Memorial Institute (RPMI) 1640 medium (Pan Biotech, Aidenbach, Germany) supplemented with 5% foetal bovine serum (FBS, PAA Laboratories, Linz, Austria) and Glutamax (Thermo Fischer Scientific, Waltham, MA, USA). 4T1 cells were transfected with pcDNA3.1(+)/Luc2 = tdT plasmid using Lipofectamine 2000 (Thermo Fischer Scientific) and underwent single-cell cloning, after sorting of red fluorescent cells using a BD FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA, USA). The media of tdTomato-4T1 cells contained 500 μg/ml G418 (Thermo Fischer Scientific) for further selection and maintenance of red fluorescence. Emerald GFP-expressing EmGFP-4T1 cells were obtained by retroviral transfection and selected on blasticidin S (Sigma-Aldrich, St. Louis, MO, USA). All cell lines were regularly tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland). Only mycoplasma-negative cultures were used for experiments.
Venus-YFP-expressing primary mouse brain endothelial cells (MBECs) were isolated from 6- to 8-week-old FVB/Ant:TgCAG-yfp_sb #27 female mice (obtained from Institute of Experimental Medicine, Budapest, Hungary). After collection of the brains, the meninges were removed and cerebral cortices were cut into small pieces and digested in two steps with collagenase and collagenase/dispase. Microvessel fragments were collected after 10 min 1000 · g centrifugation on Percoll (Sigma-Aldrich) gradient, and plated onto fibronectin/collagen-coated dishes. Endothelial cells growing out of the microvessels were cultured in DMEM/F12 (Thermo Fisher Scientific), 10% plasma-derived serum (PDS, First Link, Birmingham, UK) and growth factors. In the first two days, 4 μg/ml puromycin (Sigma-Aldrich) was added to remove contaminating cells.
YFP-MBECs and tdTomato-4T1 cells were used for endothelial-tumour cell co-cultures. First, we cultured endothelial cells on the abluminal side of the filter inserts (Corning-Costar Transwell Clear, Corning, NY, USA, #3450) coated with collagen. Tumour cells were seeded on the luminal side in a number of 4.5 · 104/cm2 and co-cultured for 48 h.
Experimental animals and surgeries
All surgeries were carried out on 8-week old female BALB/c (The Jackson Laboratory) or FVB/Ant:TgCAG-yfp_sb #27 mice. Before every procedure, mice were anaesthetized via inhaled isoflurane 4% (v/v) in oxygen for induction and 1–2% (v/v) for maintenance, from a precision vaporizer (Open Circuit Isoflurane Tabletop System, Stoelting, Dublin, Ireland). Depth of anaesthesia was monitored by toe pinch tests.
For all intravital experiments, cranial windows were used to obtain optical access to the cortex. Briefly, anaesthetized animals were mounted on a stereotaxic frame incorporating a heating pad. Craniotomy (d = 3.5 mm) was performed over the right parietal cortex with a micro drill (H.MH-170, High Speed Rotary Handpiece, Foredom, Blackstone Industries, Bethel, CT, USA) fitted with a 0.5 mm burr, followed by the removal of the dura. In some experiments, astrocytes were labelled by topical application of 10 μM SR101 (Sigma-Aldrich) in Ringer-HEPES solution for 2–3 min before the window installation. A coverslip of 5 mm diameter was then placed over the exposed brain and the edge of the glass was sealed with cyanoacrylate glue. An aluminium plate was glued onto the skull for head fixation. The exposed bone and the aluminium bar were covered with cyanoacrylate glue and dental cement (Unifast III, GC Europe, Leuven, Belgium) to increase stability. A recovery period of at least one month was allowed between implantation of the cranial window and intravital microscopy observation of endothelial-tumour cell interactions. Astrocyte-tumour cell interactions were investigated in a time frame of 30 min to 2 h after cranial window installation due to the temporary astrocyte staining. After recovery, either 106 tdTomato-4T1 cells were inoculated into the right common carotid artery or 3 · 106 tdTomato-4T1 cells were injected intracardially into FVB/Ant:TgCAG-yfp_sb #27 female mice with chronic cranial window for two-photon microscopy, or without craniotomy for ex vivo investigations. BALB/c female mice were intracardially injected with 3 · 106 EmGFP-4T1, then 1 day, 5 days or 10 days later cranial window was installed right before two-photon microscopy imaging.
For ex vivo observations, surgically untouched FVB/Ant:TgCAG-yfp_sb #27 mice received 3 · 106 tdTomato-4T1-cells intracardially. Certain animals were subjected to in vivo proliferation assay and treated intraperitoneally with 5-ethynyl-2′-deoxyuridine (EdU, Thermo Fisher Scientific, 100 mg/kg), a thymidine analogue, 24 h before tissue collection. After 1, 3, 5, 8 or 10 days, animals were anaesthetized and transcardially perfused with phosphate buffered saline (PBS, 10 mM, pH = 7.4), then with Karnovsky’s fixative (for electron microscopy) or 3% paraformaldehyde (for immunofluorescence) in PBS. Brains were removed and post-fixed by immersion in the same fixative at 4 °C overnight. The following day, the fixative was replaced with PBS (for vibratome section) or 30% sucrose solution in 0.1 M phosphate buffer (PB, for frozen sections), and the brains were stored at 4 °C until further processing.
Intravital two-photon imaging
Mice were anaesthetized with isoflurane and kept on a heating system-incorporated stereotaxic stage. The head was immobilized and positioned via the attached aluminium bar. This stable positioning and the unique pattern of the superficial pial vasculature allowed us to image the same cortex volume over days. Intravital microscopy was carried out with a FEMTO2DAlba microscope (Femtonics, Budapest, Hungary) using a 20x or 60x large working distance water immersion objective using MES software (v4.6.2336, Femtonics). Two-photon excitation was performed using a Mai Tai HP Ti-sapphire laser (Spectra-Physics, San Jose, CA, USA) at 810 nm, which was found optimal for EmGFP and CellTracker Red CMTPX excitation and also adequate for SR101 and at 900 nm, which optimally excited both tdTomato and Venus-YFP. Laser power was set to 10–40% depending on the depth of imaging (0–400 μm from the brain surface). Emission wavelengths were collected by GaAsP photomultipliers. Larger volumes (x: 500 μm; y: 500 μm; z: 250 μm) were recorded with 3 μm vertical steps to evaluate the cell number changes and dynamics of tumour cell intravascular location in the first 48 h following inoculation. To acquire sufficient red fluorescence signal, tdTomato-4T1 cells were also stained with CellTracker Red CMTPX (Thermo Fisher Scientific) for this experimental setup. ImageJ’s “3D Object Counter” plugin was used to assess the changes [1]. High magnification z-stack images (x: 120 μm; y: 120 μm; z: 120 μm, with 1 μm steps) were recorded for studying cell morphology changes and transmigration and no additional labelling of 4T1-tdTomato was applied. Image stacks were auto-levelled, merged and converted to RGB colour in Fiji [28].
Immunofluorescence and fluorescence microscopy
After fixation, the whole brain was mounted for freezing microtome (Reichert-Jung, Leica Biosystems, Wetzlar, Germany) or vibratome (Leica Biosystems) sectioning and sliced coronally. 50 μm brain sections were collected and stored in PBS with 0.05% sodium azide. Antigen retrieval was either omitted or performed by incubating slices at 85 °C for 60 min in PBS. Permeabilization was performed with 0.5% TritonX-100 in PBS for 30 min at room temperature, followed by blocking with 3% BSA (bovine serum albumin) in PBS. Primary antibody solutions were prepared in 3% BSA and 0.5% TritonX-100-containing PBS. Sections were incubated overnight under slow nutation. The following antibodies were used on vibratome sections: anti-AQP4 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-20,812), anti-claudin-5 (1:100, Thermo Fisher Scientific, #35–2500), anti-cleaved caspase-3 (1:50, Cell Signaling, Boston, MA, USA, #9661,), anti-collagen IV (1:100, Abcam, Cambridge, UK, #ab6586) and anti-PECAM-1 (1:120, Novus Biologicals, Centennial, CO, USA, #NB100–2284); on frozen sections: anti-fibronectin (1:100, Abcam, #ab2413), anti-GFAP (1:100, Abcam, #ab7260) and anti-Iba-1 (1:100, Abcam, #ab5076). Sections were extensively washed in PBS, and the secondary antibody solution was afterward applied on them for 60 min at room temperature in the dark. Alexa Fluor 488, 594, 647 anti-rabbit, anti-mouse and anti-goat IgG (Jackson ImmunoResearch, Cambridgeshire, UK and Thermo Fisher Scientific) and STAR RED anti-mouse IgG (Abberior, Göttingen, Germany) were used as secondary antibodies in a dilution of 1:300–1:600 in 3% BSA-containing PBS. Sections were then washed with PBS, counterstained with a colour compatible nuclear staining (Hoechst 33342, Sigma-Aldrich) for 5 min, washed again with PBS, rinsed in water and mounted with an aqueous fluorescent mounting solution, FluoroMount-G media (SouthernBiotech, Birmingham, AL, USA). Visualization of nuclei with DNA synthesis was performed on sections from EdU-treated animals using Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (Thermo Fischer Scientific) following the manufacturer’s instructions. ImageJ’s “3D Object Counter” plugin was used for manual assessment of EdU-positive tumour cells.
Immunohistochemistry and immunofluorescence were visualized with Leica SP5 and Leica SP8 confocal laser scanning microscopes with 63x and 100x oil immersion objectives or a STED (stimulated emission depletion) super-resolution-capable STEDYCON (Abberior Instruments, Göttingen, Germany) built on an Axio Observer Z1 inverted epifluorescence microscope (Zeiss, Oberkochen, Germany) equipped with an alpha Plan-Apochromat 100x/1.46 oil immersion objective.
Preparation of ultrathin sections and transmission electron microscopy (TEM)
TdTomato-4T1-bearing FVB/Ant:TgCAG-yfp_sb #27 mouse brains were sectioned using a VT1000S (Leica Biosystems) vibratome. 100 μm sections were collected and post-fixed for 5 h in Karnovsky’s fixative at room temperature. After post-fixation, sections were cut into 1–2 mm2 pieces. Specimens containing tdTomato-4T1 cells were selected for further processing. Samples were rinsed and post-fixed in 2% OsO4. After dehydration with a graded series of ethanol, the samples were embedded in epoxy resin (Durcupan ACM, Sigma-Aldrich) and polymerized at 55 °C for 48 h. Ultrathin sections (50 nm) were prepared with an Ultracut UCT (Leica Biosystems) and contrasted with 2% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% lead citrate (Electron Microscopy Sciences), then analysed with a JEM-1400Flash transmission electron microscope (JEOL, Tokyo, Japan) fitted with an 8 MP Matataki Flash CCD camera (JEOL).
Western-blot
4T1-tdTomato cells and YFP-Venus MBECs were co-cultured for 48 h on the two sides of the filter inserts and lysed separately in radioimmunoprecipitation assay buffer. After 30 min incubation on ice, cell lysates were centrifuged at 13,000 · g for 15 min at 4 °C. Protein concentration was determined with bicinchoninic acid (BCA) (Santa Cruz Biotechnology). Laemmli buffer was added to the samples, followed by heating at 95 °C for 3 min. Prepared samples were electrophoresed using standard denaturing SDS-PAGE procedures and blotted on polyvinylidene difluoride (0.2 μm pore size from Bio-Rad, Hercules, CA, USA and the 0.45 μm pore size from BioTrace, Pall Corporations, Port Washington, NY, USA) membranes (β-actin and fibronectin, respectively). Afterwards, the non-specific binding capacity of the membranes was blocked with 3% BSA or 5% non-fat milk in TBS-T (Tris-buffered saline with 0.1% Tween-20). Membranes were incubated with primary antibodies diluted in TBS-T: anti-β-actin (1:1000, Santa Cruz Biotechnology, #sc-47,778) or fibronectin (1:1000, Abcam, #ab2413). Blots were washed in TBS-T three times for 10 min. Horseradish peroxidase (HRP)-conjugated secondary antibodies were diluted in TBS-T as follows: 1:3000 anti-rabbit IgG and anti-mouse IgG (Jackson ImmunoResearch) and added for 1 h and then washed again in TBS-T. Immunoreaction was visualized with Clarity Chemiluminescent Substrate (Bio-Rad, Hercules, CA, USA) in a ChemiDoc MP System (Bio-Rad). Densitometry analysis was performed with the Image Lab Software, version 5.2 (Bio-Rad).