Response of the neurovascular unit to brain metastatic breast cancer cells

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.

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.

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.

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].

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.

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).

As a unique feature of brain metastasis formation [40], tumour cells spend several days in cerebral capillaries before proceeding to diapedesis through the vessel wall [19]. During this phase [15], metastatic cells may change their initial resting position in the vascular lumen. By monitoring the same brain regions in living mice using intravital two-photon microscopy, we explored the number of tumour cells/cell clusters disappearing, changing position and remaining in resting phase in the first 48 h after inoculation of triple negative, tdTomato-expressing 4T1 breast cancer cells. The relatively large volume scanned with the consequently lower magnification, and the lack of nuclear staining did not allow for clear detection of morphology changes and determination of the number of tumour cells in an intravascular cell cluster. However, we could still assess that metastatic cells gained an elongated morphology already within 1 h after the injection of tumour cells into the arterial circulation (Fig. 1a). Some cells remained in the same position for two days, while others changed their initial location and disappeared from or appeared in the scanned volumes (Fig. 1a and b). The number of initially arrested tumour cells/cell clusters (imaged at 1 h after tumour cell inoculation) decreased to 74.21 ± 21.95% at 5 h, to 38.21 ± 31.60% at 24 h and to 21.64 ± 5.59% at 48 h (Fig. 1b), as detected by monitoring the same three regions in the parietal cortex of three mice each by two-photon microscopy. We next wanted to assess the time of final arrest of the tumour cells, i.e. when they reached their resting position, from where they did not move inside the capillary lumen anymore. We found that 33.09 ± 26.97% of the cells adhered in the first hour, 31.33 ± 30.23% arrested between 1 h and 5 h and the remaining 35.58 ± 28.18% of the cells attached to the luminal side of the endothelium between 5 h and 24 h, and remained in the same location by 48 h post-inoculation (Fig. 1b). The surviving cells did not change their position after 24 h. These numbers indicate that the firm cell arrest happens in the first 24 h and, most probably, the majority of the cells find their long-term intravascular location in the initial hours.

Tumour cells arrested preferentially in vascular branching points (Fig. 1c) and left the junctions intact in the first 4 days, as assessed by PECAM-1 (platelet and endothelial cell adhesion molecule) (Additional file 1: Figure S1) and claudin-5 immunostainig (Fig. 1d) and TEM (Additional file 1: Figure S2). Breast cancer cells have been previously found to arrest exclusively in microvessels (capillaries and post-capillary venules) of the brain [19]. Outside the brain parenchyma, we could find breast cancer cells stuck in subarachnoid large vessels; however, these cells never elongated and disappeared after a few days without extravasation (Fig. 1e).

After 24–48 h spent in the lumen of parenchymal vessels, mammary carcinoma cells induced vasoconstriction and formation of endothelial plugs, which protruded into the lumen both upstream and downstream of the cancer cells (Fig. 2a). High-resolution confocal microscopy images indicated both partial and complete obstructions of the vascular lumen in the neighbourhood of the tumour cells (Fig. 2b). We could clearly see complete vessel obstruction by the tumour cell-induced plug in living animals as well, in a series of two-photon microscopy optical cross-sections (Fig. 2c). When tumour cells arrested at vascular branching points, vessels in all directions were obstructed by plugs and constrictions (Figs. 1d and 2d).

By examining 6–9 cancer cells/animal in the brains of 10 tumour-inoculated mice using confocal microscopy, we assessed the prevalence of vascular obstructions in the neighbourhood of the cancer cells on day 4 after inoculation. Majority of the tumour cell-containing microvessels were completely (61.03%) or partially (23.90%) closed by endothelial plugs. In addition, 9.01% of them were obstructed by vasoconstriction and only 6.06% remained open. These numbers indicate that it is a general phenomenon in brain capillaries that tumour cells are isolated by endothelial cells from the circulation.

Plugs of protruded endothelial bodies contained the nuclei of the endothelial cells (Fig. 2e). We hypothesized that these plugs were formed by endothelial division; however, endothelial nuclei in the plugs were EdU negative (Fig. 2f). As a positive control, we detected EdU-positive endothelial cells not involved in plug formation and not coming in contact with carcinoma cells (Additional file 1: Figure S3). Therefore, tumour cell-associated plugs were formed as the result of endothelial reorganization and not cell division. In addition, overexpression of abnormally localized claudin-5 and PECAM-1 was observed in plug-forming endothelial cells (Additional file 1: Figure S4 and S5), suggesting junctional reorganization and possibly compromised endothelial polarity. Interestingly, a large part of the intraluminal tumour cells was EdU positive (Fig. 2f) at each time point assessed: 62.50% at 24 h, 51.85% at 3 days and 40.54% at 5 days after tumour cell injection. Despite the high prevalence of EdU-positive intraluminal tumour cells, no increase in tumour cell number was observed from 24 h to days 3 or 5. Supposedly, some of the intravascular tumour cells became polyploid/multinucleated, which could help their survival and metastasis formation [22]. These large cells formed in the capillary lumen after day 3, dilated the vessel, as shown in Fig. 1d.

In the vicinity of the plugs and intravascular tumour cells, accumulation of matrix proteins was observed in the lumen, like type 4 collagen (Fig. 2g) and fibronectin (Fig. 2h). Besides the possible contribution of circulating matrix proteins [38], fibronectin was also overexpressed in the tumour cells (Fig. 2i and j). Tumour cells expressed even more fibronectin after completing the diapedesis (Additional file 1: Figure S6).

After 3–8 days spent attached to the luminal surface of the cerebral endothelium, typically on day 4 or 5, metastatic cells contracted and migrated through the vessel wall into the brain parenchyma. Interestingly, we observed a few transmigrating tumour cells that were proliferating/EdU positive (Additional file 1: Figure S7).

In contrast to the long time spent intravascularly, the diapedesis itself was relatively rapid, completed in a few hours and was accompanied by resolution of the obstructions (Fig. 3a). Our two-photon and confocal microscopy investigations revealed two types of transvascular migration. Metastatic breast cancer cells either diapedesed through small pores (Fig. 3a, b and c) or large discontinuities opened on the vessel wall (of 10 μm diameter in average) (Figs. 2d and 3d). By observing 14 transmigrations in the brains of living animals using two-photon microscopy, we counted 10 diapedesis events which occurred through pores < 5 μm (71.43%) and 4 through large fenestrations (28.57%).

Using in vitro models, we have previously shown that breast cancer cells are able to migrate transcellularly, leaving the TJs intact [10]. Here we explored this possibility in our mouse model, and found that continuity of TJs, represented by claudin-5 staining, remained intact in the vicinity of transmigrating cells (Fig. 3e). This suggests that breast cancer cells are able to take the transcellular route of transmigration through the BBB in vivo.

Another phenomenon that we observed was that cancer cells induced formation of additional vascular channels (Fig. 3f-h). Multiluminal vessels containing several channels or chambers (ranging from a few up to 15) were observed in 5 of 14 (35.71%) transmigrations tracked in two-photon microscopy. Smaller transmigration pores were formed on the thinned wall of one of the tubes of the multichannelled endothelium. Besides splitting of the capillary, rarely, we could see the growth of a collateral bypassing the tumour cell-bearing vessel (Additional file 1: Figure S8).

Migration of the tumour cells through the cerebral endothelium was frequently accompanied by blebbing of metastatic and/or endothelial cells (Fig. 4a-h). Intensive blebbing of intravascular tumour cells (Fig. 4a-c) was frequent, observed in 11 of 14 transmigrations (78.57%) imaged in two-photon microscopy. During extravasation, cancer cells usually protruded their blebbing membrane part through the transmigration pore (Fig. 4a and b), while the nucleus was left behind in the lumen and got through the vessel wall in the terminal stage of the diapedesis. Blebbing tumour cells often released extracellular vesicles (Fig. 4c), resembling large oncosomes [21].

In the neighbourhood of extravasating cancer cells, endothelial blebbing (Figs. 2g, 3e and 4d-h) was similar in frequency to tumour cell blebbing, present in 11 of 14 events observed by two-photon microscopy (78.57%) and in 10 of the 11 cases of tumour blebbing (90.91% overlap). Ranging from a few extraluminal blebs (Figs. 2g, 3e, 4e and f) to moderate or intensive vacuolization (Fig. 4d, g and h), blebbing of CECs was always linked to the presence of the tumour cells. Endothelial blebs were claudin-5 positive, while the endothelium still showed a continuous claudin-5 staining marking the intact TJs (Fig. 4f). Even severe endothelial blebbing could be reversed and the vessels were restored in 1–2 days after the tumour cells completed transendothelial migration (Fig. 4g).

Besides the non-apoptotic blebbing of the cerebral endothelium described above, which is most probably an important element of the active involvement of CECs in metastatic extravasation, signs of apoptotic blebbing and endothelial cell death could also be detected. On the one hand, we observed degenerated mitochondria in blebbing endothelial cells (Fig. 4h). On the other hand, we saw cleaved caspase-3-positive endothelial cells in the neighbourhood of carcinoma cells (Fig. 4i and j). However, this was a relatively rare event, observed in 5 of 21 cases in day 4. Endothelial active caspase-3 staining usually appeared in vesicle-like structures, showing the typical morphology of cell death. We assume that endothelial cell death might provide passage for the tumour cells through the formation of large openings on the vessel wall. The majority of tumour cells were usually cleaved caspase-3 negative (Fig. 4i); however, in 5 of 21 cases, we detected apoptotic tumour cells as well in day 4 (Fig. 4j).

As a next step, interactions of tumour cells with astrocytes and especially astrocyte end-feet were investigated during and after the extravasation. We observed that the extravasated part of breast cancer cells passed the glia limitans perivascularis right after breaching the endothelial wall, as our in vivo (Fig. 5a) and ex vivo (Fig. 5b-d) observations indicate. During extravasation, carcinoma cells opened the glia limitans formed by an almost continuous layer of astrocyte end-feet covering the extraluminal surface of the vessels (Fig. 5a). Tumour cells reaching an extraluminal position remained attached to the vessel wall, incorporating astrocyte end-feet, as shown in TEM (Fig. 5b) and confocal microscopy images (Fig. 5c and d). Both the astrocyte-specific glial fibrillary acidic protein (GFAP) and the end-foot marker aquaporin-4 (AQP4) were localized between the partly or completely extravasated tumour cell and the capillary endothelium.

After migrating through the capillary wall, tumour cells started to divide by co-opting one or two capillaries, which developed an abnormal, disorganized, bent morphology (Additional file 1: Figure S9). Tumour cells proliferated rapidly; however, endothelial proliferation was almost never observed (Additional file 1: Figure S10), suggesting that blood supply of the tumours was primarily provided by vascular co-option and not neo-angiogenesis. As metastatic lesions were growing, astrocytes were expelled from the tumour mass (Fig. 5e and Additional file 1: Figure S11). In parallel, reactive astrocytes surrounding metastatic lesions gradually retracted their end-feet from the vessels to the parenchymal border of the tumour, which gained a discontinuous end-foot coverage (Fig. 5f). It should be noted that the tumour-covered endothelium still showed a continuous claudin-5 staining, indicating the presence of complete TJs in the absence of the perivascular astrocyte sheath (Fig. 5g). We have also observed activated microglia [30] surrounding transmigrating and extravasated, but not intravascular cancer cells (Additional file 1: Figure S12).