Circulating tumor cells shed large extracellular vesicles in capillary-sized bifurcations

Circulating tumor cells (CTCs) and their clusters are the drivers of metastasis, but their interactions with capillary beds are poorly understood. Using microfluidic models mimicking human capillary bifurcations, we observed cell size- and bifurcation-dependent shedding of nuclei-free fragments by patient CTCs, CTC-derived explant cells and numerous cancer cell lines. Shedding reduced cell sizes up to 61%, facilitating their transit through bifurcations. We demonstrated that shed fragments were a novel class of large extracellular vesicles (LEVs), whose proteome was associated with immune-related and signaling pathways. LEVs were internalized by endothelial and immune cells, disrupted endothelial barrier integrity and polarized monocytes into M2 tumor-promoting macrophages. Cumulatively, these findings suggest that CTCs shed LEVs in capillary beds that drive key processes involved in the formation of pre-metastatic niches.


Scanning electron microscopy
Cells and their shed LEVs were collected as described above, and the solution was centrifuged at 180 x g for 5 min to separate cells (pellet) and retain LEVs in the supernatant (described below in more detail).LEVs were transferred onto 10 x 10 mm square glass slides that were positioned into wells of a 24 well plate.After 1 day, the media was removed and the glass slides were fixed with 4% formaldehyde (diluted in PBS) for 15 min at room temperature to crosslink proteins.Then, glass slides were chemically treated with 1% (v/v) Osmium tetroxide (Agar Scientific, UK) to crosslink lipids.The fixed samples were dehydrated with a sequence of washes with increasing concentrations of ethanol, starting from 30% and continuing with 40%, 50%, 60%, 70%, 80%, 90% and 100% (v/v) ethanol.After dehydration, the samples were chemically dried, by using Hexamethyldisilazane (Merck, UK) for 3 mins.A sticky carbon tape was attached to each square glass slide and silver-containing glue was brushed onto the samples to enhance conductivity.Finally, a 15 μm thick layer of Nickel was deposited onto the samples using a sample sputter coater (Q150TS, Quorum technologies, UK), to improve image resolution.Prepared samples were loaded into the stage holder of the scanning electron microscope (Zeiss Auriga 40 Cross beam scanning electron microscope, Zeiss, Germany).5 kV of acceleration voltage (using secondary electron detection) was applied to the sample.Samples were scanned to identify LEVs and images at 1000-8000x magnification.

Post transit cell proliferation
To evaluate the proliferative capabilities of MDA-MB231 cells after transit, 5 x 10 5 cells were flowed through a microfluidic device, the effluent collected, and cells separated from LEVs as described above.Cells were enumerated using a hemocytometer (as above).250 μl of cell suspension of 0.5x10 6 cells/ml density were added to wells of a 48-well plate.Equal numbers of cells that did not transit through devices were added to separate wells as controls.Images were taken 4 hr after seeding and then every day up to 7 days post-seeding.In other experiments, post-transit cells and control cells were left to grow for 3 days in 48 well plates and were then harvested and enumerated using a hemocytometer.

Flow cytometry
MDA-MB 231 cells, HUVECs, monocytes or purified LEVs (the latter derived from MDA-MB 231 cells as described above) were stained as described above (see also Supplementary Table 5).500 μl of each sample (10x10 5 cells/ml or 20x10 4 LEVs/ml) were loaded into an Amnis Cell Stream flow cytometer (Luminex, US) and samples were run at a flow rate of 20 μl/min.At least 1000 events per sample and condition were recorded.Excitation and emission wavelengths used for obtaining relevant dot plots are depicted in Supplementary table 6.For all histograms generated, antibody fluorescence intensity was represented in the x axis and frequency in the y axis.As a general approach, unstained samples and single-color controls were used for setting gating thresholds.
For LEVs internalization studies, pre-stained cells with a red cell tracker (HUVECs or monocytes or M1 macrophages) were co-cultured independently with pre-stained LEVs with green cell tracker for 16 hr.Internalization frequency was defined as the number of double positive events, cells that had internalized LEVs compared to overall number of cells.Where required, cells were pre-treated with Cyto-D, EIPA or combination, as described elsewhere and then LEVs were added.In each case, unstained cells, untreated cells (stained) and LEVs alone (stained), were used as controls for gating the limits for double positive dot plots (cells that had internalized LEVs) and samples were analyzed as above.For relevant experiments, fluorescent beads of 5-and 10 μm (microParticles GmbH, Germany) were used.

Protein concentration & gel electrophoresis
Initially, suspensions of each fraction were washed 1x with chilled (4 o C) PBS and lysed using 300 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fischer Scientific, UK) for 15 min on ice in a plate shaker.All fractions were centrifuged at 18900 x g for 15 min to remove debris and the supernatant containing the lysate was collected.All other previous centrifugation steps were at 180 x g for 5 min and at 9600 x g for 30 min, for cells and LMPs, respectively.The protein lysates were stored at -80 o C or placed on ice for immediate use.
Protein quantity of MDA-MB 231 control cells, cells post-transit (bifurcated capillary devices) and LEVs were measured using Bicinchoninic acid (BCA) assay kit (Life Technologies, UK), per manufacturer's instructions.The absorbance of each sample at 562 nm was analyzed using a Varioskan Flash plate reader (ThermoFischer Scientific, UK).
For qualitative protein analysis, gel electrophoresis was performed.The separating gel was prepared by mixing 1.6 ml PBS, 2 ml Acrylamide/Bis-acrylamide 30% (v/v) solution, 1.3 ml 1.5 M Tris (Life Technologies, UK), 50 μl 10% (v/v) Sodium Dodecyl Sulfate (Thermo Fischer Scientific), 50 μl 10% (v/v) Ammonium Persulfate (Bio Rad, UK) and 2 μl tetramethylethylenediamine (Bio Rad, UK).The stacking gel was prepared by mixing 1.4 ml PBS, 0.33 ml Acrylamide/Bis-acrylamide 30% (v/v) solution, 0.25 ml 1.5 M Tris, 20 μl 10% (v/v) Sodium Dodecyl Sulfate, 20 μl 10% (v/v) Ammonium Persulfate and 2 μl tetramethylethylenediamine.Both gels were left to polymerize at room temperature for 20 min.Both the separating and stacking gel were enclosed in a glass cassette (Bio Rad, UK) and incorporated in the electrophoresis chamber (Bio Rad, UK).250 μg/ml of protein samples (15 μl) are mixed with 5μl Laemmli protein buffer (Bio Rad, UK) and heated for 10 min at 95 o C. 12 μl of each sample and protein ladder solution (Bio Rad, UK) are added across the wells of the gel.The electrophoresis chamber was filled with 10% (v/v) Sodium Dodecyl Sulfate and the samples were run for 120 min at 125 V and 400 A. The gel was then removed carefully from the enclosed glass cassette and stained with 50 ml Coomassie blue (Bio Rad) in a glass sealed container for 1 hr at room temperature on a plate shaker (Thermo Fischer Scientific, UK).The staining solution was removed and the gel was destained using 50 ml of Coomassie brilliant blue R-250 destaining solution (Bio rad, UK) for 1 hr at room temperature on a plate shaker.
Images were obtained with a mobile phone (Redmi, China).

HUVEC permeability assays
15x10 3 HUVECs were cocultured in wells of a 96 well plate with 5x10 3 purified LEVs derived from MDA-MB 231 cells as described above.Equal numbers of HUVES but cultured without LEVs were used as negative controls.HUVECs were pre-stained with a CMFDA green cell tracker.At 0hr, both fluorescent and brightfield images were obtained at 10x and 20x magnification.After 30hr, similar images were obtained again to characterise the extent of HUVEC monolayer disruption.In other experiments, HUVECs were stained with VE-cad and Hoechst instead of CDFMA cell tracker as described above.Cell-free vs. cell-containing areas were extrapolated using ImageJ.
HUVEC monolayer disruption was further characterized by performing a dextran permeability assay.Millicell hanging cell culture inserts of either 0.4 μm or 8 μm pores (Merck, UK) were transferred in wells of a 24-well plate.The well was filled with 900 μl around the cell culture insert and 200 μl of HUVECs (75x10 3 cells/ml) were then transferred inside the cell culture inserts and left for a day to attach and form the monolayer.Then, HUVECs were treated with 5x10 3 purified LEVs derived from MDA-MB 231 cells (as described elsewhere) for 30 hr.Then, 100 μl of fresh media was added to 0.4 μm or 8 μm, containing either 10,000 molecular weight Dextran Alexa Fluor TM 647 (ThermoFischer Scientific, UK) or 10x10 4 cells/ml pre-stained MDA-MB 231 cells (green cell tracker), respectively, for 24 hr. 100 μl of media was collected from the bottom of the well at 24 hr and fluorescence was measured using a Varioskan Flash plate reader (ThermoFischer Scientific, UK) at 650/668 nm values of peak excitation/emission.The fluorescence values obtained at 24hr were normalized initially based on blank samples (cellfree media) and baseline subtracted using fluorescence at 0 hr.

Monocyte differentiation
CM was collected from untreated HUVECs or HUVECs cocultured with LEVs (derived from MDA-MB 231 cells as above), as described above.15x10 3 THP-1 monocytes were transferred into wells of a 96-well plate and 100% of 200 μl of CM from above conditions was added for 30 hr.Additional experimental conditions included the addition of either 5x10 3 LEVs or CM collected from LEVs on monocytes for 30 hr.Untreated monocytes were used as a negative control.After 30hr, media was removed and monocytes were stained via immunocytochemistry for CD206 and TNF-a, as described above.Multifluorescent and brightfield images were obtained at 20x magnification, using the confocal microscope.
Alternatively, 10x10 4 THP-1 monocytes were added in wells of a 24-well plate and all above experimental conditions were repeated.500 μl from each sample was collected and analyzed via flow cytometry as described above.The frequency of CD206+ events was calculated to estimate the differentiation percentage of monocytes to M2 macrophages.

Monocyte adhesion
15x10 3 monocytes were stained with a CMTPX red Cell Tracker and were then transferred in wells of a 96 well plate.Then, monocytes were treated independently with 5x10 3 purified LEVs (derived from MDA-MB 231 cells as above), with 100% CM collected from LEVs and 100% CM collected from MDA-MB 231 cells (as described above) for 30 hr.Untreated monocytes were used as a negative control.Fluorescent images of each well were obtained before and after media removal (removing non-adhered monocytes) and the percentage of remaining adhered cells was calculated.
Alternatively, 15x10 3 HUVECs were added in wells of a 96-well plate and left to grow for 1 day.In the meantime, 30x10 3 THP-1 monocytes were cultured in wells of a 48-well plate, left to grow for 1 day and then stained with a CMFDA green cell tracker.HUVECs or THP-1 monocytes were treated with 5x10 3 or 10x10 3 purified LEVs (derived from MDA-MB 231 cells as above) for 30hr, respectively.Negative control of both untreated HUVECs and untreated THP-1 monocytes were used.After 30hr, media was removed both from HUVECs and monocytes to remove non-internalized LEVs and four experimental conditions were performed: a) untreated THP-1 monocytes were added to untreated HUVECs monolayers, b) untreated THP-1 monocytes were added to LEV-pre-treated HUVECs monolayers, c) LEV-pretreated THP-1 monocytes were added to untreated HUVECs monolayers, and d) LEV-pretreated monocytes were added to LEV-pre-treated HUVECs monolayers.Using a Varioskan flash plate reader, the green fluorescence of monocytes at 0 hr was measured at 492/517 nm values of peak excitation/emission.After 4hrs of co-culture, the media was removed from each well and fresh media was added.Fluorescence of monocytes was measured by plate reader as described above.Cell-free media was used for normalization and the percentage of remaining fluorescence after media removal (removing non-adhered monocytes) was estimated.

Monocyte proliferation assay
12x10 3 monocytes were added in each well of a 96-well plate and left to grow for 1 day.Then, they were treated independently with 4x10 3 purified LEVs (derived from MDA-MB 231 cells as above) or 100% CM collected from LEVs (as described above) for altogether 48 hrs.Untreated monocytes were used as a negative control.To evaluate monocyte proliferation the MTT cell proliferation assay kit (Abcam, UK) was used, per manufacturer's instructions.The absorbance at 590 nm of each well was read using a Varioskan flash plate reader and normalized as described elsewhere.