Supplementary data from THE PRESENCE OF CX43 IN EXTRACELLULAR VESICLES REDUCES
THE CARDIOTOXICITY OF THE ANTI-TUMOR THERAPEUTIC APPROACH WITH DOXORUBICIN
Tania Martins-Marques, Maria Joao Pinho, Monica Zuzarte, Carla Oliveira, Paulo Pereira, Joost
Sluijter, Celia Gomes, Henrique Girao
Supplementary Materials and Methods
Characterization of Extracellular vesicles (EVs) by transmission electron microscopy (TEM)
EVs were fixed with 2% paraformaldehyde (PFA) and deposited on Formvar-carbon coated grids
(TAAB Laboratories). Samples were rinsed with PBS, fixed with 1% glutaraldehyde for 5 min, followed
by washing with distilled water. Grids were contrasted with uranyl-oxalate pH 7, for 5 min, and
transferred to methyl-cellulose-uranyl acetate, for 10 min on ice (8). Images were obtained in a Tecnai
G2 Spirit BioTWIN electron microscope (FEI) at 80 kV.
EV loading
5 µg EVs were incubated with 150 µM DMNPE-caged-D-luciferin for 1 h, at 37°C, protected from light.
Luciferin was released from the DMNPE group by UV-B (365 nm) photolysis (5 min, on ice) using an
UV transilluminator. Non-incorporated luciferin was removed using Exosome Spin Columns (Life
Technologies). Luciferin-loaded EVs were added to 4T1luc2 seeded in 96-well plates, or intratumorally
injected (i.t.) into mice, followed by BLI. 10 µg EVs were mixed with 100 µg dox (DOXO-cell®) in PBS
supplemented with 5 mM EDTA, and electroporated at 35 V and 150 µF, using 0.4 cm cuvettes, in a
Gene Pulser II Electroporator (BioRad) (11,22). Empty EVs were also electroporated. Nonincorporated dox was washed out with PBS, by ultracentrifugation (120,000g, 70 min). Loaded dox
was quantified by fluorescence detection in a Biotek Synergy HT microplate reader (Emission: 594
nm; Excitation: 480 nm) with Gen 5 software (Biotek). Fluorescence readings were compared with a
standard curve of free dox. On average, yield of dox encapsulation was 10%.
Determination of cell viability, proliferation, motility and colony formation in vitro
4T1luc2 cells were treated with 2 μM free dox or dox-loaded EVs for 24h. Vehicle-treated cells were
used as controls. To assess metabolic activity, the 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl
tetrazolium bromide (MTT, Sigma) assay was performed (18). For cell viability assessment, cells were
either trypsinized and stained with Trypan blue, before counting using an hemocytometer, or stained
with propidium iodide (PI) for 10 min and further analyzed by flow cytometry in a FACSCalibur cell
(Becton Dickinson). Assessment of cell proliferation by BrdU incorporation was performed according
to manufacturer’s instructions (Roche Diagnostics), following incubation in BrdU-containing medium
for 10h. Where indicated, cells were treated with 0.25 mg/ml gap26 (Caslo) for 1h. Staining of ki-67
positive cells was performed by immunofluorescence (1:100, AB16667, Abcam) (23). Images were
obtained using an Axio Observer.Z1 (Zeiss) and analyzed with Image J (National Institutes of Health).
For cell cycle analysis, cells were trypsinized, washed in PBS and fixed in 70% ice-cold ethanol for 30
min. Fixed cells were washed, and incubated with a PI/RNase staining solution (Immunostep) for 15
min. Data acquisition was immediately performed by flow cytometry and analyzed with ModFit LT
software (24). Cell motility was analyzed by time-lapse video microscopy. After a 6h pre-treatment,
as indicated, cell movement was monitored for an additional 6h, using an Axio Observer.Z1 (3 min
acquisition interval). Cells were maintained under an atmosphere of 5% CO2 at 37ºC. Manual cell
tracking analysis was performed with Image J (25). For colony formation assay, 3x103 cells were
seeded in 6-well plates and treated with 1 μM free dox or dox-loaded EVs, for 48h. Then, medium was
replaced by a drug-free medium and cells were allowed to grow for 7 days. At the end of the
experiment, cells were fixed in 1% PFA, followed by 15 min staining with 0.1% crystal violet (SigmaAldrich) (26). Microscopy images were obtained using an Axio Observer.Z1.
Western blot (WB)
Cell and tissue lysates were prepared in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 5mM EDTA, 1%
NP-40 and 0.1% SDS, pH 7.5) supplemented with protease/phosphatase inhibitors (protease inhibitor
cocktail (Roche), 2 mM phenylmethanesulfonyl fluoride (PMSF), 10 mM iodoacetamide and 2 mM
sodium ortovanadate). Tissue homogenization was performed in a tissue grinder (Polytron PT1200E,
Kinematica), followed by sonication, 30 min incubation on ice and centrifugation (1000g, 5 min and
10000g, 20 min). Total protein was determined [DC Protein Assay (BioRad)], samples were denatured
in Laemmli buffer and heated at 95ºC, for 5 min. EV pellets were lysed in a non-reducing loading
buffer. Protein samples were resolved on SDS-PAGE gels, transferred to nitrocellulose membranes,
probed with antibodies against CD63 (1:500, AB0047, Sicgen), CD81 (1:250, Sc9158, SantaCruz
Biotechnology), Cx43 (1:2500, AB0016, Sicgen), Calnexin (1:5000, AB0041, Sicgen), phosphorylated
and total p44/42 (1:500, 4377S and 9102, Cell Signaling Technology), Bax (1:250, Sc6236, SantaCruz
Biotechnology) and Bcl2 (1:250, Sc7382, SantaCruz Biotechnology), followed by incubation with HRPconjugated secondary antibodies (1:10000, BioRad). Images were visualized in a VersaDoc system
(BioRad). Densitometric quantification was performed in unsaturated images using ImageJ.
Supplementary Figures
Figure S1 – Cx43 mediates the communication between EVs and tumor cells in a dosedependent manner. A. Equal amounts of EVs containing or not Cx43 (EVCx43+ or EVCx43-) were loaded
with 150 µM D-luciferin and added to 4T1-luciferase expressing cells (4T1luc2). Lysed EVs were used
as controls. Bioluminescence images (BLI) were acquired in a IVIS Lumina XR system and the signal
intensity was calculated in a region of interest (ROI) drawn around each spot and expressed in
radiance (p/sec/cm2/sr). Graph depicts radiance levels in each condition (n=3, **p<0.01). B. 2.5 or 5
µg EVCx43+ or EVCx43- were loaded with 75 µM or 150 µM D-luciferin, and added to 4T1luc2 cells. Graph
depicts quantitative analysis of the BLI obtained 5 sec after the addition of EVs. 5 µg empty EVCx43+
or EVCx43- were used as background control.
Figure S2 - EVs loaded with dox decrease the metabolic activity and clonogenic potencial of
tumor cells in vitro. A. 10 µg EVCx43- or EVCx43+ were loaded with 100 µg dox in PBS supplemented
with 5 mM EDTA, by electroporation. Not incorporated dox was washed out with PBS by
ultracentrifugation. Values of EV-loaded dox (~10% EV pellet) were quantified by fluorescence
detection (Emission: 594 nm; Excitation: 480 nm) and are depicted on graph. Fluorescence reading
of similar amounts of free dox was used as control (n=10). B. 4T1luc2 cells were treated with either 1,
2 or 4 μM free doxorubicin (dox) or dox loaded into EVs containing or not Cx43 (EVCx43+dox or EVCx43dox) for 24 h. Equal amounts of EVCx43+ or EVCx43- were used for dox loading, and vehicle-treated cells
were used as controls. Metabolic viability was assessed by MTT reduction (n=2). C. Representative
images of colony formation assays performed in cells treated with 1 μM free or EV-loaded dox, as
indicated. Fresh media was added after 48 h and the cells cultured for an additional 7 days. At the
end of the experiment, cells were stained with crystal violet. Insets show microscopic photographs.
Scale bars, 200 μm (n=3).
Figure S3- Cx43 enhances the communication and transfer of cargo between EVs and tumor
cells in vivo. Tumor growth was induced by bilateral inoculation of 4T1luc2 cells into Swiss nude mice.
At day 4, BLI of tumors was performed by intraperitoneal (i.p.) injection of D-luciferin (150 mg/kg). At
day 5, BLI of tumors was performed after intratumoral (i.t.) injection of 150 µM D-luciferin-loaded
EVCx43+ or EVCx43- into opposed flanks of tumor-bearing mice. Graph represents radiance levels at day
5 normalized for the levels at day 4, and are expressed as fold increase over tumors stimulated with
EVCx43+ (n=3, *p<0.05).
Figure S4 – Intratumoral injection of empty EVs does not affect tumor growth in vivo. A.
Representative image of dox fluorescence in tumor-bearing mice, after i.t. injection of free dox (0.2
mg/kg) in the right posterior flank. Contralateral tumor was injected with equivalent volume of PBS.
TissueAF: auto-fluorescence. B. Representative BLI of tumors developed by Swiss nude mice 5, 8
and 11 days after bilateral subcutaneous 4T1luc2 cells inoculation. Equal amounts of Cx43-positive or
negative EVs (10 μg EVCx43+dox or EVCx43-dox, respectively) were injected (i.t) at days 5 and 8. Images
were obtained after intraperitoneal (i.p.) injection of D-luciferin (150 mg/kg).