Ovarian cancer cells lines SKOV3 (HTB-77), OVCAR3 (HTB-161), breast cancer cell lines MDA-MB231 and MCF7 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM high glucose (Hyclone, Thermo Scientific) supplemented with 20% FBS (Hyclone, Thermo Scientific), 1% Penicillin-Streptomycin-Amphotericyn B solution (Sigma), 2 mM L-glutamine (Sigma), 1X Non-Essential Amino-Acid (Hyclone, Thermo Scientific).

The E4ORF1⁺ ECs (E4⁺ECs) were generously provided by Shahin Rafii 32 . The cells were grown in M199 (Gibco, Life Technologies) supplemented with 20% FBS, 1% Penicillin-Streptomycin-Amphotericyn B solution (Sigma), 4 mM L-glutamine, 50 μg/ml heparin (Sigma) and 30 μg/ml β EC growth factor (βECGF, Upstate Biotechnology, Lake Placid, NY, USA). These cells express green fluorescence protein (GFP).

Mesenchymal stem cells (MSCs) were purchased from Stem Cells, Inc (MSC-001 F, Vancouver, CA) maintained and expanded in culture using MesenCult® MSC Basal Medium completed with Mesenchymal Stem Cell Stimulatory Supplements (Stem Cell Inc, Vancouver, CA). Their ability to differentiate in adipocytes, osteoblasts and chnodrocytes was verified as instructed by the supplier (data not shown). All cultured cells were incubated at 37°C under a water-saturated 95% air-5% CO₂ atmosphere. Shaking cultures were performed similarly using a rocking plates at low speed.

Cells were dissociated into single cell suspension by trypsinization and further sieving through 40-um cell strainers and subsequently resuspended in 3D media containing DMEM F-12 supplemented with 2% B27 (Invitrogen), 20 ng/mL VEGF (Peprotech), 20 ng/mL bFGF (Peprotech), and 5 ug/mL insulin (Sigma). The cells were plated at 5000 cells per well of ultralow attachment 24-well plates (Costar, Corning) and were grown in a humidified incubator at 37°C and 5% CO₂. The media was changed every second day. Primary spheres started to form at day 3 and continued to be cultured for up to 5 days.

Tumor material from patients presenting advanced ovarian carcinoma Stage IIIC were included in this study (IRB Number: #9161/2010, “Isolation and characterization of cancer stem cells”). During debulking surgery metastatic nodules were removed and processed as follows. Upon serial washing with PBS and red blood cell Lysis buffer (eBiosciences, San Diego, USA) nodules were minced and cultured with or without feeders (EC⁺E4) in DMEM high glucose (Hyclone, Thermo Scientific) supplemented with 20% FBS (Hyclone, Thermo Scientific), 1% Penicillin-Streptomycin-Amphotericyn B solution (Sigma), 2 mM L-glutamine (Sigma), 1X Non-Essential Amino-Acid (Hyclone, Thermo Scientific).

To trace intercellular exchange of mitochondria, cells were separately labeled with MitoTracker DeepRed and Green (Invitrogen) according to the manufacturer’s protocol 26 . Briefly, cells were resuspended in prewarmed (37°C) staining solution containing the MitoTracker® probe (50 nM) for 30 minutes under growth conditions appropriate. After staining, cells were washed 3 times in PBS and resuspended in fresh prewarmed medium. Cells were plated in mono or co-culture according to the experiment.

Live-cell TnTs imaging using previously published protocols was performed 20 . Cells were labeled with 1 mg/ml Alexa Fluor® 594 conjugated wheat germ agglutinin (WGA, Invitrogen SARL, Cergy Pontoise, France) at 5 μg/ml for 10 minutes at 37°C in the dark. WGA is a probe for detecting glycoconjugates, which selectively bind to N-acetylglucosamine and N-acetylneuraminic acid residues of cell membranes. Imaging was performed using a Zeiss confocal Laser Scanning Microscope 710 (Carl Zeiss). Post-acquisition image analysis was performed with Zeiss LSM Image Browser Version 4.2.0.121 (Carl Zeiss).

MPs from eGFP-E4⁺ECs were isolated as previously described 16 . Briefly, 48-h-supernatants of 80% serum-starved confluent tumor or endothelial cells were collected and sequentially centrifuged (4°C) at 300 g for 10 min, 800 g for 10 min, and at 3000 g for 15 min. MPs were pelleted at 100,000 g for 1 h, and washed once in PBS and centrifuged again at 100,000 g for 1 h. The final pellet containing purified MP was labeled with MitoTracker DeepRed to assess mitochondria transfer by microparticles.

After co-culture, cells were trypsinized, resuspended and washed with PBS. Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences). Data were processed with FACSDiva 6.3 software (BD Biosciences). Doublets were excluded by FSC-W x FSC-H and SSC-W x SSC-H analysis; eGFP fluorescence was acquired with 488 nm blue laser and 510/50 nm emission, Mitotracker Deep Red was acquired with 640 nm red laser and 670/14 nm emission. Figures display the median of fluorescence intensity (mfi) relative to control. Single stained channels were used for compensation and fluorophore minus one (FMO) controls were used for gating. 20,000 events were acquired per sample.

Viability of cells was examined with an MTT assay. 24 hours after treatment with doxorubicin, 10% of MTT reagent was added to each well to a final concentration of 500 μg/ml, and the cells were incubated for 4 hours at 37°C. 100 μl of DMSO were added to each well. Optical density was read at 570 nm versus 630 with an EnVision multilabel reader (PerkinElmer, Massachusetts, USA). 3 triplicates were performed per condition.

All quantitative data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using SigmaPlot 11 (Systat Software Inc., Chicago, IL). A Shapiro-Wilk normality test, with a p = 0.05 rejection value, was used to test normal distribution of data prior to further analysis. All pairwise multiple comparisons were performed by one way ANOVA followed by Holm-Sidak posthoc tests for data with normal distribution or by Kruskal-Wallis analysis of variance on ranks followed by Tukey posthoc tests, in case of failed normality test. Paired comparisons were performed by Student’s t-tests or by Mann–Whitney rank sum tests in case of unequal variance or failed normality test. Statistical significance was accepted for p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). All experiments were repeated at least three times.

Previous studies reporting the occurrence of TnTs in vitro mainly focus on a specific cell types. Here we determined the presence of TnTs between various cancer and stromal cells under the same culture conditions. Four cancer cell lines were assessed (MCF7 and MDA derived from breast cancers, and SKOV3 and OVCAR3 derived from ovarian cancers). Homo-cellular TnTs were observed in all cancer cell lines studied (Additional file 1: Figure S1A). Similarly TnTs were also found in non-malignant E4⁺ECs and MSCs (Additional file 1: Figure S1B-C). To study the role of TnTs in interactions between stromal and cancer cells, M-Orange-MSCs and eGFP-E4⁺ECs were co-cultured with different cancer cells. TnTs were found connecting cancer cells and M-Orange-MSCs or eGFP-E4⁺EC (Figure 1A-B).

All observed structures displayed the specific characteristics of TnTs: (i) they had no contact to the substratum and hover in the extracellular medium (Additional file 1: Figure S2A to C, Additional file 2: Movie 1) (ii) they exhibited a typical width of less than 0.5 μm and a length over several cell diameters (Additional file 1: Figure S2D-E), (iii) they contained F-actin (Additional file 1: Figure S2F).

We then studied explants from ovarian cancer metastatic nodules to test whether TNTs could occur in a tumor context. We cultured these in a 3D setting and were able to visualize TnTs-like structures at the periphery of the explants (Figure 1C and Additional file 1: Figure S3A). Recently there has been an emphasis on other cell culture conditions such as spheroid formation assay. These are performed in low-adherent culture plates and consist in the formation of floating cellular aggregates that evolving in cell spheroids. We therefore assessed the occurrence of TnTs in a 3D anchorage independent spheroid co-culture system. Confocal microscopy imaging of three days-old mixed spheres (stromal-cancer cells) displayed TnTs between: (i) cancer cells, (ii) cancer and stromal cells (Figure 1D and E and Additional file 1: Figure S3B and C).

Several models have been proposed for the formation of TnTs, mainly describing the connection between projections of two cell membranes 30 33 . We observed, in our culture conditions, such projections of TnTs between two cells (Figure 2A and Additional file 3: Movie 2). However, using time-lapse studies, we mainly observed the following succession of events: cell migration, large intercellular adhesion, cell separation, occurrence and/or persistence of TnTs, disruption of TnTs as the cells separated further (Figure 2B-D and Additional file 3: Movies 2, Additional file 4: Movie 3, Additional file 5: Movie 4 and Additional file 6: Movie 5). This emphasizes the role of large membrane adhesions before the formation of TnTs.

We used co-culture experiments to investigate a potential exchange of cytosolic content through TnTs. In many instances, we observed TnT-mediated transfer of GFP or M-Orange between cancer and stromal cells. Then, our time-lapse studies confirmed specific movement of dyes along a nanotube suggesting cytoplasmic components transfer from stromal to cancer cells (Figure 3A-B, Additional file 7: Movie 6 and Additional file 8: Movie 7). During phase contrast microscopy analysis, punctual densities were also observed, potentially corresponding to subcellular organelles (Figure 3C-D, Additional file 3: Movie 2). Nanotube mediates transfer of mitochondria between endothelial and endothelial progenitor have been previously described 34 . Therefore, we subsequently investigated the transfer of eGFP-E4⁺ECs mitochondria to cancer cells using the organelle-specific dye Mitotracker. Stained mitochondria could be visualized in the TnTs connecting eGFP-E4⁺ECs to various cancer cells (Figure 4A). Confocal time-lapse imaging demonstrated transfer of mitochondria stained by Mitotracker (Figure 4B and Additional file 9: Movie 8). We also investigated the transfer of eGFP-E4⁺ECs mitochondria to tumor explants. Indeed, we cultured ovarian cancer tumor explants on eGFP-E4⁺ECs feeder stained with Mitotracker-Red. After 48 h of culture, tumor explants were removed and confocal analysis showed uptake of eGFP-E4⁺ECs mitochondria (Figure 4C).

To investigate whether organelle transfer displayed cell specificity MCF7 and eGFP-E4⁺ECs or M-Orange-MSCs co-cultures were established and mitochondria transfer from stromal cells was monitored. TnTs seemed similarly distributed among cell types. However, at a ratio of 1 MCF7 /1 stromal cell, only endothelial cells were able to transfer mitochondria to MCF7s (Figure 5A-B). MSCs mitochondria transfer to cancer cells could only be visualized using an increased ratio (1/10) of MCF7/MSCs (Additional file 1: Figure S4). We then established a tri-culture system (MCF7, MSCs and E4⁺ECs). TnTs were observed between all different cell types, however, in this setting, MCF7 only acquired E4⁺ECs mitochondria (Figure 5C). We could also detect uptake of E4⁺ECs mitochondria by M-Orange-MSCs. Our data provided evidence for a preferential transfer of mitochondria from endothelial cells under our culture conditions.

One of the most common criticisms of such experiment is the ability of the dyes to diffuse. Several findings argued against such bias. The presence of MCF7 cells with different level of Mitotracker after 48 hours of co-culture argued against passive dye transfer. In our tri-culture set-up, the preferential transfer ruled out the possibility of passive diffusion. Indeed it is quite unlikely that dyes would only leak form one cell type. Switching dyes used between E4⁺ECs and MSCs did not modify these findings (data not shown). Flow cytometry evaluation of mitochondria uptake at different ratio of eGFP-E4⁺ECs/MCF7 ranging from 1/1 to 1/10 demonstrated that exchange of mitochondria was related to the number of stromal cells (Figure 5D-E).

Among other mechanisms involved in the cross-talk between cancer et stromal cells, microparticles (MPs) have been involved in the transfer of organelle 35 . We investigated the implication of MPs in the transfer of mitochondria between endothelial and cancer cells. We stained MPs extracted from eGFP-E4⁺ECs using Mitotracker. While cancer cells were able to uptake endothelial MPs, the uptake of mitochondria by MCF7 through MPs was negligible (Additional file 1: Figure S5A-B). Similar results were obtained if endothelial cells were stained before MPs isolation. This indicated that mitochondria transfer from eGFP-E4⁺ECs under our cell culture conditions did not occur through microparticles. We also used a transwell experiment setting to confirm the role of direct cell contact in the transfer of mitochondria. Confocal and flow cytometry analysis could not detect any Mitotracker-Red uptake in the transwell experiments (Additional file 1: Figure S6A-B). In order to rule out the possibility that diffusing Mitotracker into the medium could be adsorbed by the filter of the transwell chambers, we seeded MCF7 on the bottom or the top of the transwell chamber and mitotracker on the other side. Flow cytometry showed constant staining of MCF7 suggesting that passive diffusion could occur through the membrane (Additional file 1: Figure S6C). Finally shaking cultures that do not allow the formation of TnTs were performed 36 . Under these conditions no Mitochondria transfer was observed compare to controls (Additional file 1: Figure S7). These experiments excluded the possibility of massive dye transfer and strongly suggested that TnTs participated to mitochondria transfer.

Mitochondria transfer between two cells have been associated to phenotypic modification 37 , however a direct functional benefit of TnT mediated material exchange has not yet been demonstrated in the cancer setting. We hypothesized that MCF7 acquiring endothelial mitochondria would display greater chemoresistance. We previously described TnT mediated transfer of P-glycoprotein from resistant to sensitive cells 29 . We first verified that both eGFP-E4⁺ECs and MCF7 did not express ABCB1 (Data not shown).

To assess functional gain, fluorescence minus one was used to set up the cell-sorting gate as stringent as possible (Figure 6A). Cells having acquired eGFP-E4⁺ECs mitochondria (Mt⁺MCF7, red) and cells did not acquire any mitochondria (Mt^-MCF7, black) were sorted. Mt^-MCF7 population allowed us to control for other factors such as endothelial secreted factors or direct cell to cell contact without mitochondria transfer. We finally sorted cells having only acquired cytoplasmic (excluding mitochondria) content represented by the GFP⁺MCF7 population (green). We controlled the purity of our cell sorting by confocal microscopy (Figure 6B) and subjected the different cell populations to a MTT viability assay in presence of doxorubicin; MCF7 cells were used as a control (grey) (Figure 6C). Mt^-MCF7 and GFP⁺MCF7 displayed chemoresistance (from 1.31 to 1.89 fold compare to MCF7 control). However MCF7 having acquired eGFP-E4⁺ECs mitochondria (Mt⁺MCF7) displayed significantly more chemoresistance compared to MCF7 control (1.91 to 3.19 fold, p < 0.05). These results were confirmed using LiveDead staining (Figure 6D). These experiments indicated that while co-culture and transfer of cytoplasmic content induced chemoresistance, the acquisition of mitochondria significantly increased chemoresistance of MCF 7 to doxorubicin.

IRB was obtained at Hamad Medical Corporation.