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Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer

Significance Increasing evidence suggests that extracellular vesicles (EVs) can transfer genetic material to recipient cells. However, the mechanism and role of this phenomenon are largely unknown. Here we have made a remarkable discovery: EVs can harbor the full mitochondrial genome. These extracellular vesicles can in turn transfer their mtDNA to cells with impaired metabolism, leading to restoration of metabolic activity. We determined that hormonal therapy induces oxidative phosphorylation-deficient breast cancer cells, which can be rescued via the transfer of mtDNA-laden extracellular vesicles. Horizontal transfer of mtDNA occurred in cancer stem-like cells and was associated with increased self-renewal potential of these cells, leading to resistance to hormonal therapy. We propose that mtDNA transfer occurs in human cancer via EVs.


Electron Microscopy (EM)
Cells were washed with serum-free media or appropriate buffer. Both EVs and cells were fixed with a modified Karmovsky's fix of 2.5% glutaraldehyde, 4% paraformaldehyde and 0.02% picric acid in 0.1M sodium caocdylate buffer at pH 7.2.
Following a secondary fixation in 1% osmium tetroxide and 1.5% potassium ferricyanide, samples were dehydrated through a graded ethanol series and embedded in an epon analog resin. Ultrathin sections were cut using a Fiatome diamond knife (Diatome, USA, Hatfield, PA) on a Leica Ultracut S ultramicrotome (Leica, Vienna, Austria). Sections were collected on copper grids further contrastated with lead citrate and viewed on a JEM 1400 electron microscope (JEOL, USA, Inc., Peabody, MA) operated at 120 kV.
Images were recorded with a Veleta 2K x2K digital camera (Olympus-SIS, Germany).

Nucleic acid extraction
DNA extraction: cell pellets and EVs (ultracentrifugation of 10 12 cells) were resuspended in 25µl of 1X PBS followed by the addition of 450µl of DNA extraction buffer (SDS 0.5-1%, Tris-HCl 50mM pH 8.0, EDTA 0.1M) and 0,1mg/ml proteinase K 20mg/ml (ThermoFisher Scientific) and incubated O/N at 56°C. 500µl of phenol/chloroform (ThermoFisher Scientific) was added to each sample and centrifuged at 13,000 rpm for 5' at room temperature. The upper phase, containing the DNA, was transferred to a new tube where 500µl of chloroform were added. Samples were centrifuged at 13,000 rpm for 5' at room temperature; the DNA was washed a second time by repeating this step.
The upper phase was transferred to a new tube with 450µl of isopropanol and 50µl of NaAc 3M. The samples were centrifuged at 13,000 rpm for 10' at 4°C. The supernatant was discarded and the pellet washed with 750µl 70% EtOH and centrifuged at 13,000 rpm for 5' at 4°C. The DNA pellet was air dried, resuspended in 20µl of DEPC H 2 O and incubated at 37°C for 30'. DNA concentration was measured by loading 1µl of DNA on a Thermo Scientific NanoDrop™ 1000 Spectrophotometer and stored at -20°C until further analyzed. For RNA extraction we used trizol (Invitrogen). EVs were suspended in 500µl of Trizol and vigorously mixed. The samples were centrifuged at 12,000xg for 30 seconds. 200µl of chloroform were added, mixed by inversion and incubated for 2-3' at RT. After a centrifugation at 12,000xg for 15' at 4°C, the upper phase was transferred to a new tube. 400µl of isopropanol and 3µl of glycogen were added to the sample and incubated O/N at -20°C; the samples were then centrifuged at 12,000xg for 10' at 4°C.
The supernatant was discarded and the pellet washed in 750µl of cold 75% EtOH. The RNA was pelleted with a centrifugation at 8,000xg for 20' at 4°C, air dried and suspended in 10µl of DEPC H 2 O. The RNA concentration was measured with a Thermo Scientific NanoDrop™ 1000 Spectrophotometer and treated for 1 hour at 37°C with 1U (for EVs) or 2U (for cells) of Baseline-ZERO™ DNase (Epicentre ® ). RNA was stored at -80°C.

Reverse Transcription PCR (RT-PCR) and microarray analyses
cDNA was obtained by retro transcribing 1µg of RNA -previously treated with 1U (EVs) or 2U (cells) of Baseline-ZERO™ DNase0 (Epicentre ® )-and using iScript™Select cDNA synthesis Kit (Bio-Rad). The cDNA was kept at -80°C for further analysis. For microarray analysis 250ng of RNA was processed, assayed and run on Illumina GX Human HT12 platform prepared according to the manufacture's' protocol at the Genomic Molecular Core Facility (MSKCC). Data were analyzed, submitted at GEO (GSE84104).

Rolling Circle PCR and canonical PCR
DNA was isolated using phenol/chloroform (ThermoFisher Scientific). Each amplification reaction was performed on a total of 20ng of DNA using the GeneAmp® PCR System 9700, version 2.5. The amplification program was the following: (i) Polymerase activation (2 min at 95°C), (ii) amplification stage (35 cycles, with each cycle consisting of 30 seconds at 95°C, 30 seconds at 60°C, and 60 seconds at 72°C), and (iii) extension stage (5 min at 72°C). All amplification reactions were performed using the GoTaq®Flexi DNA Polymerase kit (Promega). PCR products were resolved on a 2% agarose gel. All primers used for this assay are listed at the end of the methods section. Rolling Circle amplification (RCA) was performed in EV-DNA according to the manufacturer's protocol (GE Healthcare Life Sciences): 1) EV-DNA was first digested with Plasmid Safe ATP-DNAse (Epicentre ® , 1U for 1 hour at 37°C) in order to degrade all non circular DNA 2) 10ng of previously digested EV-DNA was amplified using RCA methodology 3) 2ng of post-RCA amplicons were then amplified using qPCR with specific mtDNA and housekeeping DNA primers ( Fig. S3D and Table S4).

Real time PCR
DNA and cDNAs were amplified by quantitative PCR (qPCR) using the Applied and analyzed using Nikon software (EZ-C1 3.6).

Semi-Quantitative Mass Spectrometry analysis of CAF EV
Mass spectrometry analyses of EV were performed at the Rockefeller University Proteomics Resource Center (New York, NY, USA) using 10µg of CAF-EV protein.
Samples were denatured using 8M urea, reduced using 10mM dithiothreitol, and alkylated using 100mM iodoacetamide, followed by proteolytic digestion with

Primers
The reference sequences used for primers design are NC_010339 for the murine mitochondrial DNA/RNA, NC_012920 for the Human mitochondrial DNA/RNA (SI Appendix, Table S4).

Exit from dormancy via CAF-EVs
Generation of HTD cells was performed by treating ER+ (GFP+) breast cancer cell lines Calcein AM assay (InVitrogen). HTD were cultured in low attachment (Corning) 24-well plates. These were treated with 3x10 9 mCAF-derived EVs (wt-mtDNA hi or ρ0-mtDNA lo ) weekly x4 in the presence with HT (fulvestrant, 10µM/weekly). After 40 days, mammosphere number (bright field microscopy) was determined after 40 days. In vivo assays: Dormant ZR751 cells (HTD) were isolated and 10 5 cells were injected in the IV inguinal MFP of NOD/SCID mice. All mice were subsequently injected with 3x10 9 mCAFderived EVs (wt-mtDNA hi or ρ0-mtDNA lo ) weekly x8 (n=5 mice/group). Tumor growth was determined by BLI and after necropsy. Cat#2012) was performed for three minutes. TSG101 localization was confirmed using immunofluorescence before electron microscopy processing.

Electron Microscopy Processing
Sections were post-fixed in 1% Osmium tetroxide in 0.1M sodium cacodylate for 60 minutes on ice. After washing with water, cells were placed in 1% aqueous uranyl acetate for 30 minutes, dehydrated in a graded series of ethanol and embedded in Eponate. Sections were cut at 70nm, transferred to formvar coated slot grids and imaged on a JEOL 100CX at 80kVwith an AMT XR41 digital imaging system (Rockefeller University Electron Microscopy Core Center).     Table S3. Mass Spectrometry analysis of mCAF derived EVs, mCAFs were isolated from HTR xenografts (Figure 2), cultured in vitro and EVs were isolated. Identified peptides were filtered using