Reagents
Chemical hypoxia was induced with Cobalt Chloride (CoCl2, Merck, Darmstadt, Germany).
ER stress was induced with HA15 (Selleckchem, Zurich, Switzerland), Thapsigargin (THA, Enzo LifeSciences, Lausen, Switzerland), or Tunicamycin (TUN, Enzo LifeSciences, Lausen, Switzerland).
ER stress was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF; Sigma, Darmstadt, Germany), Melatonin (Mel; Sigma, Darmstadt, Germany), STF-083010 (STF; Selleckchem, Zurich, Switzerland), 4μ8C (Selleckchem, Zurich, Switzerland), GSK2656157 (GSK; Selleckchem, Zurich, Switzerland) or Salubrinal (SAL; Tocris Biosciences, Bristol, UK).
Cancer cell lines and culture
The human ovarian cancer cell lines COV318 (ECACC, Sigma-Aldrich) and SKOV3 (courtesy of Dr. Florence Delie, University of Geneva, Switzerland) were cultured at 37 °C and 5% CO2 in DMEM or RPMI (Gibco, Invitrogen, Basel, Switzerland) respectively, supplemented with 10% fetal bovine serum (FBS, Biochrom AG, Oxoid AG, Basel, Switzerland) and 0.05 mg.mL− 1 gentamycin (Invitrogen, Basel, Switzerland).
Generation of PGCCs by cell fusion
Cytoplasmic staining and FACS sorting: SKOV3 or COV318 cells were stained with either green or far-red cytoplasmic dyes, CellTrace CFSE (green; Life Technologies, Carlsbad, USA), and CellTrace Far-Red (far-red, Life Technologies, Carlsbad, USA), according to the manufacturer’s instructions. For each cell line, 3.5 × 105 green cells and 3.5 × 105 far-red cells were co-seeded immediately after staining in a T175 flask. Twenty-four hours after seeding, cells were treated with 300 μM CoCl2 (Merck, Darmstadt, Germany) for 48 h. Cells were then analyzed by flow cytometry with FACS Aria II (Becton Dickinson, Franklin Lakes, USA) for -GFP and far-red signals. Double-positive cells were sorted and seeded in a regular culture medium. Characteristic pictures of the sorted cells were done 16 h after seeding, using a fluorescence microscope (EVOS FL, Life Technologies, Carlsbad, USA).
Transfection and selection: SKOV3 cells were transfected with a pH2B-mCherry-IRES-puro2 plasmid or with pEGFP-CenpA-IRES-neo (Addgene, LGC Standards, Teddington, UK) using JetPEI as the transfection reagent (Polyplus transfection, Illkirch, France) according to the manufacturer’s instructions. Two days after transfection, cells were selected using selection antibiotics (1 μg.mL− 1 puromycin, InvivoGen, San Diego, USA for cells transfected with pH2B-mCherry-IRES-puro2 plasmid = SKOV3-Red, and 500 μg.mL− 1 G418, Roche Life Science, Penzberg, Germany for cells transfected with pEGFP-CenpA-IRES-neo = SKOV3-Green) for 2 weeks. Cells were then cultured in RPMI medium supplemented with 0.1 μg.mL− 1 puromycin for SKOV3-Red cells or with 50 μg.mL− 1 G418 for SKOV3-Green cells.
SKOV3-M cell line establishment: SKOV3-Red and SKOV3-Green were mixed (1:1) and cultured for 72 h before adding 0.2 μg.mL− 1 puromycin and 100 μg.mL− 1 G418 in RPMI. The culture medium was renewed every 2 days.
Cell treatments
UPR modulators- ER stress was induced in ovarian cancer cells by adding either 100 nM THA, 1 μg.mL− 1 TUN or 1 μM HA15 in culture medium. Cells were treated for 48 h before processed for analysis.
UPR pathways were inhibited in ovarian cancer cells by adding either 17.5 μM SAL, 0.3 μM GSK, 8 μM 4μ8C, 2 μM STF, 200 μM AEBSF or 1 mM Mel, in combination with 100 nM THA or 1 μg.mL− 1 TUN. Cells were treated for 48 h before processing for analysis.
Paclitaxel treatment- SKOV3 cells were treated with 0, 1, 10, and 100 nM Paclitaxel for 48 h before processing for analysis (immunofluorescence, cell viability and fusion index).
SKOV3-Red and SKOV3-Green (cell ratio 1:1) were treated with 10 nM Paclitaxel for 48 h before changing the culture medium (RPMI, supplemented with 10% FBS and gentamycin) every 2 days for 10 days.
Zymography
Twenty-four hours after the seeding of SKOV3-M, SKOV3-Red and SKOV3-Green in a 12-well plate, cell culture medium was replaced by a culture medium without FBS and the cells were incubated for 48 h. The supernatants of culture were collected and the proteolytic activity of culture supernatants (n = 3) were assayed using gelatin-substrate gel electrophoresis as described previously [26].
Western blot
Western blot analyses were performed as described by Bastida-Ruiz et al. [24]. Briefly, whole-cell extracts were fractionated by SDS-Page 10% and transferred to nitrocellulose membrane for immunoblot analysis. The membranes were first incubated with the primary antibodies rabbit anti-GRP78 (GL-19, 1∶5000 dilution from Sigma) and mouse anti-GAPDH antibodies (1∶60,000 dilution from Millipore), followed by incubation with the secondary antibodies goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated (170-6515, 1:3000 dilution, from Bio Rad) and goat anti-mouse IgG HRP-conjugated (sc-2005, 1:3000 dilution, Santa Cruz Biotechnologies).
Quantification of GFP-positive, mCherry-positive, and double-positive cells
SKOV3-Green, SKOV3-Red, SKOV3-M, and untransfected cells (ct-SKOV3) were resuspended in PBS. Percentages of negative cells, GFP-positive cells, mCherry-positive cells, and GFP and mCherry double-positive cells were measured by flow cytometry analysis (LSR Fortessa, Becton Dickinson, Franklin Lakes, USA). Data were analyzed with FlowJo software (FlowJo LLC, Ashland, USA). This experiment was repeated 3 times.
Polyploidy index / fusion index
Non-transfected cells: SKOV3 and COV318 cells were fixed in paraformaldehyde (PFA) 4% at 4 °C for 10 min, washed three times in PBS, and stained with Hematoxylin (Sigma, Darmstadt, Germany) for 1 min. Cells were then washed twice with warmed tap water before bright-field imaging was conducted (EVOS, Life Technologies, Carlsbad, USA).
Transfected cells: Cells were seeded either in Nunc Lab-Tek II Chamber slides (Life Technologies, Carlsbad, USA) for confocal microscopy or in μ-Slides 8 Well (Ibidi, Madison, USA) for fluorescent microscopy, and processed for treatment. Cells were then fixed in PFA 4% at 4 °C for 10 min, washed three times in PBS, and incubated in PBS-3% bovine serum albumin (BSA) for 30 min to eliminate non-specific binding. Cells were then stained with Alexa Fluor 647 phalloidin (1∶40 dilution, from Invitrogen, Thermo Scientific) for 20 min at room temperature and rinsed three times in PBS. Nunc Lab-Tek II Chamber slides were then mounted with Vectashield with DAPI (Vector Laboratories, Burlingame, USA) and sealed before imaging with a confocal fluorescent microscope (LSM 800 Airyscan, Zeiss, Iéna, Germany). μ-Slides 8 Well were imaged with a fluorescent microscope (EVOS FL, Life Technologies, Carlsbad, USA). Images were processed using ImageJ freeware.
For both transfected and non-transfected cells, the polyploidy or fusion index expressed in percent was calculated as follows: [(N-S)/T] × 100, where N equals the number of nuclei in syncytia, S equals the number of syncytia and T equals the total number of nuclei counted [27]. This index was calculated for three independent experiments, run in triplicate.
Cell cycle analysis
SKOV3-Green, SKOV3-Red, and SKOV3-M were resuspended in PBS with 5 μg.mL− 1 Hoechst 33342 (Life Technologies, Carlsbad, USA), and incubated for 20 min at 37 °C. The amounts of cells in G0-G1 and G2 phases were then assessed by flow cytometry analysis (LSR Fortessa, Becton Dickinson, Franklin Lakes, USA). Data were analyzed with FlowJo software (FlowJo LLC, Ashland, USA). This experiment was repeated 2 times.
Cell proliferation
On day 0, SKOV3-Green, SKOV3-Red, and SKOV3-M were resuspended in Hanks’ Balanced Salt Solution (Life Technologies, Carlsbad, USA) and stained with CellTrace Far Red (Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. The cells were immediately processed for far-red signal analysis by flow cytometry (Gallios, Beckman Coulter, Brea, USA) (D0). Far-red signal dilution was then measured daily for 4 days (D1, D2, D3, and D4). Data were analyzed with FlowJo software (FlowJo LLC, Ashland, USA). This experiment was repeated 3 times.
Cell viability (MTT assay)
SKOV3, SKOV3-Red, SKOV3-Green, and SKOV3-M cells were seeded in 96-well plates (15′000 cells/well) and incubated for 48 h. The culture medium was then replaced by 100 μL of MTT reagent (1 mg.mL− 1) for 2 h. Acidic isopropanol solution (150 μL) was added, and then each well was vigorously mixed to dissolve the precipitated formazan. UV–visible absorption was measured at 540 and 690 nm (as blank). These experiments were carried out in triplicate, three times.
Invasion assay
Cell invasion assay was performed in an invasion chamber as described elsewhere [28]. Briefly, 1.5 × 104 cells (SKOV3-Red, SKOV3-Green, and SKOV3-M) in 100 μL of RPMI supplemented with 10% FBS and 1% gentamicin were added to the upper compartment of the transwell chambers. RPMI supplemented with 20% FBS and 1% gentamicin (400 μL) was added to the lower chamber for 48 h at 37 °C in a CO2 (5%) incubator. After incubation, viable cells that invaded collagen were stained with crystal violet, and measurement was performed at 540 nm. This assay was repeated three times and each experiment was run in triplicate. Data were normalized by proliferation values (MTT assay) and expressed as AU (invasion)/ AU (MTT).
Tumor development on chick chorioallantoic membrane (CAM)
Tumor development on CAM was performed as previously described [29]. Briefly, fertilized eggs (animal facility of the University of Geneva, Geneva, Switzerland) were incubated at 38 °C with 80% relative humidity and periodic rotation. On egg development day (EDD) 4, the eggs were drilled at their narrow apex, and the hole was closed with adhesive tape. The eggs were then incubated at 38 °C with 80% relative humidity without rotation. On EDD8 the hole in the eggshell was enlarged to allow access to the CAM. After gently scratching the membrane with a needle tip, SKOV3-Red, SKOV3-Green, or SKOV3-M cells suspension (2 × 106 cells in 30 μL of geltrex, Thermo Scientific) was inoculated, and the hole was hermetically covered with Parafilm®. Eggs were returned to the incubator to allow tumor growth. Tumor growth was then monitored at EDD10.5 and 13.5 using a Wild Heerbrugg M3Z microscope at 10x magnification with a Lumenera INFINITY2-1 CDD camera with Infinity Capture Software.
Microarray
Microarray-based transcriptome profiling was performed at the iGE3 Genomics core-facility of the faculty of medicine of the University of Geneva. Briefly, 100 ng of total RNA were extracted from SKOV3-Red, SKOV3-Green, and SKOV3-M at two different passages and were used as input for the preparation of single-strand cDNA using the WT PLUS reagent kit from Thermofisher Scientific. Targets were then fragmented and labeled with the Affymetrix GeneChip WT Terminal Labeling Kit and hybridized on Human Clariom S arrays according to the manufacturer’s recommendations.
The data were analyzed with the Transcription Analysis Console (TAC) software (ThermoFisher Scientific), selecting the genes which expression was modified at least 2-fold with a p-value < 0.001.
Statistical analysis and reproducibility
Data were represented as means ± SEM. Statistical differences between samples were assessed by the Student’s t-test. A p-value < 0.05 was considered significant. GraphPrism software was used to perform the different statistical analyses. The number of repeated experiments was described in each method description.