MCF-7 cells (ATCC HTB-22) were cultured in RPMI (Gibco, Life Technologies) supplemented with 1µmol/L insulin (Sigma Aldrich), 10% fetal calf serum (FCS) (Gibco, Life Technologies) and 1% penicillin/streptomycin (100 U/ml, Gibco, Life Technologies) in a humidified atmosphere of 5% C02 at 37 °C. For time-lapse microscopy imaging, culture medium was replaced by OPTIMEM + Glutamax (Gibco by Life Technologies) supplemented with 1µmol/L insulin, 10nmol/L ß-estradiol, 20ng/ml epidermal growth factor (Invitrogen), B-27 Supplement (1X, Invitrogen), 1% penicillin/streptomycin (100 U/ml, Gibco, Life technologies).
To study the impact of paclitaxel and vinorelbine, cells were incubated in culture medium containing 100 nM paclitaxel or 20 nM vinorelbine for 24 hours, then trypsinized and seeded for aggregation assay in culture medium containing the same concentrations of paclitaxel and vinorelbine. For synchronization in mitosis, cells were incubated with 200 ng/ml nocodazole for 20h to accumulate in an abnormal pro-metaphase state. Cells were then incubated in culture medium containing 200 ng/ml nocodazole and 25 µM MG132 for 30 min, and then in medium containing only 25 µM MG132 for 1.5 hours, according to the protocol described by Cazales et al. . Addition of MG132 blocked cells in metaphase by inhibiting sister chromatid separation. Mitotic shake-off  was used to select only mitotic cells that were used for the clustering and aggregation assays. For control experiments, MG132 (25µM) or nocodazole (200ng/ml) were added to the culture medium just before seeding for aggregation assays. For actin cytoskeleton inhibition, latrunculin A (200 nM, Sigma) and CK666 (150 µM, Sigma) were added to the culture medium just before seeding for aggregation assays.
Cells were grown on coverslips coated with poly-L-lysine. Cells were washed in PBS, fixed for 10min in formalin (Sigma) then washed and permeabilized in PSB/0.25% Triton X-100 for 5 min at room temperature, and incubated in PBS/1%BSA 30min at room temperature. Coverslips were then incubated at 37°C with anti-tubulin antibodies (1:2000, Sigma #T5168) in PBS/0.1%BSA for 1 hour. After washes in PBS, goat anti-mouse Alexa 488 antobodies (1/800, Molecular probes # A-11001) were applied at room temperature for 1 hour. DNA was stained with DAPI at 1µg/ml at room temperature for 10 min.
LifeAct-mCherry-expressing MCF-7 cell line
The 17-amino acid LifeAct coding sequence fused to GFP2 was excised from the pLifeAct-TagGFP2 vector (Ibidi; catalog number#60101) and cloned in the pTRIP lentiviral shuttle vector in frame with the cDNA encoding the mCherry fluorescent protein. The resulting plasmid (pTRIP LifeAct mCherry) was used to produce lentiviral particles in 293FT embryonic kidney cells (Life Technologies) after calcium chloride transfection with the pGag/pol and pVSV-G plasmids (provided by the Vectorology platform, INSERM U1037). At 7 hours post-transfection, DMEM + Glutamax (Gibco by Life Technologies) complemented with 10% FCS was washed off and replaced with serum-free OPTIMEM + Glutamax (Gibco by Life Technologies). Lentiviral particles were harvested 48 hours later and the viral titer was quantified by flow cytometry (BD Accuri C6) in HT1080 cells (ATCC) transduced with serial dilutions of lentiviruses. MCF-7 cells (ATCC HTB-22) were then transduced in the presence of 4μg/ml protamine sulfate in OPTIMEM + Glutamax. The medium was replaced 7 hours later by RPMI (Gibco by Life Technologies) supplemented with 10% FCS and 1µM insulin (Sigma-Aldrich, Ref. I0516). The generated stable LifeAct-mCherry-expressing MCF-7 cell line underwent two rounds of cell sorting (Cytometry and Cell Sorting platform, INSERM UMR 1048) followed by single-cell clonal isolation in 96-well plates.
Flow cytometry analyses
Trypsinized cells were collected and fixed in 4% formalin solution (Sigma-Aldrich) for 10 min, then washed and permeabilized in PBS/1% BSA containing 0.25% Triton X-100 on ice for 5 min. Mitotic cells were detected with the 3.12.i.22 antibody  diluted (1:10000) in PBS/0.1% BSA. After a wash in PBS, cells were incubated with a goat anti-mouse Alexa Fluor 488 antibody (Molecular Probes) at room temperature for 1 h. After DNA staining with 10μg/mL propidium iodide (Sigma Aldrich) at room temperature for 20 min, cells were analyzed with an Accuri™ C6 Flow Cytometer (BD Science) and the Accuri software.
This assay was performed essentially as previously described [15,16]. Cells (500 cells/well) were seeded in low-attachment round-bottomed 96-well plates (Costar®), except in the 36 peripheral wells to avoid edge effects. Plates were centrifuged at 400g for 4 min, and then cell aggregation in each well was followed by time-lapse video-microscopy. Images were acquired with an inverted widefield Zeiss Axio Observer microscope fitted with a 0.3 N.A. 10X objective and a CoolSNAP CDD camera (Roper scientific) in bright-field for at least 5 h (1 acquisition/15 min). At each time point and position, 20-µm spaced z-stacks over 160µm depth (8 stacks) in brightfield were acquired. A custommade MATLAB procedure was used to monitor and measure cell cluster formation over time. The main steps of the workflow were: (1) image processing at each time point and for each cluster by focus stacking to merge images of multiple focal planes into one in-focus image; (2) binarization and edge detection with a Sobel filter to define the boundaries of each cluster and of holes inside the cluster (to exclude them); (3) saving the projection, segmentation and image overlay; and (4) calculation of the typical parameters (perimeter, area, normalized area: Area T0/Area T(x)).
Evaluation of aggregate cohesion - Flush assay
Aggregates formed in each well were mechanically dissociated directly in the wells by making 1, 3, 5, or 8 flushes. In each flush, 50µl of cells and medium were gently aspirated with a multi-channel micropipette and vigorously flushed back. Cells were then allowed to sediment for 10 min before quantifying the number of dissociated individual cells in 10µL. Quantification was done in triplicate for each experimental condition.
Single-cell clustering assay in dedicated PDMS micro-wells
The PDMS pre-polymer was mixed with the polymerization agent Sylgard 184 (10:1 ratio), degassed in a vacuum chamber, and poured in a silicon wafer (RENATER facility of LAAS, CNRS, France). After a second degassing, PDMS was cured at 60°C overnight. Arrays of nine PDMS micro-wells (see Additional file 2: Fig. S2) were cut, peeled off, and glued in each compartment of CELLviewTM cell culture dishes (Greiner Bio-one). Micro-wells were incubated with 20 mg/ml Pluronic-F127 (Sigma) overnight to prevent cell adhesion, and then rinsed twice before use.
LifeAct-mCherry-expressing MCF-7 cells were distributed in the compartments at a density that allowed the sedimentation of approximately 20 cells per micro-well. Cluster formation was followed by time-lapse video-microscopy using an inverted widefield Zeiss Axio Observer microscope fitted with a 0.3 N.A. 10X objective. Images were acquired for 3h (one acquisition every 10s) and processed with lmageJ software packages . Before automated analysis, images were manually corrected. Specifically, parts of other cells, staining background and debris were removed using the clearing function of ImageJ. Then, the lmageJ macro was used for image segmentation and calculation of the shape descriptors (circularity and aspect ratio).
Data were analyzed with GraphPad Prism version 6.00 (GraphPad soft- ware, La Jolla California USA, www.graphpad.com)