Supplementary Table 1: Antibodies Used in RPPA

Supplementary information

Supplementary table

Supplementary Table 1: Antibodies used in RPPA

Supplementary Figure legends

Supplementary Figure 1: Analysis of LIMK1 expression in different breast cancer cell lines.

A. TS/A-pGL3, MCF7, MDA-MB-231 and MDA-MB-231-ZNF217rvLuc2 lysates (10 µg of proteins) were blotted for the indicated proteins.

B. Quantification (arbitrary units) of the LIMK1 western blot staining using ImageJ software.

Supplementary Figure 2: Effect of PTX on the viability of breast cell lines.

TS/A-pGL3, MDA-MB-231 and MDA-MB-231-ZNF217rvLuc2 cells were incubated for 48 hours with PTX (0 to 10µM). The percentage of viable cells was calculated using the MTT assay.

Supplementary Figure 3: Weight and behavior monitoring of mice used in the TS/A-pGL3 xenografts experiment.

All mice from vehicle and Pyr1 groups presented normal body weight development. Mice from PTX group began to lose weight between day15 and day 21.

Supplementary Figure 4: Pharmacokinetic analysis of Pyr1 and its metabolite M1

A single dose of Pyr1(10mg/Kg) was injected (i.p.) and plasma concentrations of Pyr1 and its metabolite M1 were measured using HPLC at the indicated times, as described in the methods section.Pyr1 was not detectable at the first time point (25 min.).

Supplementary Figure 5: RPPA results of tubulin acetylation, tubulin detyrosination and cofilin phosphorylation levels in tumors of mice treated with vehicle, Pyr1 and PTX.

Detyrosinatedtubulin, acetylated tubulin and phosphorylated cofilin levels were assessed by RPPA in TS/A-pGL3 tumor allografts. Boxplots show the distribution of expression in mice treated with Pyr1 (A) or PTX (B) and compared to the vehicle group. Boxes represent the 25–75th percentile; brackets: range; black line: median; black dots: outliers, * p <0.05.

Supplementary Figure 6: Pyr1 effect on tumor cell density.

Hematoxylin and eosin staining was carried out in tumor sectionsfrom mice treated with vehicle, Pyr1 10mg/Kg or PTX 10mg/Kg, as indicated. A representative area of the tumorof each mouse is shown. Bar, 100µm.

Supplementary Figure 7: In vivo analysis of thePyr1effect on LIMKs activity markers.

A. Lysates of MDA-MB-231 tumor xenografts (30µg) treated with vehicle, Pyr1 or PTX were blotted for detyrosinated tubulin, phosphorylated cofilin (P-cofilin) and actinas indicated.

Signal intensities were quantified using ImageJ software and the ratios of detyrosinated tubulin/ actin(B) and phosphorylated cofilin/actin(C)were calculated. Bars = SEM, **p<0.01, ***p<0.001.

Supplementary Movie 1: FRAP analysis of actin dynamics in invadopodia treated with vehicle or Pyr1. Representative movie of an invadopodia in a MEF SrcY527F cell expressing Actin GFP treated with 0.25% DMSO(left)or 25µM Pyr1 (right). Regions of invadopodia were bleached. Recovery of fluorescent intensities within the bleached area was measured over 2 min with a 40x oil immersion objective.

Supplementary Movie 2: MDA-MB-231 Dendra2 tumors followed by intravital microscopy after 8 days of vehicle treatment.

Representative movie of a tumor of a mouse treated with vehicle. Images were taken using a 23x objective every 15min for a total of 135 minutes. Movie is played at 10 frames/sec.

Supplementary Movie 3: MDA-MB-231 Dendra2 tumors followed by intravital microscopy after 8 days of Pyr1 treatment.

Representative movie of a tumor of a mouse treated with 10mg/Kg Pyr1. Images were taken using a 23x objective every 15min for a total of 105 minutes. Movie is played at 10 frames/sec.

Supplementary methods

Antibodies: The primary antibodies used were detyrosinated-Tubulin (L4 (7)), LIMK1 (Cell Signaling Technology, 3842).

The references of the antibodies used in RPPA analysis are listed in Supplementary Table 1.

Cell lines and cell culture: For the intravital microscopy experiment, MDA-MB-231 cells obtained from the ATCC were modified in Dr. van Rheenen's team. They were infected with lentivirus carrying pLV.CMV.puro.Dendra2 pasoA and then selected with 2mg/ml puromycin. For culture and amplification these cells were grown in DMEM + 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin at 37°C and 5% CO2.

MEF (mouse embryonic fibroblasts) cells were a generous gift of Dr. Richard Hynes (Koch Institute for Integrative Cancer Research, MIT, USA). The expression of the constitutive active form of the Src kinase (SrcY527F) was performed via retroviral infection using the pBabe vector (provided by Dr. M. Humphries University of Manchester, Manchester, United Kingdom). cDNA was delivered via retroviral transduction after packaging in Phoenix-Eco (American Type Culture Collection, Manassas, VA). Supernatant containing viral particles from transduced cells was harvested, filtered, and after addition of 8g/ml polybrene (Sigma-Aldrich) was used to infect fibroblasts. Cells were grown in DMEM containing glutamine and supplemented with 10% (v/v) FCS and 1% (v/v) penicillin/streptomycin.

RPPA: total protein extract was isolated from TS/A-pGL3 tumor allograft. Tumors were lysed using mechanic tissue lyser (MagNAlyser, Roche) and suspended in RPPA buffer (50 mMTris pH=6.8, 2% SDS, 5% glycerol, 2 mM DTT, 2.5 mM EDTA, 2.5 mM EGTA, Phosphatase inhibitors, Protease inhibitors, 4 mM Sodium Orthovanadate, 20 mM Sodium Fluoride) at a final concentration of 1mg/mL.

Total protein extract was isolated from TS/A-pGL3 tumor allograft. Tumors were lysed using mechanic tissue lyser (MagNAlyser, Roche) and suspended in RPPA buffer (50 mMTris pH=6.8, 2% SDS, 5% glycerol, 2 mM DTT, 2.5 mM EDTA, 2.5 mM EGTA, Phosphatase inhibitors, Protease inhibitors, 4 mM Sodium Orthovanadate, 20 mM Sodium Fluoride) at a final concentration of 1mg/mL.Extracts were then boiled for 10 min at 100°C, sonicated to reduce viscosity and centrifuged 10 min at 15000 rpm. The supernatant was harvested and stored at -80°C. Protein concentration was determined (Pierce BCA reducing agent compatible kit, ref 23252). Samples were deposited onto nitrocellulose covered slides (Supernova, Grace Biolabs) using a dedicated arrayer (2470 arrayer, Aushon Biosystems). Five serial dilutions, ranging from 1000 to 62.5 µg/ml, and three technical replicates per dilution were printed for each sample. Arrays were labeled with 25 specific antibodies (see supplementary Table 1 for a complete list antibodies references) or without primary antibody (negative control), using an Autostainer Plus (Dako). Briefly, slides were incubated with avidin, biotin and peroxydase blocking reagents (Dako) before saturation with TBS containing 0.1% Tween-20 and 5% BSA (TBST-BSA). Slides were then probed overnight at 4°C with primary antibodies diluted in TBST-BSA. After washes with TBST, arrays were probed with horseradish peroxidase-coupled secondary antibodies (Jackson ImmunoResearch Laboratories, Newmarket, UK) diluted in TBST-BSA for 1 h at RT. To amplify the signal, slides were incubated with Bio-Rad Amplification Reagent for 15 min at RT. The arrays were washed with TBST, probed with Alexa647-Streptavidin (Molecular Probes) diluted in TBST-BSA for 1 h at RT and washed again in TBST. For staining of total protein, arrays were incubated 15 min in 7% acetic acid and 10% methanol, rinsed twice in water, incubated 10 min in Sypro Ruby (Invitrogen) and rinsed again. The processed slides were dried by centrifugation and scanned using a GenePix 4000B microarray scanner (Molecular Devices). Spot intensity was determined with MicroVigene software (VigeneTechInc). All primary antibodies used in RPPA have been previously tested by Western Blotting to assess their specificity for the protein of interest.

RPPA data were normalized using Normacurve (Troncale et al., 2012) followed by a median regression approach (data are scaled by array and median regression is applied on the proteins across all arrays. Residues are set as the final values). Groups of samples were compared using a Student’s T-test.

Pharmacokinetics analysis of Pyr1 and its metabolite M1

6 female CD-1mice (mean weight 30g) were intraperitoneally injected with 10 mg/Kg of Pyr1. At different times (25 min, 57 min, 1h 05 min, 2 h, 4h, 8h) samples of blood were collected alternatively (3 mice per time point) and the plasma concentration of Pyr1 and its metabolite M1 was measured using HPLC. Due to sample processing time and known degradation of Pyr1 into the plasma, Pyr1 concentration may be underestimated whereas M1 concentration may be overestimated.

Hematoxylin and Eosin staining of MDA-MB-231 tumors: MDA-MB-231 tumors were isolated and embedded in OCT tissue freezing medium (Jung) and stored at -20°C before sectioning. OCT embedded tumor sections (6µm) were stained withhematoxylin and eosin.

Mammary imaging window surgery: to intravitally trace the migration of MDA-MB-231 Dendra2 cells over time, a mammary imaging window was inserted (for details see Kedrinet al.(1); Gligorijevicet al. (2); Ritsma et al. (3)). Briefly, approximately one monthaftercell injection, whentumorswere palpable (diameters of approximately 7 mm), micewereanesthetizedusing 2% isoflurane. Under aseptic conditions, an incision was made through the skin overlying the tumor and an imaging window was inserted. The imaging window was secured using a non-absorbable non-woven purse-string suture (4-0 prolene suture).

Tumoral cells migration tracking: three-dimensional tile scans of tumor areas (3 x 3 x 0.1 mm [XYZ] with 5µm Z steps) were acquired every fifteen minutes during 2 hours. Five tile scans were collected for each mouse for 2 weeks. Data were saved using Leica Application Suite AF software. All tile scans were processed using custom software (developed by J. van Rheenen). ImageJ software (NIH, Bethesda, MD, USA) was used to quantify cells migration. Briefly, fifty cells were tracked by following the displacement of cell centroid over time. At least three z-stacks per tile scan were quantified and five tile scans per mice were recorded. Velocity, total displacement (total path length traveled by the cell during the time lapse) and persistence (ratio of the net to total path length traveled by the cell during the time lapse) were calculated. Three mice in the control group and four mice in the treated group were analyzed.

Quantification of Dendra2 fluorescent signal in MDA MB 231 Dendra2 tumors: This quantification was done after at least 8 days of treatment.Dendra2 fluorescent areaswere quantified using the “measurement” tool of ImageJ software (NIH, Bethesda, MD, USA). To that aim, tile scans were converted to 8-bit grey scale images. A percentile threshold was applied to delimit Dendra2 fluorescent areas from the non-fluorescent areas. Five tile scans have been analyzed for each mouse. Three control mice and four Pyr1-treated mice were included in the study.

Quantification of the lung metastasis of MDA-MB-231 Dendra2 tumors: Lungs were isolated and fixed in PLP buffer O/N at 4°C. On the following day, the fixed tissues were washed twice with PLP buffer and placed for at least 6 hours in 30% sucrose at 4°C. The tissues were then embedded in OCT tissue freezing medium (Jung) and stored at -20°C before sectioning. Fluorescent Dendra2 areas from OCT embedded tumor sections (14µm) were quantified using ImageJ software as described for the quantification of Dendra2 fluorescent signal in tumors. A total of six fields were examined and quantified for each tumor in the control and treated groups.

Supplementary References

1. Kedrin D, Gligorijevic B, Wyckoff J, Verkhusha V V, Condeelis J, Segall JE, et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods. 2008;5:1019–21.

2. Gligorijevic B, Kedrin D, Segall JE, Condeelis J, van Rheenen J. Dendra2 photoswitching through the Mammary Imaging Window. J Vis Exp. MyJoVE Corporation; 2009

3. Ritsma L, Steller EJA, Ellenbroek SIJ, Kranenburg O, Borel Rinkes IHM, van Rheenen J. Surgical implantation of an abdominal imaging window for intravital microscopy. Nat Protoc. 2013;8:583–94.