Supplementary Table I: List of primers used for qpcr

Transcription

1 Supplementary Table I: List of primers used for qpcr Primer ABCG2 F ABCG2 R β Actin F β Actin R ALDH1A1 F ALDH1A1 R ALDH1A1 promoter F ALDH1A1 promoter R CD24 F CD24 R CD44 F CD44 R CXCR1 F CXCR1 R CXCR4 F CXCR4 R E Cad F E Cad R EGFR F EGFR R GAPDH F GAPDH R N Cad F N Cad R NOTCH4 F NOTCH4 R OCT4 F OCT4 R Snail1 F Snail1 R Snail2 F Snail2 R TGFB2 F TGFB2 R Twist F Twist R Vimentin F Vimentin R Sequence AGCTCAGATCATTGTCACAGTCGT GAACCCCAGCTCTGTTCTGG CATCCACGAAACTACCTTCAACTCC GAGCCGCCGATCCACAC GGAGTCACTCAAGGCCCTCAG GGCCTCCTCCACATTCCAG TTCTGATTCGGCTCCTGG TTGCTCTGAGTTTGTTCATCC CCCAAATCCAACTAATGCCAC AGAGTAGAGATGCAGAAGAGAG ATCAGATGGACACTCACATGGGAG ATGCCAAGATGATCAGCCATTCT TGGCCGGTGCTTCAGTTAG CATGTCCTCTTCAGTTTCAGCAAT ACGGACAAGTACAGGCTGCAC CCCAGAAGGGAAGCGTGA TACGCCTGGGACTCCACCTA CCAGAAACGGAGGCCTGAT GCTCTACAACCCCACCACGTA ACGCACGAGCCGTGATCT ACCCACTCCTCCACCTTTGAC CATACCAGGAAATGAGCTTGACAA TCAAAGCCTGGAACATATGTGATG GTATACTGTTGCACTTTTTCTCGTACAA GCCCCTCTGGTTTCACAGG AGTTGGCCTTGTCTTTCTGGTC CCTGCACCGTCACCCCT GGCTGAATACCTTCCCAAATAGAAC ATGCCGCGCTCTTTCCT GCTGGAAGGTAAACTCTGGATTAGA GGCTGGCCAAACATAAGCA GCTTCTCCCCCGTGTGAGT CGTCTCAGCAATGGAGAAGAATG CGCTGGGTTGGAGATGTTAAA GCAGGGCCGGAGACCTAG TTTTTAGTTATCCAGCTCCAGAGTCTCTA CTCCGGGAGAAATTGCAGG AGACGTGCCAGAGACGCATT

2 SUPPLEMENTARY FIGURES Supplementary figure 1: CSC subpopulation reaches stable percentage overtime. MCF7 and HCT116 tdtomato + cells were sorted by FACS and cultured during 14 passages and up to 200,000 cells. The percentage of tdtomato + cells was progressively restored to the initial percentage found in MCF7 (A) and HCT116 (B) cell lines before sorting. (C) Once the percentage of tdtomato+ cells (CSC) is restored it remains stable in culture in successive rounds of isolation and culture, reproducing the original cell line. FACS images obtained every 3 passages are shown.

3 Supplementary figure 2: HCT116-ALDH1A1/tdTomato CSC model. (A) Expression of tdtomato in colon HCT116 cancer cells after transfection with the reporter vector ALDH1A1/tdTomato. (B) CSC quantification and sorting by FACS showed positive tdtomato expression in 12.31±0.72 % of cell population. (C) Expression of stemness markers in tdtomato + cells (CSC) by q-pcr showed overexpression of ALDH1, ABCG2, CD44, CXCR4 and PUF5F1 with a respective fold change of 2.61 ± 0.42, 1.47 ± 0.10, 1.33 ± 0.08, 1.66 ± 0.25 and 2.33 ± 0.36 compared to tdtomato- cells (non-csc). At least three biological replicates are reported as mean±sem. Differences were regarded as statistically significant (unpaired Student's t-test) when p-values were smaller than 0.05 (*) and 0.01 (**).

4 Supplementary figure 3: Expression levels of CD44 and EGFR in tumor cell lines. (A) Expression of CD44 by qpcr in various breast cancer cell lines. (B) Expression of EGFR in various colon cancer cell lines. The expression levels of CD44 and EGFR from SKBR3 and MCF7 cell lines were respectively used as reference controls. At the protein level, the expression of CD44 (C) and EGFR (D) was confirmed in CSC sorted from MCF7 and HCT116 cell lines, by immunostaining and flow cytometry.

5 Supplementary figure S4: Storage stability of PTX micelles. (A) The stability of PTX in PLGA-co-PEG micelles at room temperature was controlled overtime. The initial concentration of PTX (day 0) remained constant until day 7 (14.3 ± 0.37μg/mL). At day 21, the amount of PTX was reduced to 12.4 ± 0.15 μg/ml (87% of the initial amount). After 30 days, the total amount of drug dropped to 84% (10.22 ± 0.11 µg/ml) and then remained constant for 60 days. (B) TEM micrographs showed monodisperse micelles with a spherical shape.

6 SUPPORTING INFORMATION Information S1: Cell cultures MDA-MB-231, HCT116 and HCT8 cells were cultured in RPMI medium (Lonza) supplemented with 10% FBS (Lonza), 6mM L-Glutamine (Lonza), 0,1 mm Non Essential Amino acids (NEAA) (Lonza) and and 1% penicillin-streptomycin. MCF7 cells were cultured in DMEM F12 complete medium (Life Technologies) supplemented with a 10% of fetal bovine serum (FBS) (Lonza) and 1% penicillin-streptomycin. Blasticidin (10 µg/ml) (Life Technologies) was used as a selective antibiotic for ALDH1A1/tdTomato cell lines. All cell lines were maintained in atmosphere with 5% of CO2 at 37ºC. MCF7 cells were cultured as mamospheres in serum-free media containing DMEM F12 (Life Technologies), 30% of glucose (Sigma), 20 μg /ml of human recombinant EGF (Sigma-Aldrich), 10 μg/ml human recombinant bfgf (BD Biosciences), 1% penicillin-streptomycin (Life Technologies), 1% L-GlutaMAX (Sigma), 100 mg/ml Heparin (Sigma) and 10% Hormone Mix (Glucosis, Putrescin, Apo-transferrin, Insulin, Selen, Progesterone) in Ultra-Low attachment surface plates (Cultec). Information S2: Fluorescence-Activated Cell Sorting (FACS) In order to sort the tdtomato + CSC, MCF7-ALDH1A1/tdTomato and HCT116- ALDH1A1/tdTomato cells were washed with PBS (Lonza) and trypsinized with 1X trypsin- EDTA (Life Technologies). Then they were resuspended in PBS +10%FBS, and DAPI was added to marked non-viable cells. First, cell debris and doublets were removed by forward and side scatter gating and just alive cells (DAPI negative cells) were admitted for sorting. TdTomato

7 fluorescence was detected through the CYG-A channel and sorted using the cytometer FACSAria (BD Biosciences). Information S3: Flow cytometry The amount of tdtomato + cells in continuous culture was assessed by flow cytometry using Fortessa instrument (BD Biosciences). First, cell debris and doublets were removed by forward and side scatter gating and just alive cells (DAPI negative cells) were evaluated. tdtomato fluorescence was detected through the EYG-A channel. For each sample, at least 10,000 individual cells were collected and the mean fluorescence intensity was evaluated. To assess the therapeutic effect of naked PTX or PTX loaded in micelles on CSC, cells were seeded in different quantity for 24 h to allow adhesion. For control samples, treated with vehicle or empty nanoparticle, cells were seeded and for samples treated with PTX and targeted / non-targeted PLGA-co-PEG nanoparicles cells were seeded. Cells were then treated for 72 h and let to recover in complete medium for 48 h subsequently. Cells were trypsinised and tdtomato fluorescence was detected by Fortessa (BD Biosciences) as described above. To ensure the therapeutic effect of the treatments, the cell proliferation and cell viability was controlled during all the experiment. Proliferation and viability of cells was assessed by Trypan Blue exclusion and cell counting with the automated cell counter (Countess). At least three biological replicates, each involving at least two technical replicates were involved in final results expressed as the mean±sem. Statistical analysis was performed using the unpaired Student's t-test. Differences were regarded as statistically significant when p-value were smaller than 0.05.

8 For internalization study, cells were seeded in complete medium in for 24 h to allow adhesion. 500 μg/ml micelles were added to samples at different time points: 3, 10, 30, 60, 120 min or over-night (O/N) depending on the experiment. Then, cells trypsinized and resuspended in PBS supplemented with 10% FBS and analyzed by FACSCalibur system (BD Biosciences). For each sample, at least 10,000 individual cells were collected and the mean fluorescence intensity was evaluated. Information S4: Real time quantitative PCR Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen) and obtained RNA was reverse transcribed with High Capacity cdna Reverse Transcripton Kit (Applied Biosystems) according to the manufacturer's instructions. The cdna was amplified with specific primers (Table S1) by qpcr using SYBR Green on a 7500 Real Time PCR System (Applied Biosystems). Transcriptional quantification relative to both, Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and actin housekeeping genes (Table S1) was performed using Qbase software, based on the ΔΔCt method, calculating relative normalized quantities (NRQ) of mrna expression (26). At least three biological replicates, each involving at least two technical replicates were involved in final results expressed as the mean±sem. Statistical analysis was performed using the unpaired Student's t-test. Differences were regarded as statistically significant when p-value were smaller than 0.05 Information S5: Mammospheres forming assay Cells were seeded in 6-well ultra-low attachment plates at low density (10,000 cells/well) in serum free medium and were treated with the naked PTX or targeted/no-targeted PLGA-co-PEG

9 micelles. After 72 h of incubation the medium was replaced by new drug- free medium and mammospheres were formed within additional 5 days. When they were 60 nm of size, mammospheres were transferred into complete medium with FBS in conventional cell dish. After attached colonies visible to the unarmed eye were formed, cells were fixed with methanol: acid acetic (3:1) and stained with 0.1% crystal violet. At least three biological replicates, each involving at least two technical replicates were involved in final results expressed as the mean±sem. Statistical analysis was performed using the unpaired Student's t-test. Differences were regarded as statistically significant when p-value were smaller than 0.05 Information S6: In vivo tumorigenic capacity assay Female NOD.CB17-Prkdc scid/j mice (Charles River, Barcelona, Spain) were kept in pathogenfree conditions and used at 6 weeks of age. Animal care was handled in accordance with the Guide for the Care and Use of Laboratory Animals of the Vall Hebron University Hospital Animal Facility, and the experimental procedures were approved by the Animal Experimentation Ethical Committee at the institution. MCF7.Fluc2-ALDH1A1/tdTomato + (CSC) and MCF7.Fluc2-ALDH1A1/tdTomato - (Non-CSC) at 10,000, 1,000 and 100 cells in 50 µl sterile PBS:Matrigel (1:1) were inoculated orthotopically into the right mammary fad pad (i.m.f.p.). Tumor growth was monitored twice a week by conventional caliper measurements (D d 2 /2, where D is the major diameter and d the minor diameter). At the end of the experiment, animals were euthanized by cervical dislocation and subjected to gross necropsy. Tumors were collected and analyzed by ex vivo bioluminescence imaging (BLI) to determine tumor bioluminescence intensities. Ex vivo BLI was performed using the IVIS Spectrum Imaging System, and images and measurements of bioluminescent signal

10 were acquired and analyzed using Living Image software (PerkinElmer Life Science, Inc., Boston, MA, USA). All in vivo experiments were performed at the Molecular Imaging Platform (PIM) of the CIBER- BBN In Vivo Experimental Platform located at Vall d Hebron Research Institute (VHIR) (Barcelona, Spain) Information 7: Confocal microscopy Cells were cultured in glass slides coated with gelatin in for 24 h to allow adhesion. Then, cells were incubated 2 h with nanoparticles (500 μg/ml) and lysosomes were stained with Lysotracker red DND-99 (Life Technologies). Cells were then fixed in 4% paraformaldehyde followed by DAPI nuclei staining. Cells were viewed under a Spectral Confocal Microscope MFV1000 (Olympus). Minimal single optical sections were collected for each fluorochrome sequentially and analyzed with the FV10-ASW 3.1 Viewer software (Olympus). Information 8: MTT cell viability assays 5000 cells/well were seeded in 96-well in microtiter plate and after they attached, they were incubated in the presence or absence of increasing concentrations of unloaded nanoconjugates, drug-loaded nanoconjugates, standard solution of PTX and vehicle control for 72 h. After the incubation, 0.5 mg/ml 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to each well. Plates were incubated for an additional 4 h at 37ºC and 180 μl of Dimethyl sulfoxide (DMSO) (Sigma) was added to each well. The absorbance at 580 nm of each well was read on a microplate reader. Cell viability was calculated a minimum of 3 biological replicates with 6 technical replicates for each assay. Final results are expressed as the

11 mean±sem. Statistical analysis was performed using the unpaired Student's t-test. Differences were regarded as statistically significant when p-value were smaller than 0.05.