Cell culture and flow experiments
Human Mammary Epithelial Cells (HMECs) and BT-474 cells were respectively obtained from Lonza (Walkersville MD, USA) and ATCC (American Type Culture Collection, Manassas VA, USA) and cultured per recommendations. MCF-7 and SK-BR-3 cells were provided by Dr. Carrie Shemanko (Department of Biological Sciences, University of Calgary) while MDA-MB-231 cells were provided by Dr. Don Morris (Tom Baker Cancer Center, University of Calgary). MCF-7, SK-BR-3, and MDA-MB-231 cells were grown in Dulbecco's Modified Eagle's Medium (Life Technologies Inc., ON, Canada) supplemented with 10% fetal bovine serum, 1% L-Glutamine and 1% Penicillin Streptomycin. MCF-7, BT-474, MDA-MB-231, and SK-BR-3 cell lines are representative cell lines of luminal A, luminal B, basal, and HER2-enriched subtypes of breast cancer, respectively45. Disease-relevant differences between breast cancer subtypes have been described elsewhere36,37,38.
For bioreactor culture, cell monolayers were cultured on glass plates pre-coated with 145 µg/mL Rat Tail collagen I (Life Technologies Inc., ON, Canada) and grown to confluence. Some slides were exposed to fluid flow by setting up the parallel-plate flow chamber as previously described15, with the addition of pulse dampeners to create steady flow. Some glass plates were grown as static controls, and their culture media was replaced at the same time as flow was set up. The magnitude of shear stress on cell monolayers was calculated using the Navier-Stokes equation for a Newtonian fluid in parallel plate geometry as previously reported15. Cells were exposed to an average shear stress of 1 Pa for 20 hours, and flow was provided by a Masterflex peristaltic pump and tubing (Cole Parmer, Montreal, QC, Canada). For each cell line, a minimum of three independent experiments were conducted for each condition.
Cells from flow experiments and static cultures were harvested as previously described15. Total RNA was isolated using the EZNA Total RNA Kit (Omega Bio-Tek Inc., Norcross GA, USA) as per the manufacturer’s instructions. Hydrated RNA samples were stored at – 80oC until analysis. RNA was assayed using the Quant-iT™ RiboGreen RNA Assay Kit (Life Technologies Inc., Burlington ON, Canada) and standard curves generated using a microplate reader.
Real-time quantitative PCR: 1 µg of RNA was converted to complementary DNA (cDNA) using the RT2 First Strand Kit (SA Biosciences, Mississauga ON, Canada) according to the manufacturer’s protocol. cDNA for each sample was loaded onto the RT2 Profiler PCR Arrays for Human Epithelial-to-Mesenchymal Transition (SA Biosciences, Mississauga ON, Canada) along with SYBR Green ROX qPCR Mastermix (SA Biosciences, Mississauga ON, Canada). Quantitative PCR was carried out on a ViiA 7 Real Time PCR System (Life Technologies, Foster City CA, USA) under the following conditions: 95 oC for 10 min and 40 cycles of 15 s at 95 oC and 60 s at 60 oC. Actin-beta (ACB), beta-2-microglobulin (B2M), and glutaraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes. Relative gene expression was calculated using the comparative cycle threshold method39. Three independent replicates were run for each condition. Statistical significance for differential expression of genes between conditions was determined using the Student’s t-test.
For individual gene expression quantification by PCR, cDNA was synthesized using the qScript™ cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg MD, USA) according to manufacturer’s protocol. A total reaction volume of 20 µl was then prepared by adding 2 µl cDNA, 10 µl TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, Ottawa, ON, Canada), and 8 µl mixture of primers, probes, and nuclease-free water. Quantitative PCR was carried out on a ViiA 7 Real Time PCR System (Life Technologies, Foster City CA, USA). PCR experiments were run in triplicates and B2M was used as the reference gene. The following cycling conditions were employed for target gene amplification: 50 oC for 2 min, then 95 oC for 20 s, followed by 40 cycles of 1 s at 95 oC and 20 s at 60 oC. Three independent replicates were run for each condition and relative gene expression was calculated using the comparative cycle threshold method39. Statistical significance for differential expression of genes between conditions was determined using the Student’s t-test.
EMT was induced by adding StemXVivo EMT Inducing Media Supplement (R&D Systems, Minneapolis, MN, USA) and monitored per manufacturer’s recommendations40. Briefly, cells were gently detached from culture dishes using a dissociation solution, centrifuged, and re-suspended in warmed culture media containing StemXVivo EMT Inducing Media Supplement. Cell cultures were incubated at 37 oC and 5% CO2, and media changed every two days. EMT induction was completed five days after plating.
Protein expression analysis by immunocytochemistry was performed using the Human EMT 3-Color Immunocytochemistry kit (R&D Systems Inc., Minneapolis MN, USA). Briefly, cells were fixed in 4% paraformaldehyde, incubated in a blocking buffer made up of 1% bovine serum albumin (BSA), 10% rabbit serum, and 0.3% Triton X-100 in 1X PBS. Confluent areas on slides were marked using PAP pens. Samples were further incubated in blocking buffer containing conjugated antibodies to human Snail, E-Cadherin, and Vimentin, diluted according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst dye (Life Technologies Inc., Burlington ON, Canada) and mounted with Prolong Gold Antifade Reagent (Life Technologies Inc., Burlington ON, Canada). Slides were allowed to dry in the dark overnight, and the samples were imaged using an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus Inc., Waltham, MA, USA). A minimum of three independent experiments were performed for each condition. Normalized fluorescence intensity was calculated using ImageJ (v1.48, U.S. National Institute of Health) as previously described41.
GeneChip PrimeView Human Gene Expression Microarrays (Affymetrix, Santa Clara CA, USA) were used to quantify changes in gene expression. Signal intensity files were pre-processed using robust multichip average normalization42. Microarray data was analyzed using Biometric Research Branch – ArrayTools Version 4.5.0 (National Cancer Institute, USA)43 and Partek Genomics Suite Version 6.6 (Partek Incorporated, Missouri, USA). For each cell line, microarrays were run for 3 independent replicates of static and flow-exposed cells and differentially expressed genes (fold change ≥ 1.5 and p-value ≤ 0.01) were identified by analyzing class comparisons between static and flow samples. Similarly, gene set enrichment analyses were performed against gene sets from the Molecular Signature Database (Broad Institute, USA), with at least 5 differentially expressed genes in a particular Gene Ontology category at a p-value ≤ 0.05 considered statistically significant44.
Interaction network analysis
The Cytoscape (version 3.0) software platform was used to visualize gene interaction networks45,46. Differentially expressed genes between static and flow-exposed breast cancer cells were mapped onto a human interactome network obtained from integrated complex traits networks (iCTNet), Version 1.046. The BiNGO and MCODE plugins were used to assess enrichment of nodes for biological processes recorded in the Gene Ontology database47,48.
Clinical patient data analysis: Level 3 mRNA expression data from The Cancer Genome Atlas (TCGA) was obtained from Synapse (http://synapse.org:syn1461151)49. Gene expression data was measured using the Illumina HiSeq 2000 RNA Sequencing version 2 analysis platform (RNA-Seq by Expectation Maximization, RSEM). This dataset contains whole-genome expression data for 104 healthy volunteers, 317 luminal A, 93 luminal B, 26 HER2, and 81 basal breast cancer patients. Breast cancer subtype classification was based on immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal receptor 2 (HER2)50,51. Gene expression was compared between subtypes using box plots showing the median and interquartile ranges. Prognostic value of genes differentially expressed upon flow stimulation was evaluated using Kaplan–Meier curves (http://kmplot.com/analysis/) to compare relapse-free survival times in a data set containing 2,878 breast cancer patients52. Statistical significance was determined by the log-rank test.
A Human Umbilical Vein Endothelial Cell (HUVEC) monolayer was obtained by seeding 250,000 cells per well in gelatin-coated 6-well culture dishes, grown for two days and activated with 150 ng/mL of TNF-α for 5 hours. HUVECs were obtained from Lonza (Walkersville MD, USA) and cultured per recommendations. MDA-MB-231 and MCF-7 cells were cultured, and flow preconditioned as described above. Flow-stimulated and unstimulated cells were non-enzymatically detached from glass plates, re-suspended in serum free media, and added to the HUVEC monolayers at a density of 200,000 cells per well. Cancer cells were left to adhere on the endothelial monolayer for 90 minutes, after which non-adherent cells were carefully washed twice with HBSS and cells were fixed with 4% PFA for 10 minutes. Adherent breast cancer cells from four independent wells (five fields per well) were counted and used to quantify relative adhesion as the ratio of adherent cancer cells per endothelial cells present in a field.
Adhesion experiments under flow
A HUVEC monolayer was cultured on glass slides by seeding 200,000 cells per slide and grown for two days. Slides were pre-coated with 145 µg/mL Rat Tail collagen I (Life Technologies Inc., ON, Canada). Following activation with 150 ng/mL of TNF-α for 5 hours, slides were embedded in a parallel-plate flow chamber as previously described15. Flow-stimulated and unstimulated cells (200,000cells/ml) were suspended in serum free media, loaded onto a syringe, and mounted on an infusion pump (Harvard Bioscience, Holliston, MA, USA) connected to the endothelial monolayer. Cancer cells were pumped over the endothelial monolayer at a shear rate of 16 sec− 1 for 10 min. Each experiment was replicated four times. Pictures were taken at several time points and several locations on the slides and used to quantify relative adhesion as the ratio of adherent cancer cells per endothelial cells present in a field.