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Supplementary Materials and Methods
Functional fractionation of cancer cells by invasive assays
Through four serial passages (p4), human lung cancer A549 cell-derived spheres were
transferred back to adhesive tissue culture plates, after which they migrated back onto plates and
reformed a monolayer with morphological heterogeneity [and were then collected as lowmotility (LM) cells] (1). To establish an EMT/metastasis cell model, LM cells were seeded onto
Matrigel-coated Boyden chamber membranes. After a 24-h incubation period, the cells that had
invaded the Matrigel were collected as high-motility1 (HM1) cells, signifying one passage
through the basement-membrane matrix. Subsequently, these cells were regrown and passed 19
more times through the invasion-selection procedure. The cells harvested from each subsequent
round of selection were designated HM2 to HM20. A graphic depicting the approach to derive
invasive subpopulations is provided in Supplementary Fig. S1A
Supplementary Results
Functional fractionation of cancer cells by invasive assays
Supplementary Fig. S1B shows the morphologic changes in cells after invasion selection, as
seen under a phase-contrast microscope. The LM cells had a typical epithelial-like morphology,
i.e. flat with a multiangular shape in a monolayer culture. By the 20th invasion selection,
however, the cells were fibroblast-like and more rounded in culture compared with LM cells
(Supplementary Fig. S1B; HM20). As anticipated, the resulting HM20 cells acquired a
mesenchymal appearance, and they exhibited downregulated expression of epithelial marker
transcripts (Supplementary Fig. S1C) and proteins (Supplementary Fig. S1D) (such as E-
cadherin, P-cadherin (P-cad), and -catenin), and upregulated expression of mesenchymal
marker transcripts (Supplementary Fig. S1C) and proteins (Supplementary Fig. S1D) (such as
Vimentin and fibronectin). Furthermore, the expression levels of these EMT-related transcription
factors in HM20 cells resembled the levels seen in cells that have undergone EMT
(Supplementary Fig. S1C and D). To confirm protein expression, the selected cells were
immunostained for E-cadherin, -catenin, and Vimentin. We found that, after serial invasion
selection, E-cadherin/-catenin expression was strongly reduced and Vimentin expression was
substantially increased (Supplementary Fig. S1E and F).
Reference
1.
Su YJ, Lai HM, Chang YW, Chen GY, Lee JL. Direct reprogramming of stem cell
properties in colon cancer cells by CD44. EMBO J 2011;30:3186-99.