Figure 6
From: The isolation and characterization of CTC subsets related to breast cancer dormancy
In vitro characterization of 3D CTC tumorspheres.
(a) Generation of CTC tumorspheres over time in culture. Tumorsphere assays were performed in FACS sorted (CD45−/CD44+/CD24−/EpCAM-negative/uPAR+/−/int β1+/−) in vitro 3D CTC subsets derived from no BCBM patient. Trypsinized 10-15 3D CTC tumorspheres were cultured in 96-well plate coated with 1% soft agar and quantified at successive weeks under phase contrast microscopy (Zeiss, Inc.); (b) CTCs cell proliferation assays (WST-1, Roche Life Sciences, Inc.) over time in culture were performed in FACS-sorted in vitro 3D CTC subsets containing uPAR/int β1 combinatorial expression. Trypsinized 10-15 3D CTC tumorspheres were cultured in 96-well plate coated with 1% soft agar. Absorbance was measured at 450 nm and 690 nm wavelength at 8 hrs after adding WST-1 reagent at different time points. All data are representative of at least three independent experiments with mean standard deviation (±). Student paired type 2 t-test was performed and p-value* (<0.01) were calculated and found to be significant; (c) CTC adhesion assays. Four CTC subsets with combinatorial expression of uPAR and int β1 were aliquoted into 96 well flat-bottom plates coated with Trevigen® PathClear Basement Membrane Extract® (BME) and incubated for 96 hours at 37 °C for adhesion assay.
