Fig 2: The cBCSC and CAIX populations are inhibited following treatment with U-104 under suspension. (A) MB-231 and (B) MCF7 cells were cultured in a monolayer (D0) or suspension condition for 3 or 7 days and then treated with 0, 10, 50, or 100 µM of U-104 for 24 h. Treated cells were then analyzed for CD44, CD24, and CAIX surface expression using MIFC. The CAIX+ and the CD44+CD24− decreased in a dose-dependent manner with U-104 treatment. (n = 3, * p < 0.05, ** p < 0.01).

Fig 3: Identification of a panel of breast cancer cell lines with emergent anoikis-resistant cancer stem cell populations. (A) A panel of seven breast cancer anoikis-resistant cell lines was evaluated for cancer stem cell markers CD44, CD24, and Sox2 following culturing in either a monolayer (day 0) or non-adherent suspension condition for up to seven days using immunoblotting. Total protein expression for CD44 and Sox2 was increased over the seven days of culturing relative to the monolayer condition. CD24 was decreased in the MCF7 and T47D cell lines, and low expression was maintained in the MB-231 cell line. Immunoblot images are representative of 3 biological replicates. (B) The surface expression of CD44 and CD24 was analyzed using multispectral imaging flow cytometry (MIFC), and the CD44+CD24− population was quantified from the bivariate graph (C). The data shown are the percentages of CD44+CD24− cells out of the total population. Bivariate charts were gated on the isotype controls for each marker. (n = 3, * p < 0.05 relative to the monolayer culture (D0)). (D) MIFC images were taken of individual cells at each time point, and representative images for MB-231, MCF7, and T47D cells at days 0 (D0) and 7 (D7) of suspension culturing are shown. Assigned cell number of population is shown on the brightfield images.