Figure 1.
Morphologic and immunohistochemical analysis of tumourspheres cultured from a panel of basal-like/claudin-low and luminal-like breast cancer cell lines.
(A) Light microscope images of cell lines grown in adherent and sphere-promoting suspension conditions 7 d after seeding (images taken at 40× magnification, insets 100x magnification). H&E analysis of sections of formalin-fixed, paraffin embedded spheres are also shown. Arrows indicate wide lumina within MCF7 spheres and cleft-like lumina within KPL-1 spheres. Scale bars 100 µm. (B) Average sphere diameter measured by digital image measurement of the long axis of spheres. Data represent median diameter of spheres ± SD measured in 10 random fields, representative of two biological replicates. (C) Light microscope images of structures formed by MCF7 and SUM-159-PT cells grown in matrigel. Images taken at 100x magnification, scale bar 100 µm. (D) Representative images from immunohistochemical analysis of the indicated antigens on FFPE preparations of spheres from basal-like (i, iii, ix, xv, xvii: SUM-159-PT; v, vii, xi, xiii: HBL100) and luminal-like cell lines (ii, vi, xii, xiv: MCF7; iv, viii, xvi, xviii: KPL-1; x: BT-474). Complete data set available in Figure S1. Images taken at 200x magnification (black triangle, 400x). Scale bar 100 µm. Black arrows indicate intermittent laminin1/2.
Table 1.
IHC methods.
Figure 2.
Multiparametric flow cytometry analysis of mammary epithelial and stem cell markers in matched adherent and sphere cultures of breast cancer cell lines.
(A) Experiment design. Matched adherent and sphere cultures of luminal-like and basal-like/claudin-low cell lines were generated in triplicate from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates. Panel 1: MUC1-FITC, HER2-PE and the LIVE/DEAD® red cell viability stain; Panel 2: CD49f-Pacific Blue, Aldefluor® reagent (for ALDH1 activity), CD24-PE, LIVE/DEAD® red, AC133-APC, EpCAM-PerCP Cy5.5 and CD44-APC-Cy7. (B) Gating strategy for simultaneous detection of cell surface markers. Acellular particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity (live cells designated ‘LDR live’). For each experiment, gates were placed based on autofluorescence of unstained adherent or sphere control samples to account for differences in the autofluorescence of cells grown in the different formats. For Aldefluor® (ALDH1 activity), gates were placed based on the fluorescence of the DEAB negative control (refer to supp methods). Population frequencies were determined for individual parameters (MUC1+, HER2+, AC133+, EpCAM+, ALDH1+, CD44+, CD24+, CD49f+). For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (see Fig. 3).
Figure 3.
Differences in the frequencies of functional mammary epithelial subpopulations in parallel sphere and adherent cultures of breast cancer cell lines assessed by multiparametric flow cytometry analysis.
Adherent and sphere cultures were dissociated, stained with fluorescent antibody conjugates and analysed as described in Fig. 2. Heat maps show changes in subpopulation frequencies within the live cell population. Red shades = higher frequency in spheres compared to matched adherent cultures; blue shades = lower frequency in spheres; empty = not consistent and/or not statistically significant across biological replicates; diagonal line = not determined. Statistical significance was determined using two-way ANOVA and 2-tailed, paired t-tests. Results are depicted only where the trend was directionally consistent and statistically significant over biological replicates (refer to methods). Significance levels are colour-coded: light pink/blue: P = 0.05–0.001; mid-pink/blue: P = 0.001–0.0001; dark red/blue: P<0.0001). Dotted boxes indicate changes of interest: significant enrichment with AC133+ cells in 3/6 cell lines (A), and frequent shifts in CD49f+/CD24+ phenotypes with sphere culture (B).
Figure 4.
In vitro functional analysis of progenitor cell content in breast cancer cell line tumourspheres.
(A) Sphere forming efficiency (SFE) of cell lines through serial sphere passage.–14 replicates per dilution, 4 dilutions/cell line/passage), expressed as a percentage of the original number of cells seeded, then averaged across the dilutions. Bars represent the mean SFE ± SD from 2–3 independent experiments. Two-tailed, unpaired t-tests demonstrated no significant difference in SFE between passage 1 and 5 for any cell line (ns). (B) Correlation between stem cell symmetric division rates of matched adherent and sphere cultures. Ten breast cancer cell lines (MCF7, KPL-1, BT-474, SKBR-3, T47D, SUM-159-PT, MDA-MB-436, HBL100, Hs578T and BT-20) were seeded in triplicate at equal densities in adherent or sphere-promoting conditions, and the total number of cells was calculated every 5–7 d for 2–8 passages. Mean fold expansion and the rates of long-term proliferating cell symmetric division (Kll) were calculated for each culture [30] and plotted for correlation analysis (solid line, linear correlation; dotted lines 95% confidence interval). The relationship between adherent and sphere KII rates was statistically significant (linear correlation analysis (post-test for linear trend); P = 0.0016). (C) Primary clonogenicity of MCF7 and SUM-159-PT cells in different growth formats. Clonogenicity in adherent growth conditions, matrigel overlay and sphere-promoting conditions was calculated as the number of colonies (adherent or non-adherent) that grew after 7 d, as a percentage of cells seeded. Data shown are means ± SE, representative of at least three separate experiments performed in triplicate. (D) Secondary clonogenicity of MCF7 and SUM-159-PT cells derived from spheres and matrigel structures. Clonogenicity was calculated after dissociating and reseeding cells derived from spheres and matrigel structures into adherent conditions at limiting dilution. Data are means ± SE, representative of one of three separate experiments performed in triplicate. P values in C and D were generated using two-tailed, unpaired t-tests.
Figure 5.
Mammospheres derived from MUC1+ or CD10+ progenitors from reduction mammoplasties comprise hollow and solid structures consistent with luminal- and myoepithelial-like morphologies.
(A) Strategy for isolating luminal and myoepithelial progenitor-enriched subpopulations from reduction mammoplasty tissue. Tissues were physically and enzymatically processed to epithelial-rich, single cell suspensions, then cultured for 7d in mammary epithelial growth medium to generate enough cells for fluorescence-activated cell sorting (FACS) and sphere culture. Primary cultures were stained with fluorescent antibody conjugates (MUC1-FITC, CD10-PE-Cy5), the Sytox® Blue viability stain and ‘Lineage’ cocktail (‘Lin’: CD140b-PE, CD45-PE, CD31-PE; markers of stromal fibroblasts, leukocytes and endothelia respectively). CD10 and MUC1-sorted cells were placed in sphere-promoting culture for 10d before analysis. (B) Gating strategy for enrichment of luminal- and myoepithelial-like progenitor cells. Acellular particles and dead cells were excluded based on low light scatter and Sytox® blue positivity. Non-epithelial, PE+ cells were also excluded, then MUC1+ and CD10+ cells were collected for sphere culture. (C) Light microscope images (i,iii) and H&E histological sections (ii,iv–vii) of spheres generated from CD10+ or MUC1+ primary breast epithelial cells. Open arrows, MUC1+-derived spheres often had limited lateral connections giving a petal-like appearance. Solid arrows, shows single cell with signet ring secretory morphology. (D) Immunophenotypic analysis of MUC1+ and CD10+ progenitor-derived primary breast mammospheres. Spheres were generated as described in A/B. Representative images from immunohistochemical analysis of the indicated antigens on FFPE preparations of spheres are shown. Images taken at 200× magnification. Scale bar 100 µm. Black arrows indicate apical membranous staining of E-cadherin and EGFR in luminal-like spheres. Red arrows indicate the mesenchymal-like spheres found rarely amongst the dominant structures formed in both CD10+ and MUC1+ sorted populations.
Figure 6.
Relative frequencies of luminal and myoepithelial/bipotent human mammary epithelial cells in MUC1+-derived mammosphere cultures.
MUC1+ sphere cultures were prepared as described in Fig. 5A, in parallel with matched MUC1+ adherent cultures, then dissociated and used in a colony forming cell (CFC) assay to determine the clonogenicity of luminal and myoepithelial/bipotent progenitor compartments (which give rise to colonies with luminal and mixed/myo morphologies respectively). (A) Light micrographs of Geimsa-stained colonies grown from dissociated spheres and parallel adherent cultures. Representative colonies are shown for adherent- (i-iii) and sphere-derived (iv-vi) cells. This includes examples of luminal (i, iv and v), mixed (ii, iii) and myoepithelial (vi) colony morphologies. (B) Overall clonogenicity (colonies formed as a percentage of total cells seeded) of spheres and parallel adherent cultures. (C) Comparison of luminal and myo/bipotent progenitor cell clonogenicity of spheres and parallel adherent cultures. Data are from four biological replicates (specimens from 4 patients), each performed in triplicate. Statistical tests used were paired, two-tailed students t-tests (P values indicated; ns = not significant).