In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity
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.