Heterogeneous Cell Types in Single-cell-derived Clones of MCF7 and MDA-MB-231 Cells

Variations in cell phenotype in a single-cell-derived clone may result from asymmetric cell divisions that lead to different cell fate in a homogenous microenvironment. The aim of this study was to demonstrate the extent of cell variety in single-cell-derived clones and propose a different strategy in treating cancer by observed phenotypic heterogeneity in cellular types. Additionally, the role of metabolic enzyme and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in cellular phenotype was evaluated in two breast cancer cell lines.

Two GAPDH-overexpressing breast cancer cell lines, MCF7-GAPDH-RFP (MCF7-RFP) and MDA-MB-231-GAPDH-RFP (MDA-RFP), were established. Microscopic recordings were made at 12-h intervals in single-cell-derived clones of both cell lines up to 8-10 days. Crystal violet and Hoechst 33342 (Hoechst), as well as specific cell-type (epithelial and mesenchymal) antibodies, were used for cytochemical and immunohistochemical staining analyses, respectively, at 3, 6 and 9 days during cell growth.

Three types of clones with distinct morphologies were identified as holo-, mero- and paraclones. The rates of colony survival during cell growth were 8.3 and 41.7% in MCF7-RFP and MDA-RFP, respectively. Although no significant difference was found in the colony forming efficiency (CFE) of both MCF7 and MDA-MD-231 wild-type cells, a markedly significant difference was seen in the CFE of MCF7-RFP and MDA-RFP cells (p=0.001). Wild-type cell-derived holoclones of both cell lines showed drug resistance to doxorubicin (Dox). However, levels of proliferating cell nuclear antigen (PCNA) and vimentin (VIM) marginally decreased in Dox-treated clones. Furthermore, high level of intraclonal heterogeneity was found for CD44, CD140a, VIM, fibronectin (FN), focal adhesion kinase (FAK), paxillin (PXN) and vinculin (VCL) in MCF7 and MDA clones during different stages of clonal development. Expressions of CD140a, FN, VIM and FAK were induced in GAPDH-red fluorescent protein (RFP)-tagged clones of both cell lines.

The GAPDH-RFP recombinant protein played an important role in morphological heterogeneity detection in early stages of clonal development. Moreover, phenotypic heterogeneity in clones, caused by the cells expressing specific antigens, such as CD44, CD140a, FN, VIM, FAK and VCL, can be the right target for therapeutic drugs.