The ability to simultaneously identify, recover, and study cellular subsets within a tumor has remained a challenge for all except the hematopoietic lineages, due in part to the limited tools available for the analyses of adherent cells. The recognition that distinct cellular elements, such as cancer stem cells, within tumors confer biological characteristics has increased the need for the development of platforms to permit studies of rare cellular subsets contained within heterogeneous cell populations.
The micropallet array platform is a platform consisting of micro-scale pedestals, termed micropallets arrayed on a glass slide. Each micropallet holds a single adherent cell in culture and can be selectively recovered with minimal perturbation to the cell permitting isolation, selection, and recovery of single cells. We proposed to utilize this technology to analyze a rare tumor cell subset, the putative cancer stem cell.
We made significant advancements to this technologic platform most notably incorporation of multichannel immunofluorescent confocal imaging to identify specific cellular subsets within complex mixtures, such as would be obtained from a tumor biopsy. As there is no one marker that identifies cancer stem cells, panels of cell surface markers were required. The ability to interrogate the marker expression patterns of six-molecules provided a critical functionality that enabled the identification and enumeration of single cells with defined surface phenotype. We established panels for the analysis of breast, prostate, and pancreatic epithelial tumors to demonstrate the flexibility of the multicolor panel. We provided proof of concept for gene expression analyses using recovered single cells, by both qRT-PCR and Nanostring. These advancements to the platform enabled the analysis of breast tumor specimens.
We optimized 1) the enzymatic dissociation conditions to obtain single cell suspensions, 2) selection of micropallet extracellular matrix coating, and 3) determination of an appropriate incubation time to facilitate tumor cell adherence. Finally, we demonstrated proof of concept for the analysis of breast tumor specimens from which putative single breast cancer stem cells, defined by their surface marker expression profiles were collected off the arrays and profiled for their expression of stem-cell related genes.
