Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.
Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.
Department of Chemistry, University of North Carolina at Chapel Hill , Kenan and Caudill Laboratories, 125 South Road, Chapel Hill, North Carolina 27599-3290, United States.
Andrew S
AS
Truong
Department of Chemistry, University of North Carolina at Chapel Hill , Kenan and Caudill Laboratories, 125 South Road, Chapel Hill, North Carolina 27599-3290, United States.
Christian A
CA
Lochbaum
Department of Chemistry, University of North Carolina at Chapel Hill , Kenan and Caudill Laboratories, 125 South Road, Chapel Hill, North Carolina 27599-3290, United States.
Matthew W
MW
Boyce
Department of Chemistry, University of North Carolina at Chapel Hill , Kenan and Caudill Laboratories, 125 South Road, Chapel Hill, North Carolina 27599-3290, United States.|Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , 450 West Drive, Chapel Hill, North Carolina 27599-7295, United States.
Matthew R
MR
Lockett
D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D006801:Humans; D010209:Paper; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm