Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR

Abstract

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.

Assessing chemotherapeutic effectiveness using a paper-based tumor model

“…21,30 Sheets of Whatman 105 lens paper were wax-patterned with a ColorQube 8570 printer (Xerox) and sterilized with ultra-violet (UV) light overnight prior to usage. Two paper designs were used in this study: i) a “9-zone scaffold,” which contained nine circular zones (2.5 μm diameter) for seeding with cell-free or cell-laden Matrigel, and ii) a “single-zone scaffold”, which contained one circular zone (2.5 mm diameter) for seeding.…”

“…21,30 Two PET designs were used for this study: i) an “open” design, which contained nine openings matching the locations of the zones in the 9-zone scaffold; and ii) a “closed” design, which prevented the exchange with medium by containing no openings. 30 …”

“…Cell-containing sheets have been analyzed with a number of different readouts, including: quantitative polymerase chain reaction (qPCR), fluorescence microscopy, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. 21–24 Due to the similarities between chemical environments in PBCs and avascular tissue, this platform has been used to investigate the invasion of breast and lung carcinomas, 21,23,25,26 cardiac ischemia, 27 and cancer metabolism. 24,28,29 …”

Paper‐based Invasion Assays for Quantifying Cellular Movement in Three‐dimensional Tissue‐like Structures

“…The holders are then placed in a 6‐well plate and incubated for a given amount of time before the individual layers of the invasion stack are physically separated and analyzed. The number of cells that invade neighboring scaffolds can be quantified by fluorescence imaging techniques, quantitative PCR, confocal microscopy, western blotting, or ELISA (Kenney et al., ; Truong et al., ; Truong & Lockett, ). These measures allow examination of invasion in terms of cell number, directionality, extent of invasion, and distance traveled.…”

“…Soluble factors (e.g., hormones, peptides, proteins, growth factors) are commonly associated with this directed cellular movement, through processes such as autocrine, paracrine, and juxtacrine signaling (Bhat & Bissell, 2014;Singh & Harris, 2005;Roussos, Condeelis, & Patsialou, 2011). Oxygen has recently been shown to be a potential chemoattractant (Mosadegh et al, 2015;Truong, Lochbaum, Boyce, & Lockett, 2015;Kenney, Boyce, Truong, Bagnell, & Lockett, 2016). Oxygen gradients are common in poorly vascularized tissues and in solid tumors.…”