Real-time imaging of cancer cell chemotaxis in paper-based scaffolds

Abstract

Cellular migration is the movement of cells, cultured as a monolayer; cellular invasion is similar to migration, but requires the cells to move through a three-dimensional material such as basement membrane extract or a synthetic hydrogel. Migration assays, such as the transwell assay, are widely used to study cellular movement because they are amenable to high-throughput screens with minimal experimental setup. These assays offer limited information about cellular responses to gradients in vivo because they oversimplify the threedimensional (3D) environment of a tissue. There are a number of invasion assays that support 3D cultures, some of which provide experimental control over the spatial and temporal gradients imparted on the culture. These assays, in their current form, are difficult to setup and maintain, and often require specialized laboratory equipment or engineering expertise. Here we describe a paper-based invasion assay in which cellular movement can be monitored in real-time with fluorescence microscopy. These assays are easily prepared and utilize materials commonly found in any laboratory: a single sheet of paper. These sheets are wax patterned to contain channels in which cells suspended in a hydrogel are seeded and cultured. Cell-containing sheets of paper are placed in a custom-built holder that allows gradients to form along the length of the channels. In this work, we compare the invasion of cells cultured in the presence and absence of an oxygen gradient. Our result support previous findings that oxygen is a chemoattractant, and selectively directs cellular movement in a 3D culture environment.

Assessing chemotherapeutic effectiveness using a paper-based tumor model

“…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 …”

Engineered Paper‐Based Cell Culture Platforms

“…As demonstrated in the last two studies, paper scaffolds hold great promise in modulating chemotaxis that is induced by oxygen gradients. Using paper scaffolds, Kenney et al studied the invasion of MDA‐MB‐231 cells with and without an oxygen gradient . In hypoxic regions, oxygen supply did not meet the metabolic demand creating a gradient directing the movement of invasive cells.…”

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.…”