A paper-based invasion assay: Assessing chemotaxis of cancer cells in gradients of oxygen

Abstract

This work describes a 3D, paper-based assay that can isolate subpopulations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O 2 ). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. Stacking the layers of paper assembles them into 3D tissue-like constructs of defined thickness and composition. The plastic holder ensures the layers of paper are in conformal contact; this geometry allows the cells to migrate between adjacent layers through the embedded hydrogel. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel (into which the cells migrate). Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients both in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates subpopulations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells. Page 3 of 35 Significance StatementThe invasion of cancerous cells from a tumor into surrounding tissues is one contribution to metastasis-a major contributor to death for patients with cancer. There is a strong link between the directed invasion of cancer cells and the gradients of molecules formed in the microenvironment of the tumor. Using a paper-based invasion assay, this work demonstrates that oxygen-a nutrient known to induce significant behavioral changes to cells within a tumor in a concentration-dependent manner-can also act as a chemoattractant, resulting in the migration of cancer cells towards higher concentrations of oxygen. This finding, and the invasion assay described, could lead to a better understanding of oxygen-based chemotaxis in cancer, and ultimately new strategies for managing metastasis.

Electrically Activated Paper Actuators

“…[2] In recent years, paper has become increasingly interesting as a material in new applications. [3][4][5] For example, we and others have used it for microfluidic [6][7][8][9][10] and electroanalytical devices as the basis for low-cost diagnostics, [11,12] as 3-D scaffolds for cell growth, [13][14][15] as a substrate for printed electronics, [16][17][18][19][20][21][22] and in micro-electromechanical systems (MEMS). [16,23,24] A missing component for paper-based devices is an electrically controlled actuator that is embedded within the paper, can be fabricated by printing, and continues to operate when the paper that supports it is creased and/or folded.…”

Redefining Chinese calligraphy rice paper: an economical and cytocompatible substrate for cell biological assays

“…Since then, the CiGiP technique has been expanded to build different platforms for cell assays and people have studied cell growth, migration and differentiation using paper-based tools. Remarkably, the CiGiP model has been applied to investigate interactions between broblasts and human lung cancer, 8 chemotaxis of cancer cells in an oxygen gradient, 9 and differentiation of human-induced pluripotent stem cells. 10 In addition, a paper sheet has been fabricated into versatile microdevices to investigate cellular phosphorylation, 11 biomineralization of articial bone tissues, 12 and high-throughput testing of the effect of soluble compounds.…”

Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system

“…The parental A549 cells is known to be less metastatic in vivo than the other two A549 variants [49]. Using sandwich-based ELISA, we found that the A549 parental cell line did not secrete a detectable level of HGF, while both A549-HGF and A549-HGF-M secreted significant levels of HGF (Table 1).…”

“…Previously described scaffolds in the Cells-in-Gels-in-Paper (CiGiP) [46,[49][50][51] and Cells-in-Gels-in-Mesh (CiGiM) [48] culture systems used sheets of paper (or mesh) that were patterned to contain an array of hydrophilic regions separated by hydrophobic borders. In this work, we used a scaffold that requires fewer steps to fabricate than the paraffin film-patterned mesh sheet, but has a hydrophobic barrier that prevents the lateral diffusion of oxygen more efficiently than wax-printed paper [46,48].…”