Abstract
3D paper-based cultures (PBCs) are easy-to-use and provide a biologically representative microenvironment. By stacking a sheet of cell-laden paper below sheets containing cell-free hydrogel, we form an assay capable of segmenting cells by the distance they invaded from the original cell-seeded layer. These invasion assays are limited to end-point analyses with fluorescence-based readouts due to the highly scattering nature of the paper scaffolds. Here we demonstrate that optical coherence tomography (OCT) can distinguish living cells from the surrounding extracellular matrix (ECM) or paper fibers based upon their intracellular motility amplitude (M). M is computed from fluctuation statistics of the sample, rejects shot noise, and is invariant to OCT signal attenuation. Using OCT motility analysis, we tracked the invasion of breast cancer cells over a 3-day period in 4-layer PBCs (160-300 µm thick) in situ. The cell population distributions determined with OCT are highly correlated with those obtained by fluorescence imaging, with an intraclass correlation coefficient (ICC) of 0.903. The ability of OCT motility analysis to visualize live cells and quantify cell distributions in PBC assays in situ and longitudinally provides a novel means for understanding how chemical gradients within the tumor microenvironment affect cellular invasion.
Highlights
Cellular invasion, a process in which cells remodel their surrounding extracellular matrix (ECM) prior to moving into neighboring tissue, plays an important role in tissue formation and maintenance as well as in disease progression
The paper-based culture (PBC) platform we developed provides several experimental advantages over other 3D invasion assays [6,7,8,9], including the ability to segment populations of cells based on the distance invaded without the need for fixation or histological slicing [10,11]
To demonstrate that M enables one to distinguish live cells from the paper fibers and background ECM, we performed optical coherence tomography (OCT) motility analysis on cell-laden and cell-free cultures, that had both been incubated for 3 days prior to analysis, where the OCT operator was blinded to the identities of the cultures
Summary
A process in which cells remodel their surrounding extracellular matrix (ECM) prior to moving into neighboring tissue, plays an important role in tissue formation and maintenance as well as in disease progression. Paper-based invasion assays are comprised of several sheets of paper stacked together, where cells from a single cell-containing sheet invade into neighboring cell-free sheets. We and others have quantified the invasion of both carcinoma and stromal cells in PBCs containing either mono- or cocultures [10,11,12,14,15]. With these invasion assays, we showed cells placed in diffusion-limited environments containing monotonic gradients of oxygen, nutrients, and waste products invade regions of higher oxygen tension preferentially [10,12]
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