Cellular Invasion Assay for the Real-Time Tracking of Individual Cells in Spheroid or Tumor-like Mimics.

Cellular invasion is the gateway to metastasis, with cells moving from a primary tumor into neighboring regions of healthy tissue. Invasion assays provide a tractable experimental platform to quantitatively assess cellular movement in the presence of potential chemokines or inhibitors. Many such assays involve cellular movement from high cell densities to cell-free regions. To improve the physiological relevance of such assays, we developed an assay format to track cellular movement throughout a uniform density of cells. This assay format imparts diffusion-dominated environments along the channel, resulting in oxygen and nutrient gradients found in spheroids or poorly vascularized tumors. By incorporating oxygen- and pH-sensing films, we quantified spatial and temporal changes in the extracellular environment while simultaneously tracking the movement of a subset of cells engineered to express fluorescent proteins constitutively. Our results show the successful invasion into neighboring tissues likely arises from a small population with a highly invasive phenotype. These highly invasive cells continued to move throughout the 48 h experiment, suggesting they have stem-like or persister properties. Surprisingly, the distance these persister cells invaded was unaffected by the density of cells in the channel or the presence or absence of an oxygen gradient. While these datasets cannot determine if the invasive cells are inherent to the population or if diffusion-dominated environments promote them, they highlight the need for further study.

Generating linear oxygen gradients across 3D cell cultures with block-layered oxygen controlled chips (BLOCCs).

Oxygen is a transcriptional regulator responsible for tissue homeostasis and maintenance. Studies relating cellular phenotype with oxygen tension often use hypoxia chambers, which expose cells to a single, static oxygen tension. Despite their ease of use, these chambers are unable to replicate the oxygen gradients found in healthy and diseased tissues. Microfabricated devices capable of imposing an oxygen gradient across tissue-like structures are a promising tool for these studies, as they can provide a high density of information in a single experimental setup. We describe the fabrication and characterization of a modular device, which leverages the gas-permeability of silicone to impose gradients of oxygen across cell-containing regions, assembled by layering sheets of laser cut acrylic and silicone rubber. The silicone also acts as a barrier, separating the flowing gases from the cell culture medium, preventing evaporation or bubble formation in experiments that require prolonged periods of incubation. The acrylic components provide a rigid framework to provide a sterile culture environment. Using oxygen-sensing films, we show the device can support gradients of different ranges and steepness by simply changing the composition of the gases flowing through the silicone components of the BLOCC. Using a cell-based reporter assay, we demonstrate that cellular responses to hypoxia are proportional to oxygen tension.

Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

Hypoxia is a common feature in solid tumors. Clinical samples show a positive correlation between the expression of the hypoxia-inducible factor HIF-1α and estrogen receptor alpha (ERα) and a negative correlation between HIF-1α and hormone sensitivity. Results from monolayer cultures are in contention with clinical observations, showing that ER (+) cell lines no longer express ERα under hypoxic conditions (1% O2). Here, we compared the impact of hypoxia on the ERα signaling pathway for T47D cells in a 2D and 3D culture format. In the 2D format, the cells were cultured as monolayers. In the 3D format, paper-based scaffolds supported cells suspended in a collagen matrix. Using ELISA, Western blot, and immunofluorescence measurements, we show that hypoxia differentially regulates ERα protein levels in a culture environment-dependent manner. In the 2D format, the protein levels are significantly decreased in hypoxia. In the 3D format, the protein levels are maintained in hypoxia. Hypoxia reduced ERα transcriptional activation in both culture formats. These results highlight the importance of considering tissue dimensionality for in vitro studies. They also show that ERα protein levels in hypoxia are not an accurate indicator of ERα transcriptional activity, and confirm that a positive stain for ERα in a clinical sample may not necessarily indicate hormone sensitivity.

Paper-based Transwell assays: an inexpensive alternative to study cellular invasion.

Cellular movement is essential in the formation and maintenance of healthy tissues as well as in disease progression such as tumor metastasis. In this work, we describe a paper-based Transwell assay capable of quantifying cellular invasion through an extracellular matrix. The paper-based Transwell assays generate similar datasets, with equivalent reproducibility, to commercially available Transwell assays. With different culture configurations, we quantify invasion: upon addition of an exogenous factor or in the presence of medium obtained from other cell types, in an indirect or direct co-culture format whose medium composition is dynamically changing, and in a single-zone or parallel (96-zone) format.

A pH-Sensing Optode for Mapping Spatiotemporal Gradients in 3D Paper-Based Cell Cultures.

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pKa of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

3D cellular invasion platforms: how do paper-based cultures stack up?

Cellular invasion is the gateway to metastasis, which is the leading cause of cancer-related deaths. Invasion is driven by a number of chemical and mechanical stresses that arise in the tumor microenvironment. In vitro assays are needed for the systematic study of cancer progress. To be truly predictive, these assays must generate tissue-like environments that can be experimentally controlled and manipulated. While two-dimensional (2D) monolayer cultures are easily assembled and evaluated, they lack the extracellular components needed to assess invasion. Three-dimensional (3D) cultures are better suited for invasion studies because they generate cellular phenotypes that are more representative of those found in vivo. This feature article provides an overview of four invasion platforms. We focus on paper-based cultures, an emerging 3D culture platform capable of generating tissue-like structures and quantifying cellular invasion. Paper-based cultures are as easily assembled and analyzed as monolayers, but provide an experimentally powerful platform capable of supporting: co-cultures and representative extracellular environments; experimentally controlled gradients; readouts capable of quantifying, discerning, and separating cells based on their invasiveness. With a series of examples we highlight the potential of paper-based cultures, and discuss how they stack up against other invasion platforms.

Investigating the impacts of DNA binding mode and sequence on thermodynamic quantities and water exchange values for two small molecule drugs.

Doxorubicin and nogalamycin are antitumor antibiotics that interact with DNA via intercalation and threading mechanisms, respectively. Because the importance of water, particularly its impact on entropy changes, has been established in other biological processes, we investigated the role of water in these two drug-DNA binding events. We used the osmotic stress method to calculate the number of water molecules exchanged (Δnwater), and isothermal titration calorimetry to measure Kbinding, ΔH, and ΔS for two synthetic DNAs, poly(dA·dT) and poly(dG·dC), and calf thymus DNA (CT DNA). For nogalamycin, Δnwater<0 for CT DNA and poly(dG·dC). For doxorubicin, Δnwater>0 for CT DNA and Δnwater<0 for poly(dG·dC). For poly(dA·dT), Δnwater~0 with both drugs. Net enthalpy changes were always negative, but net entropy changes depended on the drug. The effect of water exchange on the overall sign of entropy change appears to be smaller than other contributions.

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors.

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

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

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.