Modeling the Role of Cancer-Associated Fibroblasts in Tumor Cell Invasion

The major cause of cancer-related deaths can be attributed to the metastatic spread of tumor cells—a dynamic and complex multi-step process beginning with tumor cells acquiring an invasive phenotype to allow them to travel through the blood and lymphatic vessels to ultimately seed at a secondary site. Over the years, various in vitro models have been used to characterize specific steps in the cascade to collectively begin providing a clearer picture of the puzzle of metastasis. With the discovery of the TME’s supporting role in activating tumor cell invasion and metastasis, these models have evolved in parallel to accommodate features of the TME and to observe its interactions with tumor cells. In particular, CAFs that reside in reactive tumor stroma have been shown to play a substantial pro-invasive role through their matrix-modifying functions; accordingly, this warranted further investigation with the development and use of invasion assays that could include these stromal cells. This review explores the growing toolbox of assays used to study tumor cell invasion, from the simple beginnings of a tumor cell and extracellular matrix set-up to the advent of models that aim to more closely recapitulate the interplay between tumor cells, CAFs and the extracellular matrix. These models will prove to be invaluable tools to help tease out the intricacies of tumor cell invasion

Measuring Reaction Rate Constant in Individual Cells to Facilitate Accurate Analysis of Cell-Population Heterogeneity

We propose Cytometry of Reaction Rate Constant (CRRC) for accurate analysis of cell-population heterogeneity with respect to a specific molecular reaction. Conceptually, in CRRC, the cells are loaded with a reaction substrate, and its conversion into a product is followed by time-lapse fluorescence microscopy at the single-cell level. A reaction rate constant is determined for every cell by using a known kinetic mechanism of the reaction, and a kinetic histogram “number of cells vs. the rate constant” is built. Finally, this histogram is used to determine parameters of reaction-based cell-population heterogeneity. Here, we studied a reaction of substrate extrusion from cells by ABC transporters. We proved that sizes of subpopulations with different extrusion rates could be accurately determined from the kinetic histogram, and this determination was not significantly affected by change in substrate concentration. We foresee that CRRC will facilitate the development of reliable disease biomarkers based on parameters of reaction-based cell-population heterogeneity

Spheroid-Based Approach to Assess Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of Reaction Rate Constant

File main pdf file (CRRC of dispersed-settled spheroidal cells.pdf) describes experimental results and their interpretation for a study of tissue relevance of analyses of dispersed-settled tissues cells by Cytometry of Reaction Rate Constant (CRRC). CRRC uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Practical CRRC requires that a tissue sample be disintegrated into a suspension of dispersed cells and these cells settle on the support surface before being analyzed by CRRC. We call such cells “dispersed-settled” to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multi-drug-resistance (MRD) transport which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions, and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into practical analytical tool. Additional (zip) files contain supporting images, kinetic traces, and histograms. Their detailed descriptions are provided in the main pdf file. </div

Primary esophageal and gastro-esophageal junction cancer xenograft models:Clinicopathological features and engraftment

<p><b>A–C.</b> Voltage recordings of locomotor-related drive obtained from embryonic slow (ES) fibres of control (<b>A</b>), DETA-NO raised (<b>B</b>) and L-NAME raised (<b>C</b>) fish at 2 days post fertilisation (dpf). <b>D–F</b>. Mean end plate potential (EPP) amplitude (<b>D</b>), rise time (<b>E</b>) and decay time (<b>F</b>) measured during episodes of fictive swimming. Data in <b>D-F</b> are represented as mean ± SEM. *** p<0.001.</p