Abstract
Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid-containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.
Highlights
Our results show that this simple coverslip hypoxia variant, applied to LNCaP cells, can be used to create a microenvironment where cells become more hypoxic and extracellular pH decreases as the distance from an oxygen source increases
The morphological change we observed in LNCaP cells after coverslip treatment was accompanied by a marked increase in fibrillar actin staining, with actin fibers often extending throughout the whole cytoplasm, a pattern not seen in cells close to the oxygen source (Figure 4a)
We have modified the coverslip hypoxia method, and we have shown that in combination with the use of LNCaP cells, our variant of this method generates a microenvironment where cells become more hypoxic, extracellular pH becomes more acidic, and cell morphology becomes increasingly altered as the distance from the oxygen source increases
Summary
Capillary at which hypoxia ensues (Olive, Vikse, & Trotter, 1992). As a consequence, central regions in early tumor masses distant from the. An alternative approach is to place a coverslip on top of a cell monolayer, so that oxygen can only diffuse through the narrow space between the cells and the edges of the coverslip, a method termed coverslip hypoxia (Pitts & Toombs, 2004). Using this method in combination with oxygen‐sensitive green fluorescent protein (GFP) fluorescence shifts, it was shown that oxygen levels in cells under coverslips decreased from the edges toward the center of the coverslip (Takahashi & Sato, 2010). Our results further support the use of the coverslip hypoxia method as a simple but revealing approach to model the oxygen diffusion restriction of the early tumor microenvironment and its effects on the tumor cell phenotype
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