Abstract
BackgroundInvasiveness is a major factor contributing to metastasis of tumour cells. Given the broad variety and plasticity of invasion mechanisms, assessing potential metastasis-promoting effects of irradiation for specific mechanisms is important for further understanding of potential adverse effects of radiotherapy. In fibroblast-led invasion mechanisms, fibroblasts produce tracks in the extracellular matrix in which cancer cells with epithelial traits can follow. So far, the influence of irradiation on this type of invasion mechanisms has not been assessed.MethodsBy matrix-embedding coculture spheroids consisting of breast cancer cells (MCF-7, BT474) and normal fibroblasts, we established a model for fibroblast-led invasion. To demonstrate applicability of this model, spheroid growth and invasion behaviour after irradiation with 5 Gy were investigated by microscopy and image analysis.ResultsWhen not embedded, irradiation caused a significant growth delay in the spheroids. When irradiating the spheroids with 5 Gy before embedding, we find comparable maximum migration distance in fibroblast monoculture and in coculture samples as seen in unirradiated samples. Depending on the fibroblast strain, the number of invading cells remained constant or was reduced.ConclusionIn this spheroid model and with the cell lines and fibroblast strains used, irradiation does not have a major invasion-promoting effect. 3D analysis of invasiveness allows to uncouple effects on invading cell number and maximum invasion distance when assessing radiation effects.
Highlights
Invasiveness is a major factor contributing to metastasis of tumour cells
We demonstrate fibroblastled collective invasion in MCF-7 and BT474 tumour cells cocultivated with two different fibroblast strains (immortalized BJ1-hTert and primary human dermal fibroblasts (HDF)) in spheroids embedded in a commercially available extracellular matrix blend comprised of basement membrane extract, derived from murine EHS sarcoma cells, and collagen I, from bovine extensor tendons
MCF-7 and BT474 cells were cultivated in RPMI-1640 medium (Sigma Aldrich); SkBr3, MDAMB-231 and BJ1-hTert were cultured in DMEM (Sigma Aldrich), and HDF was cultured in Fibroblast Basal Medium (Primary Cell Solution)
Summary
Invasiveness is a major factor contributing to metastasis of tumour cells. Given the broad variety and plasticity of invasion mechanisms, assessing potential metastasis-promoting effects of irradiation for specific mecha‐ nisms is important for further understanding of potential adverse effects of radiotherapy. A Boyden chamber or transwell migration chamber is used in which a BM- or ECM-like matrix covers the porous membrane separating both chambers [6]. Tumour cells seeded on this matrix have first to invade the matrix before they can pass the pores of the separating membrane. This assay, which evaluates the number of cells able to pass the filter, has predominantly been used in in vitro studies on the effect of irradiation on invasive potential of tumour cells [5, 8,9,10]. Invasion stimulating potential of irradiation has frequently been described, while irradiation with carbon ions or alpha particles appears to have lower invasion stimulating capability than irradiation with photons [5, 8, 9, 11]
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