3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments

Abstract

Carol Box , Uwe oelfke & Gail ter Haar For multimodality therapies such as the combination of hyperthermia and radiation, quantification of biological effects is key for dose prescription and response prediction. Tumour spheroids have a microenvironment that more closely resembles that of tumours in vivo and may thus be a superior in vitro cancer model than monolayer cultures. Here, the response of tumour spheroids formed from two established human cancer cell lines (HCT116 and CAL27) to single and combination treatments of radiation (0-20 Gy), and hyperthermia at 47 °C (0-780 CEM 43) has been evaluated. Response was analysed in terms of spheroid growth, cell viability and the distribution of live/dead cells. Time-lapse imaging was used to evaluate mechanisms of cell death and cell detachment. It was found that sensitivity to heat in spheroids was significantly less than that seen in monolayer cultures. Spheroids showed different patterns of shrinkage and regrowth when exposed to heat or radiation: heated spheroids shed dead cells within four days of heating and displayed faster growth post-exposure than samples that received radiation or no treatment. Irradiated spheroids maintained a dense structure and exhibited a longer growth delay than spheroids receiving hyperthermia or combination treatment at (thermal) doses that yielded equivalent levels of clonogenic cell survival. We suggest that, unlike radiation, which kills dividing cells, hyperthermia-induced cell death affects cells independent of their proliferation status. This induces microenvironmental changes that promote spheroid growth. In conclusion, 3D tumour spheroid growth studies reveal differences in response to heat and/or radiation that were not apparent in 2D clonogenic assays but that may significantly influence treatment efficacy. Whilst monolayer cell cultures have underpinned decades of cancer research, they have been criticized for not being reliable predictors of treatment response in vivo 1,2. The culture geometry of a flat, rigid substrate limits the number of neighbouring cells, resulting in reduced inter-cellular contact and communication compared with cells in vivo. Two dimensional (2D) culture conditions cause cells to stretch, increasing the surface area directly exposed to culture medium 3-5. Cancer cells cultured in 2D are thus always provided with oxygen and nutrients. These are more restricted in the physiological microenvironment of a tumour where inefficient vasculature prevents their homogeneous supply. Despite these limitations, advantages such as low cost, high throughput, ease of application and control over microenvironmental factors have made monolayer cultures the standard technique for evaluation of cellular responses. However, there may be considerable inconsistencies between experimental results obtained in vitro in 2D culture and those observed in vivo. One possible way of bridging this gap is to use three dimensional (3D) tumour spheroid cultures. Here, cells are grown as aggregates of single or multiple cell types, ...

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