Cell line-specific efficacy of thermoradiotherapy in human and canine cancer cells in vitro

Abstract

Objective Aims were to investigate sensitivity of various human and canine cancer cell lines to hyperthermia and the influence of particular treatment conditions, and to analyze the DNA-damage response and mode of cell death in cell line radiosensitized by hyperthermia. Additionally, we were interested in the involvement of HSP70 in radiosensitization. Methods Radiosensitization by hyperthermia was determined in a panel of human and canine cancer cell lines using clonogenic cell survival assay, as well as levels of heat shock proteins (HSPs) using immunoblotting. The influence of the hyperthermia-radiotherapy time gap, different temperatures and the order of treatments on clonogenicity of hyperthermia-sensitive A549 cells was investigated. Additionally, DNA damage and cell death were assessed by Comet assay and an apoptosis/necrosis assay. Further we induced transient knockdown in A549 cells to test HSP70’s involvement in radiosensitization. Results Out of eight cell lines tested, only two (A549 and Abrams) showed significant decrease in clonogenic cell survival when pre-treated with hyperthermia at 42°C. Strong induction of HSP70 upon thermoradiotherapy (HT-RT) treatment was found in all cell lines. Transient knockdown of HSP70 in A549 cells did not result in decrease of clonogenic cell survival in response to HT-RT. Conclusion Tumor cell-type, temperature and order of treatment play an important role in radiosensitization by hyperthermia. However, hyperthermia has limited potency to radiosensitize canine cancer cells grown in a 2D cell culture setting presented here. DNA damage and apoptosis/necrosis did not increase upon combined treatment and cytosolic levels of HSP70 appear not to play critical role in the radiosensitization of A549 cells.

Role of HSP70 in response to (thermo)radiotherapy: analysis of gene expression in canine osteosarcoma cells by RNA-seq

“…5D and Supplementary Table 11). Also in this group, genes involved in cellular processes (38) were the most represented, followed by metabolic processes (13) and biological regulation (13). In the radiotherapy alone treated group (RT) only few genes were significantly induced in negative control siRNA and HSP70 knockdown cells, 25 and 12, respectively (Supplementary Tables 2 and 3).…”

“…Directly after hyperthermia (time interval between hyperthermia and irradiation was approximately 10 min), cells were transferred to radiation facility and irradiated. Prior to this study, the heating profile of the incubator used for hyperthermia treatments was characterized thoroughly by measurements of the temperature of the cell culture dish (standard 10-cm dish used for clonogenic assay) 13 . Based on data from three runs, thermal doses were calculated according to the cumulative equivalent minutes at 43 °C iso-effect model (CEM 43 ) 14 .…”

Holistic View on Cell Survival and DNA Damage: How Model-Based Data Analysis Supports Exploration of Dynamics in Biological Systems

“…Clonogenic cell survival and comet assay measurements were shown to be repeatable [14,29]; thus, it is reasonable to expect repeatable results from patient biopsies [38]. This Figure 6: Histograms of parameter values after calibration with the software set to different modes, and at the bottom, the joint distribution obtained by combining the two posterior sets is shown.…”

“…The sequence of treatments (hyperthermia followed by irradiation) and the treatment time were chosen to match the clinical practice [30]. To ensure repeatability and quantify thermodynamic effects such as heat transfer and evaporative cooling, incubators were carefully calibrated [29]. In case of an experiment without hyperthermia treatment, the cells remained in the 37°C incubator.…”

“…If a cell loses clonogenicity due to the treatment, it will not form a clone, while cells which survive the treatment (in the sense of maintaining clonogenicity) will form a colony. The dataset to model clonogenic cell survival in canine osteosarcoma Abrams cells used here was previously published, and the details of the experimental protocol are described in [29].…”