Effects of Acute and Repeated Heat Therapy on Prostate Cancer Cell Characteristics

Background The efficacy of conventional anti‐cancer treatments is dependent, in part, on tumor blood flow and oxygenation. Tumor vasculature is abnormal relative to healthy tissue, leading to reduced blood flow and oxygen delivery, and thus, treatment resistance. An emerging adjuvant to conventional treatment to overcome resistance to radiation is heat therapy. While heat therapy may sensitize tumors to radiation via increases in tumor blood flow and thus, oxygenation, we hypothesized that heat‐induced radiosensitization occurs independent, in part, of tumor blood flow or oxygenation. Methods Clonogenic cell survival, cell viability, and cell cycle distribution were assessed using human prostate cancer (PC‐3) cells in vitro . Individual tissue culture flasks of PC‐3 cells were randomized into 6 groups: normothermic non‐radiated (NT‐NR, n=8), normothermic radiated (NT‐R, n=8), acute hyperthermic non‐radiated (HTA‐NR, n=8), acute hyperthermic radiated (HTA‐R, n=8), chronic (repeated) hyperthermic non‐radiated (HTC‐NR, n=8), and chronic (repeated) hyperthermic radiated (HTC‐R, n=8). NT‐NR and NT‐R flasks were maintained in an incubator at 37° C for the duration of experiments. HTA‐NR and HTA‐R flasks were maintained in an incubator at 37° C and heated in a separate incubator to 41° C for 60 minutes prior to radiation. HTC‐NR and HTC‐R flasks were maintained at 37°C and heated to 41°C for 60 minutes every 48 hours for 3 heat treatments. Non‐radiated flasks were subjected to 0 Gy radiation, while radiated flasks were subjected to 2 Gy radiation. For clonogenic cell survival, cells were then plated in 60 mm tissue culture dishes at a density of 500 cells/plate and 1000 cells/plate in 5 replicates each per flask and allowed to grow for 8 days in an incubator at 37° C. Cell survival was assessed via counting the number of fixed and stained colonies >50 cells at the completion of 8 days of incubation. For cell viability, cells were plated into 96‐well plates and incubated for 24, 48, and 72 hours before addition of MTT reagent for quantification of absorbance. For determination of cell cycle distribution, cells were stained with propidium iodide and analyzed via flow cytometry. Data are presented as mean ± SEM. Results Clonogenic cell survival was significantly reduced between NT‐NR vs. NT‐R, HTA‐NR, HTA‐R, and HTC‐R (100 % ± 9.7% vs. 59.1 % ± 5.9 %, 72.4% ± 8.5%, 40.3% ± 3.1%, and 43.3% ± 3.4%, respectively; p < 0.05). There were no differences between NT‐R and HTA‐NR or HTC‐NR. Percentage of cells in G0/G1 was significantly increased and percentage of cells in G2/M was significantly decreased in NT‐R and HTC‐NR vs. NT‐NR and HTA‐NR. Conclusions This investigation indicates that mild‐temperature hyperthermia before radiation treatment does not enhance the efficacy of radiation treatment, but that heat therapy alone may be as effective as low‐dose radiation in vitro . Further, given that 2 Gy radiation alone (representative of one radiation fraction given clinically) was not more effective at reducing cancer cell survival, heat therapy may be an effective strategy to limit cancer progression before radiation therapy begins. Support or Funding Information NIH HL137156‐01A1, RSG‐14‐150‐01‐CCE