15. Cryosensitization approaches in cryotherapy

Abstract

Cryoablation for the treatment of prostate cancer (CaP) is increasingly utilized in the clinical arena. Due to the sensitivity of surrounding tissues to freezing, implementation of a positive freeze margin often presents challenges. Co-morbidities coupled with the risk of a zone of incomplete ablation at the periphery of a cryogenic lesion may be reduced by increasing cryotherapy’s total ablative potential via a combinatorial approach. Various agents have been utilized in vitro in an attempt to synergistically increase cancer cell death in conjunction with freezing. Previous studies have shown that chemotherapeutic drugs such as taxotere and 5-FU are effective cryosensitizers. More recently we have shown the combinatorial potential of calcitriol, the active metabolite of vitamin D3. In both human and murine models, 24 h calcitriol exposure prior to freezing to temperatures associated with the periphery (−10 °C through −25 °C) resulted in increased cell death compared to freezing alone. Specifically, in the androgen insensitive human CaP line PC-3, 24 h calcitriol pre-treatment yielded a 36% increase in cell death at the −15 °C isotherm compared to temperature-matched controls (60% viable vs. 96%, respectively). In addition to 2D in vitro modeling, studies were also conducted using a novel 3D prostate tissue engineered model (pTEM) system. CaP cells grown in this 3D matrix were frozen using a novel supercritical nitrogen (SCN) device in the presence or absence of calcitriol as a cryosensitizer. Results indicate that the zone of ablation within the iceball increased by 20% with the addition of calcitriol. These in vitro studies illustrate the potential benefits of calcitriol in combination with a cryosurgical treatment regime in androgen insensitive CaP. Additionally, we also investigated the “long-term” calcitriol cryosensitization potential using an in vitro dose escalation scheme. In order to model an oral VD3 regime we utilized a 1 nM initial dose of calcitriol followed by a 5 nM increase every 2 days, resulting in a 32-day gradual escalation to 80 nM. Calcitriol dose escalation yielded a significant increase in cell death over the 24 h pre-treatment in all temperatures examined. Additionally, maintenance of 80 nM calcitriol for 20 days after the escalation scheme resulted in complete cell death at the −25 °C isotherm with no re-growth after 3 days. Interestingly, when calcitriol treatment was discontinued after the escalation scheme this sensitization benefit was lost. These results suggest that in a prolonged oral treatment regime, vitamin D3 may provide for improved CaP sensitivity to cryoablation thereby increasing cell death while reducing the need for an enlarged positive freeze margin. The results of this study suggest a potential “benchtop to bedside” framework for future clinical translation.