Robert G. VanBuskirk

Cryoablation of renal cancer: variables involved in freezing-induced cell death.

The detection of renal tumors has increased significantly over recent years resulting in a greater demand for novel, minimally invasive techniques. Cryoablation has emerged as a valuable treatment modality for the management of renal cancer. In an effort to detail the effects of freezing in renal cancer, the human renal cancer (RCC) cell line, 786-O, was evaluated in vitro. 786-O cells were exposed to a range of freezing temperatures from −5 to −40°C and compared to non-frozen controls. The data show that freezing to −5°C did not affect 786-O cell viability, while −10°C, −15°C, and −20°C results in a significant loss of viability (23, 70, and 91%, respectively). A complete loss of cell viability was evident at temperatures of −25°C and colder. Following this analysis, variables involved in the success of cryoablation were investigated. For each of the temperatures tested, extended freeze hold times and passive thawing rates resulted in more extensive cell damage. Additionally, a double freeze-thaw cycle significantly increased cell death compared to a single cycle (62% vs. 22% at −10°C; 89% vs. 63% at −15°C, respectively). While these variables play an important part in the effective application of cryoablation, a molecular understanding of the cell death involved is critical to improving efficacy. Apoptotic inhibition afforded 12% (−10°C), 25% (−15°C), and 11% (−20°C) protection following freezing. Using fluorescence microscopy analysis, the results demonstrated that apoptosis peaked at six hours post-thaw. Next, apoptotic initiating agents including 5-FU and resveratrol (RVT) applied prior to freezing exposure resulted in a significant increase in cell death compared to either application alone. Importantly, the combination of RVT and freezing was noticeably less effective when applied to normal renal cells. The results herein demonstrate the efficacy of freezing and describe a novel therapeutic model for the treatment of renal cancer that may distinguish between cancer and normal cells.

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer

Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (−10°C) or TRAIL (500ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.

Cryoablative response of prostate cancer cells is influenced by androgen receptor expression

To investigate in prostate cancer cells the consequences of androgen‐insensitivity (AI) development on the cellular and molecular responses to freezing, as a challenge in prostate cancer treatment occurs when the androgen‐sensitive (AS) phenotype switches to an AI phenotype, the latter of which is often refractory to many therapies.

Vitamin D3 cryosensitization increases prostate cancer susceptibility to cryoablation via mitochondrial-mediated apoptosis and necrosis

Whats known on the subject? and What does the study add?

Changing paradigms in biopreservation.

The field of cryopreservation has a long and successful history of in-depth study and progress. Advances in our knowledge base and our ability to cryopreserve cells have been consequential and have led to its widespread integration into academic, clinical, and agricultural settings. While many cell systems are successfully cryopreserved today, there remains significant cell loss associated with cryopreservation. Moreover, even today some cell systems remain uncryopreservable from a practical perspective. This is due to the diversity of post-freeze responses of individual cells to the various stressors experienced during the freeze-thaw process. In 1998, several independent groups reported on the direct involvement of apoptotic and necrotic cell death following cryopreservation (Baust, et al., 1998 and Borderie, et al., 1998). In addition to those reports, a substantial literature base describing the modulation of cell death through the use of various protease inhibitors, free radical scavengers, media formulations, and other novel compounds exist. These studies have identified diverse molecular-based, cellular responses to cryopreservation and have further demonstrated the significant improvements in cell survival through the modulation of molecular events. Numerous studies have reported on the molecular-based phenomena of cryopreservation-induced delayed onset cell death, yet our understanding of the pathway activation, progression, control, and the downstream effect on cell function remains in its infancy. To this end, modulation studies, such as targeted apoptotic control (TAC), have shown promise in furthering our understanding of the activation pathways and are proving to be a critical next step in the evolution of the cryopreservation sciences. This review provides an overview of the current literature on the mechanisms of cell death associated with cryopreservation failure.

Integrin Involvement in Freeze Resistance of Androgen-Insensitive Prostate Cancer

Cryoablation has emerged as a primary therapy to treat prostate cancer. Although effective, the assumption that freezing serves as a ubiquitous lethal stress is challenged by clinical experience and experimental evidence demonstrating time–temperature-related cell-death dependence. The age-related transformation from an androgen-sensitive (AS) to an androgen-insensitive (AI) phenotype is a major challenge in the management of prostate cancer. AI cells exhibit morphological changes and treatment resistance to many therapies. As this resistance has been linked with α6β4 integrin overexpression as a result of androgen receptor (AR) loss, we investigated whether α6β4 integrin expression, as a result AR loss, contributes to the reported increased freeze tolerance of AI prostate cancer. A series of studies using AS (LNCaP LP and PC-3 AR) and AI (LNCaP HP and PC-3) cell lines were designed to investigate the cellular mechanisms contributing to variations in freezing response. Investigation into α6β4 integrin expression revealed that AI cell lines overexpressed this protein, thereby altering morphological characteristics and increasing adhesion characteristics. Molecular investigations revealed a significant decrease in caspases-8, -9, and -3 levels in AI cells after freezing. Inhibition of α6β4 integrin resulted in increased caspase activity after freezing (similar to AS cells) and enhanced cell death. These data show that AI cells show an increase in post-freeze susceptibility after inhibition of α6β4 integrin function. Further understanding the role of androgen receptor-related α6β4 integrin expression in prostate cancer cells responses to freezing might lead to novel options for neo-adjunctive treatments targeting the AR signaling pathway.

The Story of Adjuvants to Boost the Performance of Cryoablation

Cryoablation represents a disruptive therapeutic strategy, a paradigm shift in the approach to cancer control. Cancer, whether indolent or lethal, dormant, slow growing or aggressive, focal or diffuse, succumbs equally to the cascade of stresses attendant to controlled, targeted freezing. Unlike other minimally invasive therapeutic options such as radiation, chemotherapy, and hormonal ablation, where the effectiveness of each is linked to the cell cycle and specific sensitivities of the cancer cell during division, freeze lethality is independent of the cancer’s defensive strategies. Further, as a non-repetitive treatment, mutagenic adaptive responses are denied.