Cryoablation: physical and molecular basis with putative immunological consequences.

Cryoablation (CA) is unique as the singular energy deprivation therapy that impacts all cellular processes. CA is independent of cell cycle stage and degree of cellular stemness. Importantly, CA is typically applied as a non-repetitive (single session) treatment that does not support adaptative mutagenesis as do many repetitive therapies. CA is characterized by the launch of multiple forms of cell death including (a) ice-related physical damage, (b) initiation of cellular stress responses (kill switch activation) and launch of necrosis and apoptosis, (c) vascular stasis, and (d) likely activation of ablative immune responses. CA is not without limitation related to the thermal gradient formed between cryoprobe surface (∼-185°C) and the distal surface of the freeze zone (∼0°C) requiring freeze margin extension beyond the tumor boundary (up to ∼1 cm). This limitation is mitigated in part by commonly applied dual freeze thaw cycles and the use of freeze sensitizing adjuvants. This review will (1) identify the cascade of damaging effects of the freeze-thaw process, its physical and molecular-based relationships, (2) a likely immunological involvement (abscopic effect), and (3) explore the use of freeze-sensitizing adjuvants necessary to limit freezing beyond the tumor margin.

Evaluation of a New Cystoscopic Cryocatheter and Method for the In Situ Destruction of Bladder Cancer: Preliminary In Vivo Study.

Purpose: To assess the ability to deliver full-thickness bladder wall cryoablation through a cystoscopic approach using a new closed-loop 6F cryocatheter and thermal dose-controlled protocol. Materials and Methods: Evaluations were conducted using a chronic porcine model wherein 10 lesions/animal were created throughout the bladder (bladder wall, trigone region, ureteral orifice, and distal ureter). A 6F cryocatheter was passed through the working channel of a flexible cystoscope. Single 1- and 1.5-minute freeze protocols in a saline environment were evaluated and resultant lesion size was determined. A laparoscopic approach was utilized to observe the transmural extension of the ice propagation. Results: Studies demonstrated the generation of transmural lesions characterized by full-thickness histologic necrosis after freezing for 1.5 minutes regardless of tissue thickness (range 2-12 mm). All animals were found to have good overall health (maintained weight, appetite, mobility, and energy levels) throughout the recovery period. No significant deviations were noted in complete blood count and serum chemistry bloodwork except for elevated creatine kinase levels. Importantly, no fistulas or perforations were noted. Conclusions: The cryocatheter was able to rapidly and effectively freeze the bladder wall through a cystoscopic approach. The results showed the ability to consistently ablate an ∼1 cm diameter and up to 1.2 cm deep using a single 1.5-minute freeze protocol. Analysis of the ablation efficacy revealed ∼80% destruction within the frozen mass. Although further testing and refinement are needed, these studies demonstrate the potential of this new approach to provide a next-generation strategy for the treatment of bladder cancer.

The Assessment of a Novel Endoscopic Ultrasound-Compatible Cryocatheter to Ablate Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease that may be treated utilizing thermal therapies. Cryoablation is an effective, minimally invasive therapy that has been utilized for the treatment of various cancers, offering patients a quicker recovery and reduced side effects. Cryoablation has been utilized on a limited basis for the treatment of PDAC. With the recent reports on the success of cryoablation, there is a growing interest in the use of cryoablation as a standalone, minimally invasive procedure to treat PDAC. While offering a promising path, the application of cryoablation to PDAC is limited by current technologies. As such, there is a need for the development of new devices to support advanced treatment strategies for PDAC. To this end, this study investigated the performance of a new endoscopic ultrasound-compatible cryoablation catheter technology, FrostBite. We hypothesized that FrostBite would enable the rapid, effective, minimally invasive delivery of ultra-cold temperatures to target tissues, resulting in effective ablation via an endoscopic approach. Thermal properties and ablative efficacy were evaluated using a heat-loaded gel model, tissue-engineered models (TEMs), and an initial in vivo porcine study. Freeze protocols evaluated included single and repeat 3 and 5 min applications. Isotherm assessment revealed the generation of a 2.2 cm diameter frozen mass with the -20 °C isotherm reaching a diameter of 1.5 cm following a single 5 min freeze. TEM studies revealed the achievement of temperatures ≤ -20 °C at a diameter of 1.9 cm after a 5 min freeze. Fluorescent imaging conducted 24 h post-thaw demonstrated a uniformly shaped ellipsoidal ablative zone with a midline diameter of 2.5 cm, resulting in a total ablative volume of 6.9 cm3 after a single 5 min freeze. In vivo findings consistently demonstrated the generation of ablative areas measuring 2.03 cm × 3.2 cm. These studies demonstrate the potential of the FrostBite cryocatheter as an endoscopic ultrasound-based treatment option. The data suggest that FrostBite may provide for the rapid, effective, controllable freezing of cancerous pancreatic and liver tissues. This ablative power also offers the potential of improved safety margins via the minimally invasive nature of an endoscopic ultrasound-based approach or natural orifice transluminal endoscopic surgery (NOTES)-based approach. The results of this pre-clinical feasibility study show promise, affirming the need for further investigation into the potential of the FrostBite cryocatheter as an advanced, minimally invasive cryoablative technology.

Vitamin D(3) cryosensitization increases prostate cancer susceptibility to cryoablation via mitochondrial-mediated apoptosis and necrosis.

OBJECTIVES: To investigate the effect and molecular mechanisms of action of Vitamin D(3) (VD(3) ) as a neo-adjunctive agent before cryosurgery in an effort to increase treatment efficacy for prostate cancer (CaP). To eliminate the potential for disease recurrence that exists at the periphery of the freeze lesion, where temperatures may be insufficient to destroy both androgen-sensitive (AS) and androgen-insensitive (AI) CaP. METHODS: Human CaP cells, LNCaP, were each genetically altered to express the AS and AI phenotypes and subjected to VD(3) treatment and freezing in an in vitro and tissue-engineered model. Cell viability, caspase inhibitor and western blot studies were used to determine the basis of the different responses of AI and AS cells to VD(3) cryosensitization. RESULTS: VD(3) was found to be a highly effective cryosensitizer, resulting in a >50% overall increase in cell death after -15 °C freezing. Fluorescence microscopy, western blot analysis and caspase protease assays confirmed that the increased activation of apoptosis was modulated through a mitochondrial-mediated pathway. Caspase inhibition studies showed that apoptosis played an integral role in cell death, with VD(3) cryosensitivation-induced apoptotic events responsible for >30% of the overall cell death after -15 °C freezing. CONCLUSIONS: The present study suggests that the use of VD(3) as a cryosensitizer increases cryoablation efficacy through the increased activity of apoptosis as well as through necrosis. The data show that through VD(3) treatment the overall level of AI CaP cell tolerance to freezing is reduced to a level similar to that of AS CaP. VD(3) pre-treatment in conjunction with cryoablation may increase treatment efficacy and reduce disease recurrence for CaP patients.

Assessment of a novel cryoablation device for the endovascular treatment of cardiac tachyarrhythmias.

OBJECTIVES: Cryoablation is an effective alternative treatment for cardiac arrhythmias offering shortened recovery and reduced side effects. As the use of cryoablation increases, the need for new devices and procedures has emerged. This has been driven by technological limitations including lengthy periods to generate a single lesion (3-5 min), uncertain transmurality, and differential efficacy. Furthermore, due to limited ablation capacity under high heat loads, cryo has had limited success in the treatment of ventricular arrhythmias. To this end, in this study we evaluated a new cryoablation catheter, ICEolate, for the targeted ablation of cardiac tissue. METHODS: Performance assessment included calorimetry, freeze zone isothermal distribution characterization and catheter ablation capacity in a submerged, circulating, heat-loaded ex vivo tissue model. A pilot in vivo study was also conducted to assess ablative capacity of the cryocatheter in a fully beating heart. RESULTS: Ex vivo studies demonstrated ice formation at the tip of a cryocatheter within 5 s and a tip temperature of ~-150°C within 10 s. The device repeatedly generated freeze zones of 2 cm × 3 cm in less than 2 min. Tissue model studies revealed the generation of a full thickness (5-10 mm) cryogenic lesion within 1 min with an opposite (transmural) surface temperature of <-60°C under a circulating 37°C heat load. Pilot in vivo studies demonstrated the delivery of an ablative "dose," producing a continuous full thickness transmural linear lesion in <60 s at both atrial and ventricular sites. CONCLUSION: These studies suggest that the supercritical nitrogen cryodevice and ICEolate cryocatheter may provide for rapid, effective, controllable freezing of targeted tissue. The ablative power, speed, and directional freeze characteristics also offer the potential of improved safety via a reduction in procedural time compared to current cryoablation devices. These technological developments may open new avenues for the application of cryo to treat other cardiac arrhythmogenic disorders.

Development of a tissue engineered human prostate tumor equivalent for use in the evaluation of cryoablative techniques.

The study of the effectiveness of cryotherapy as a curative treatment for prostate cancer has often relied on the use of either in vitro cell culture monolayers or animal models. While the data gleaned from these studies have been valuable, each model has inherent limitations. In order to bridge the gap between in vitro studies and clinical applications, we developed a 3-dimensional, tissue engineered human prostate cancer model to simulate and assess the effects of cryotherapy and adjunctive treatments on cell viability and activation of cell death pathways throughout the thermally variable freeze zone. Human prostate cancer cells (PC3) were seeded into collagen based matrices and cryolesions were generated using an Oncura SeedNet Gold cryosurgical device with 17-gauge cryoprobes. Analyses revealed widespread necrosis diminishing towards the edge of the freeze zone, and a time-dependent wave of apoptosis starting as early as 1 hr post-thaw at low temperatures (< -40 degrees C) and moving toward the periphery (-20 degrees C) as recovery times reached 12 and 24 hr. Distal to the -10 degrees C isotherm, minimal cell death was apparent (< 20%) over controls. The adjunctive use of chemotherapeutic agents in conjunction with cryosurgery displayed a similar induction of cell death cascades, but with the zone of cryodestruction extending approximately 10 to 15 degrees C further into the freeze zone periphery. By providing an extracellular environment and a matrix to minimize innate variables, the tissue engineered model yielded a more in vivo-like, tumor-like environment supportive of a deeper understanding of the specific biological responses of cancer cells/tumors to cryotherapeutic intervention.

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 degrees C and compared to non-frozen controls. The data show that freezing to -5 degrees C did not affect 786-O cell viability, while -10 degrees C, -15 degrees C, and -20 degrees 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 degrees 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 degrees C; 89% vs. 63% at -15 degrees 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 degrees C), 25% (-15 degrees C), and 11% (-20 degrees 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.

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems.

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (-5 to -40 degrees C), and cells were thawed followed by return to 37 degrees C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At -15 degrees C, PC-3 yielded approximately 55% viability versus approximately 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze-thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching -40 degrees C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze-thaw cycles, and passive thawing to provide maximum cell destruction.

Assessment of Cryosurgical Device Performance Using a 3D Tissue-Engineered Cancer Model.

As the clinical use of cryoablation for the treatment of cancer has increased, so too has the need for knowledge on the dynamic environment within the frozen mass created by a cryoprobe. While a number of factors exist, an understanding of the iceball size, critical isotherm distribution/penetration, and the resultant lethal zone created by a cryoprobe are critical for clinical application. To this end, cryoprobe performance is typically characterized based on the iceball size and temperature penetration in phantom gel models. Although informative, these models do not provide information as to the impact of heat input from surrounding tissue nor give any information on the ablative zone created. As such, we evaluated the use of a tissue-engineered tumor model (TEM) to assess cryoprobe performance including iceball size, real-time thermal profile distribution, and resultant ablative zone. Studies were conducted using an Endocare V-probe cryoprobe, with a 10/5/10 double freeze-thaw protocol using prostate and renal cancer TEMs. The data demonstrate the generation of a 33- to 38-cm3 frozen mass with the V-Probe cryoprobe following the double freeze of which ∼12.7 and 6.5 cm3 was at or below -20°C and -40°C, respectively. Analysis of ablation zone using fluorescence microscopy 24 hours postthaw demonstrated that the internal ∼40% of the frozen mass was completely ablated, whereas in the periphery of the iceball (outer 1 cm region), a gradient of partial to minimal destruction was observed. These findings correlated well with clinical reports on renal and prostate cancer cryoablation. Overall, this study demonstrates that TEMs provide an effective model for a more complete characterization of cryoablation device performance. The data demonstrate that while the overall iceball size generated in the TEM was consistent with published reports from phantom models, the integration of an external heat load, circulation, and cellular components more closely reflect an in vivo setting and the impact of penetration of the critical (-20°C and -40°C) isotherms into the tissue. This is important as it is well appreciated in clinical practice that the heat load of a tissue, cryoprobe proximity to vasculature, and so on, can impact outcome. The TEM model provides a means of characterizing the impact on ablative dose delivery allowing for a better understanding of probe performance and potential impact on ablative outcome.