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.

Dose Escalation of Vitamin D3 Yields Similar Cryosurgical Outcome to Single Dose Exposure in a Prostate Cancer Model.

Vitamin D3 (VD3) is an effective adjunctive agent, enhancing the destructive effects of freezing in prostate cancer cryoablation studies. We investigated whether dose escalation of VD3 over several weeks, to model the increase in physiological VD3 levels if an oral supplement were prescribed, would be as or more effective than a single treatment 1 to 2 days prior to freezing. PC-3 cells in log phase growth to model aggressive, highly metabolically active prostate cancer were exposed to a gradually increasing dose of VD3 to a final dose of 80 nM over a 4-week period, maintained for 2 weeks at 80 nM, and then exposed to mild sublethal freezing temperatures. Results demonstrate that both acute 24-hour exposure to 80 nM VD3 and dose escalation resulted in enhanced cell death following freezing at -15°C or colder, with no significant differences between the 2 exposure regimes. Apoptotic analysis within the initial 24-hour period postfreeze revealed that VD3 treatment induced both caspase 8- and 9-mediated cell death, most notably in caspase 8 at 8-hour postfreeze. These results indicate that both the intrinsic and extrinsic apoptotic pathways are involved in VD3 sensitization prior to freezing. Additionally, both acute and gradual dose escalation regimes of VD3 exposure increase prostate cancer cell sensitivity to mild freezing. Importantly, this study expands upon previous reports and suggests that the combination of VD3 and freezing may offer an effective treatment for both slow growth and highly aggressive prostate cancers.

Cryopreservation: Evolution of Molecular Based Strategies.

Cryopreservation (CP) is an enabling process providing for on-demand access to biological material (cells and tissues) which serve as a starting, intermediate or even final product. While a critical tool, CP protocols, approaches and technologies have evolved little over the last several decades. A lack of conversion of discoveries from the CP sciences into mainstream utilization has resulted in a bottleneck in technological progression in areas such as stem cell research and cell therapy. While the adoption has been slow, discoveries including molecular control and buffering of cell stress response to CP as well as the development of new devices for improved sample freezing and thawing are providing for improved CP from both the processing and sample quality perspectives. Numerous studies have described the impact, mechanisms and points of control of cryopreservation-induced delayed-onset cell death (CIDOCD). In an effort to limit CIDOCD, efforts have focused on CP agent and freeze media formulation to provide a solution path and have yielded improvements in survival over traditional approaches. Importantly, each of these areas, new technologies and cell stress modulation, both individually and in combination, are now providing a new foundation to accelerate new research, technology and product development for which CP serves as an integral component. This chapter provides an overview of the molecular stress responses of cells to cryopreservation, the impact of the hypothermic and cell death continuums and the targeted modulation of common and/or cell specific responses to CP in providing a path to improving cell quality.

Biobanking: The Future of Cell Preservation Strategies.

With established techniques cryopreservation is often viewed as an "old school" discipline yet modern cryopreservation is undergoing another scientific and technology development growth phase. In this regard, today's cryopreservation processes and cryopreserved products are found at the forefront of research in the areas of discovery science, stem cell research, diagnostic development and personalized medicine. As the utilization of cryopreserved cells continues to increase, the demands placed on the biobanking industry are increasing and evolving at an accelerated rate. No longer are samples providing for high immediate post-thaw viability adequate. Researchers are now requiring samples where not only is there high cell recovery but that the product recovered is physiologically and biochemically identical to its pre-freeze state at the genominic, proteomic, structural, functional and reproductive levels. Given this, biobanks are now facing the challenge of adapting strategies and protocols to address these needs moving forward. Recent studies have shown that the control and direction of the molecular response of cells to cryopreservation significantly impacts final outcome. This chapter provides an overview of the molecular stress responses of cells to cryopreservation, the impact of the apoptotic and necrotic cell death continuum and how studies focused on the targeted modulation of common and/or cell specific responses to freezing temperatures provide a path to improving sample quality and utility. This line of investigation has provided a new direction and molecular-based foundation guiding new research, technology development and procedures. As the use of and the knowledge base surrounding cryopreservation continues to expand, this path will continue to provide for improvements in overall efficacy and outcome.

Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage.

Human mesenchymal stem cell (hMSC) research has grown exponentially in the last decade. The ability to process and preserve these cells is vital to their use in stem cell therapy. As such, understanding the complex, molecular-based stress responses associated with biopreservation is necessary to improve outcomes and maintain the unique stem cell properties specific to hMSC. In this study hMSC were exposed to cold storage (4°C) for varying intervals in three different media. The addition of resveratrol or salubrinal was studied to determine if either could improve cell tolerance to cold. A rapid elevation in apoptosis at 1h post-storage as well as increased levels of necrosis through the 24h of recovery was noted in samples. The addition of resveratrol resulted in significant improvements to hMSC survival while the addition of salubrinal revealed a differential response based on the media utilized. Decreases in both apoptosis and necrosis together with decreased cell stress/death signaling protein levels were observed following modulation. Further, ER stress and subsequent unfolded protein response (UPR) stress pathway activation was implicated in response to hMSC hypothermic storage. This study is an important first step in understanding hMSC stress responses to cold exposure and demonstrates the impact of targeted molecular modulation of specific stress pathways on cold tolerance thereby yielding improved outcomes. Continued research is necessary to further elucidate the molecular mechanisms involved in hypothermic-induced hMSC cell death. This study has demonstrated the potential for improving hMSC processing and storage through targeting select cell stress pathways.

Investigation of Lung Cancer Cell Response to Cryoablation and Adjunctive Gemcitabine-Based Cryo-Chemotherapy Using the A549 Cell Line.

Due to the rising annual incidence of lung cancer (LC), new treatment strategies are needed. While various options exist, many, if not all, remain suboptimal. Several studies have shown cryoablation to be a promising approach. Yet, a lack of basic information pertaining to LC response to freezing and requirement for percutaneous access has limited clinical use. In this study, we investigated the A549 lung carcinoma cell line response to freezing. The data show that a single 5 min freeze to -15 °C did not affect cell viability, whereas -20 °C and -25 °C result in a significant reduction in viability 1 day post freeze to <10%. These populations, however, were able to recover in culture. Application of a repeat (double) freeze resulted in complete cell death at -25 °C. Studies investigating the impact of adjunctive gemcitabine (75 nM) pretreatment in combination with freezing were then conducted. Exposure to gemcitabine alone resulted in minimal cell death. The combination of gemcitabine pretreatment and a -20 °C single freeze as well as combination treatment with a -15 °C repeat freeze both resulted in complete cell death. This suggests that gemcitabine pretreatment may be synergistically effective when combined with freezing. Studies into the modes of cell death associated with the increased cell death revealed the increased involvement of necroptosis in combination treatment. In summary, these results suggest that repeat freezing to -20 °C to -25 °C results in a high degree of LC destruction. Further, the data suggest that the combination of gemcitabine pretreatment and freezing resulted in a shift of the minimum lethal temperature for LC from -25 °C to -15 °C. These findings, in combination with previous reports, suggest that cryoablation alone or in combination with chemotherapy may provide an improved path for the treatment of LC.

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 the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model.

The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various "front end" strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.

An In Vitro Investigation into Cryoablation and Adjunctive Cryoablation/Chemotherapy Combination Therapy for the Treatment of Pancreatic Cancer Using the PANC-1 Cell Line.

As the incidence of pancreatic ductal adenocarcinoma (PDAC) continues to grow, so does the need for new strategies for treatment. One such area being evaluated is cryoablation. While promising, studies remain limited and questions surrounding basic dosing (minimal lethal temperature) coupled with technological issues associated with accessing PDAC tumors and tumor proximity to vasculature and bile ducts, among others, have limited the use of cryoablation. Additionally, as chemotherapy remains the first-line of attack for PDAC, there is limited information on the impact of combining freezing with chemotherapy. As such, this study investigated the in vitro response of a PDAC cell line to freezing, chemotherapy, and the combination of chemotherapy pre-treatment and freezing. PANC-1 cells and PANC-1 tumor models were exposed to cryoablation (freezing insult) and compared to non-frozen controls. Additionally, PANC-1 cells were exposed to varying sub-clinical doses of gemcitabine or oxaliplatin alone and in combination with freezing. The results show that freezing to -10 °C did not affect viability, whereas -15 °C and -20 °C resulted in a reduction in 1 day post-freeze viability to 85% and 20%, respectively, though both recovered to controls by day 7. A complete cell loss was found following a single freeze below -25 °C. The combination of 100 nM gemcitabine (1.1 mg/m2) pre-treatment and a single freeze at -15 °C resulted in near-complete cell death (<5% survival) over the 7-day assessment interval. The combination of 8.8 µM oxaliplatin (130 mg/m2) pre-treatment and a single -15 °C freeze resulted in a similar trend of increased PANC-1 cell death. In summary, these in vitro results suggest that freezing alone to temperatures in the range of -25 °C results in a high degree of PDAC destruction. Further, the data support a potential combinatorial chemo/cryo-therapeutic strategy for the treatment of PDAC. These results suggest that a reduction in chemotherapeutic dose may be possible when offered in combination with freezing for the treatment of PDAC.

The unfolded protein response in human corneal endothelial cells following hypothermic storage: implications of a novel stress pathway.

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4°C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4-8h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.

Morphology of hypoxia following cryoablation in a prostate cancer murine model: its relationship to necrosis, apoptosis and, microvessel density.

The aim of this study is to investigate the tumor tissue changes in terms of hypoxia and demonstrate its relationship to vascularity and apoptosis following therapeutic cryoablation in a prostate tumor murine model. Total 67 male C57BL/J6 mice were assigned into sham-operation group and cryoablation group. Murine prostate tumors (RM-9) were inoculated subcutaneously in a right hind leg and treated with cryotherapy. Of 30 mice, tumor volumes were measured for 12 days following operation. Of 37 mice, tumor tissues were harvested in 24h following operation, and histological/molecular changes were analyzed. Hematoxylin and eosin or immunohistochemical staining were utilized to quantify tumor necrosis, hypoxia (pimonidazole), vascularization (CD31), and apoptosis (cleaved caspase-3). The results showed that cryoablated tumors demonstrated significant delayed growth following treatment compared to controls. Pathological analysis revealed that the severity of hypoxia increased in the cryoablation arm compared to controls. Necrotic and apoptotic populations were also found to be increased in the cryoablation arm (P=0.028 and 0.021). Hypoxia demonstrated a positive correlation with necrosis (r=0.520, P=0.001) and apoptosis (r=0.474, P=0.003), while showing negative correlation with microvessel density (MVD) (r=-0.361, P=0.021). We concluded that in the peripheral areas from the cryoneedle impact site, strong hypoxic responses were found, which may play important role in tumor freezing injury. To our knowledge, this is the first report describing cryoablation-mediated changes of hypoxia at a molecular level in the prostate cancer murine model.

Breast Cancer Cryoablation: Assessment of the Impact of Fundamental Procedural Variables in an In Vitro Human Breast Cancer Model.

INTRODUCTION: Breast cancer is the most prominent form of cancer and the second leading cause of death in women behind lung cancer. The primary modes of treatment today include surgical excision (lumpectomy, mastectomy), radiation, chemoablation, anti-HER2/neu therapy, and/or hormone therapy. The severe side effects associated with these therapies suggest a minimally invasive therapy with fewer quality of life issues would be advantageous for treatment of this pervasive disease. Cryoablation has been used in the treatment of other cancers, including prostate, skin, and cervical, for decades and has been shown to be a successful minimally invasive therapeutic option. To this end, the use of cryotherapy for the treatment of breast cancer has increased over the last several years. Although successful, one of the challenges in cryoablation is management of cancer destruction in the periphery of the ice ball as the tissue within this outer margin may not experience ablative temperatures. In breast cancer, this is of concern due to the lobular nature of the tumors. As such, in this study, we investigated the level of cell death at various temperatures associated with the margin of a cryogenic lesion as well as the impact of repetitive freezing and thawing methods on overall efficacy. METHODS: Human breast cancer cells, MCF-7, were exposed to temperatures of -5°C, -10°C, -15°C, -20°C, or -25°C for 5-minute freeze intervals in a single or repeat freeze-thaw cycle. Samples were thawed with either passive or active warming for 5 or 10 minutes. Samples were assessed at 1, 2, and 3 days post-freeze to assess cell survival and recovery. In addition, the modes of cell death associated with freezing were assessed over the initial 24-hour post-thaw recovery period. RESULTS: Exposure of MCF-7 cells to -5°C and -10°C resulted in minimal cell death regardless of the freeze/thaw conditions. Freezing to a temperature of -25°C resulted in complete cell death 1 day post-thaw with no cell recovery in all freeze/thaw scenarios evaluated. Exposure to a single freeze event resulted in a gradual increase in cell death at -15°C and -20°C. Application of a repeat freeze-thaw cycle (dual 5-minute freeze) resulted in an increase in cell death with complete destruction at -20°C and near complete death at -15°C (day 1 survival: single -15°C freeze/thaw = 20%; repeated -15°C freeze/thaw = 4%). Analysis of thaw interval time (5 vs 10 minute) demonstrated that the shorter 5-minute thaw interval between freezes resulted in increased cell destruction. Furthermore, investigation of thaw rate (active vs passive thawing) demonstrated that active thawing resulted in increased cell survival thereby less effective ablation compared with passive thawing (eg, -15°C 5/10/5 procedure survival, passive thaw: 4% vs active thaw: 29%). CONCLUSIONS: In summary, these in vitro findings suggest that freezing to temperatures of 25°C results in a high degree of breast cancer cell destruction. Furthermore, the data demonstrate that the application of a repeat freeze procedure with a passive 5-minute or 10-minute thaw interval between freeze cycles increases the minimal lethal temperature to the -15°C to -20°C range. The data also demonstrate that the use of an active thawing procedure between freezes reduces ablation efficacy at temperatures associated with the iceball periphery. These findings may be important to improving future clinical applications of cryoablation for the treatment of breast cancer.

Investigation of Bladder Cancer Cell Response to Cryoablation and Adjunctive Cisplatin Based Cryo/Chemotherapy.

Due to a rising annual incidence of bladder cancer, there is a growing need for development of new strategies for treatment. In 2018, the World Cancer Research Fund and other groups reported that there were ~550,000 new cases worldwide of bladder cancer. It has been further estimated that >200,000 individuals die annually from bladder cancer worldwide. Various treatment options exist. However, many if not all remain suboptimal. While the preferred chemotherapeutic options have changed in the past few years there have been few advances in the bladder cancer medical device field. Cryoablation is now being evaluated as a new option for the treatment of bladder cancer. While several studies have shown cryoablation to be promising for the treatment of bladder cancer, a lack of basic information pertaining to dosing (minimal lethal temperature) necessary to destroy bladder cancer has limited its use as a primary therapeutic option. Concerns with bladder wall perforation and other side effects have also slowed adoption. In an effort to detail the effects of freezing on bladder cancer, two human bladder cancer cell lines, SCaBER and UMUC3, were evaluated in vitro. SCaBER, a basal subtype of muscle invasive bladder cancer, and UMUC3, an intermediate transitional cell carcinoma, are both difficult to treat but are reportedly responsive to most conventional treatments. SCaBER and UMUC3 cells were exposed to a range of freezing temperatures from -10 to -25°C and compared to non-frozen controls. The data show that a single 5 minute freeze to -10°C did not affect cell viability, whereas -15°C and -20°C results in a significant reduction in viability 1 day post freeze to <20%. These populations, however, were able to recover in culture. A complete loss of cell viability was found following a single freeze at -25°C. Application of a repeat (double) freeze resulted in complete cell death at -20°C. In addition to freezing alone, studies investigating the impact of adjunctive low dose (1 µM) cisplatin pre-treatment (30 minutes and 24 hours) in combination with freezing were conducted. The combination of 30 minute cisplatin pre-treatment and mild (-15°C) freezing resulted in complete cell death. This suggests that subclinical doses of cisplatin may be synergistically effective when combined with freezing. In summary, these in vitro results suggest that freezing to temperatures in the range of -20 to 25°C results in a high degree of bladder cancer cell destruction. Further, the data describe a potential combinatorial chemo/cryo therapeutic strategy for the treatment of bladder cancer.

The pathophysiology of thermoablation: optimizing cryoablation.

PURPOSE OF REVIEW: To describe the response of prostate cancer to thermal therapies with an emphasis on cryoablative techniques. RECENT FINDINGS: Long-term follow-up studies demonstrate clearly the effectiveness of the use of modern cryoablative techniques in the management of prostate cancer. Recently published American Urology Association Best Practice Guidelines identify prostate cryoablation as both primary and salvage therapies. Recent findings demonstrate the effectiveness of -40 degrees C exposure as lethal to prostate cancer genotypes following a double freeze-thaw encounter. In addition, the use of adjunctive agents to sensitize the cancer to freezing is reported. SUMMARY: Thermal therapeutic options, especially cryoablation, are of growing interest for the treatment of prostatic and renal cancers. The methods of application of cryoablative therapy and the mechanisms of cell death that are attendant to the freezing-thaw encounter are clearly understood. Research focused on the development of freeze sensitizing agents that work adjunctively is of central interest in furthering the efficacy of this therapy.

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

OBJECTIVE: 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. MATERIALS AND METHODS: PC-3 (AI) and LNCaP (AS) were each genetically altered to express the opposite phenotype and subjected to an in vitro freezing model. Viability, caspase inhibitor and Western blot studies were used to determine the basis of the differential responses of AI and AS cells. RESULTS: LNCaP high-passage cells, formed by repeated passage of LNCaP (AS) cells, were AI and showed a phenotypic shift to freeze resistance matching the freeze response of PC-3 cells (AI). While stably transfected androgen receptor (AR)-transfected cells (PC-3 AR) had a freezing sensitivity similar to that of the LNCaP (AS) cell line. Importantly, AI cell lines survived and recovered from freezing exposure to temperatures as low as -40 degrees C whereas AS cell lines did not. Caspase inhibition studies and related fluorescent probes showed an elevated level of apoptotic involvement in both AS cell lines after freezing compared with their AI counterparts. Western blot analysis showed that AR expression was modified after exposure to freezing. CONCLUSION: This study suggests that AS cancers may be far more sensitive to a freezing insult and this might be linked to elevated apoptosis and caspase activity. As such, cryoablation may prove most effective in cancer cells that have not yet progressed to a more resistant AI phenotype, but both generic variants can be fully ablated at sufficiently low temperatures.

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.

Defeating Cancers' Adaptive Defensive Strategies Using Thermal Therapies: Examining Cancer's Therapeutic Resistance, Ablative, and Computational Modeling Strategies as a means for Improving Therapeutic Outcome.

BACKGROUND: Diverse thermal ablative therapies are currently in use for the treatment of cancer. Commonly applied with the intent to cure, these ablative therapies are providing promising success rates similar to and often exceeding "gold standard" approaches. Cancer-curing prospects may be enhanced by deeper understanding of thermal effects on cancer cells and the hosting tissue, including the molecular mechanisms of cancer cell mutations, which enable resistance to therapy. Furthermore, thermal ablative therapies may benefit from recent developments in computer hardware and computation tools for planning, monitoring, visualization, and education. METHODS: Recent discoveries in cancer cell resistance to destruction by apoptosis, autophagy, and necrosis are now providing an understanding of the strategies used by cancer cells to avoid destruction by immunologic surveillance. Further, these discoveries are now providing insight into the success of the diverse types of ablative therapies utilized in the clinical arena today and into how they directly and indirectly overcome many of the cancers' defensive strategies. Additionally, the manner in which minimally invasive thermal therapy is enabled by imaging, which facilitates anatomical features reconstruction, insertion guidance of thermal probes, and strategic placement of thermal sensors, plays a critical role in the delivery of effective ablative treatment. RESULTS: The thermal techniques discussed include radiofrequency, microwave, high-intensity focused ultrasound, laser, and cryosurgery. Also discussed is the development of thermal adjunctive therapies-the combination of drug and thermal treatments-which provide new and more effective combinatorial physical and molecular-based approaches for treating various cancers. Finally, advanced computational and planning tools are also discussed. CONCLUSION: This review lays out the various molecular adaptive mechanisms-the hallmarks of cancer-responsible for therapeutic resistance, on one hand, and how various ablative therapies, including both heating- and freezing-based strategies, overcome many of cancer's defenses, on the other hand, thereby enhancing the potential for curative approaches for various cancers.

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.