Characterization of canine mastocytoma cell response to cryoablation

Mastocytoma represents 16%-21% of all skin cancers in dogs, making it the most common form of cutaneous cancer [1]. Mast cells are generated from hematopoietic stem cells, reside in connective tissues, and comprise a necessary component of the immune response system [2]. The cause of mast cell tumors (MCT’s), whether benign or malignant, is unknown. The formation of cancerous MCT’s occurs frequently in dogs with median age of onset between 8-9 years [3,4]. One of the most predictive factors in MCT development and subsequent aggressiveness is breed. The majority of reports agree that boxers, Labrador retrievers, pugs, golden retrievers and bulldog breeds present with the highest incidence of MCT’s [3-7]. Though there are a number of factors which can infl uence prognosis, including breed, c-kit mutation status and age, many veterinarians agree that the most predictive factor in MCT aggressiveness is tumor grade. Though there Abstract

can be wide variability in the physical appearance of cutaneous MCT's, grade I tumors are often surgically excised with small margins for successful outcome, while grade II and III tumors can present a greater challenge. In general, a solitary MCT with wide surgical excision margins carries a positive prognosis. However, the surgical excision can cause the release of a large amount of histamine and other cytokines which may result in complications such as systemic shock or anaphylaxis [8]. Additionally, in some cases, wide surgical margins are not possible. Therefore, other treatments such as chemotherapy and radiotherapy have been considered to achieve complete remission [1,9,10]. Surgical excision typically requires the animal to undergo general anesthesia. This procedure is both costly and can be high risk in certain geriatric patients. In these cases, the associated morbidity, risk and cost severely limits the treatment options for MCT's, which can result in elected non-treatment of the disease.
An alternative treatment option for cutaneous MCT is Cryoablation (CA). CA is the use of ultra-low temperatures to provide targeted freezing and subsequent destruction of cancerous or non-cancerous tissues. As a minimally invasive modality with less side effects than radiation and chemotherapy, CA is an attractive option for the treatment of solid tumors [11][12][13]. Cryosurgical techniques include ultrasound guided imaging to visualize ice ball formation as well as temperature monitoring by the placement of multiple thermocouples [13][14][15]. CA has been applied with a high degree of success in the treatment of skin, prostate, renal, liver, breast, and bone cancers, among others in both humans and animals [16][17][18][19][20][21][22][23]. To this end, several reports have demonstrated CA is as effective or superior to other cancer treatment options including surgery, radiotherapy and chemotherapy [12,[24][25][26]. In addition to being an effective cancer therapy, CA has the benefi t of low incidence of postoperative morbidity, low levels of pain, and is often utilized as a outpatient surgical approach [12,24,25]. CA also offers the benefi t of being a minimally invasive procedure thereby reducing and in some cases eliminating the need for general anesthesia [13,14]. Cryosurgery also has a long but limited history of application in the veterinary setting. Some of the fi rst reports of CA in small animals were in the treatment of dermatological conditions in dogs and cats [27][28][29]. Over time, the range of CA treated neoplasms has expanded to include less accessible organs. CA has been reported in numerous cases as early as 1971 for the treatment of laryngeal neoplasms [30][31][32]. Additionally, cardiac CA procedures have been performed to treat canine arrhythmias [33], as well as to test the feasibility of epicardial ablation [34]. One study performed on canine renal neoplasms determined the procedure was a safe and feasible treatment option [35] and a case report described successful CA of a canine intranasal tumor [36]. These studies illustrate the use of cryosurgery as a possible treatment option for a variety of disease states.
The reported benefi ts and effectiveness of CA in both the veterinary and human cancer therapy arenas suggests that CA may represent a viable option for MCT. While a potential option, there is little information in the primary literature as to the effectiveness and response of MCT's to CA. Numerous studies have detailed the effects of freezing in various cell systems demonstrating a differential response to similar freezing insults. Cell death due to freezing is not only a consequence of freeze rupture, but is also related to differential freeze-induced cell stress response which is believed to dictate therapeutic outcome [15,26,37]. Studies have also demonstrated that different molecular dispositions (sub-types) of the same cancer tissue type can respond differently CA [38][39][40]. We have published studies using numerous cell models investigating this phenomenon [12,26,40]. For instance, Snyder, et al., have shown that breast cancer cells tolerate freezing to -15°C, whereas cardiac cells can withstand -30°C [41,42]. Other studies have demonstrated prostate cancer cells tolerate -40°C to -80°C [12,43,44]. Studies have also established the translatability of in vitro data to clinical outcome and protocol establishment. For example, in vitro studies by Gage, et al.. [12,13,45], led to the clinical target of -40°C for prostate cancer. Studies by Snyder, et al., [41] and others [46], established -30°C as the target for cardiac ablation. More recently, in vitro cell and tissue engineered model studies proved critical to the development of the percutaneous SCN system for prostate cancer [47,48]. Given the potential of CA, we conducted a series of cellular and molecular based analyses of mast cells following exposure to sub-freezing temperatures using the C2 cell line in order to gain an understanding of MCT cell response to CA. The C2 mast cell line was derived from a canine with end-stage illness after multiple metastases were observed and thereby represents an aggressive and advanced form of MCT cancer [49]. The temperature range of -5°C to -25°C was selected for investigation as it represents the reported transitional temperature range between complete cell death and survival in a number of cancers [11,13,15,50,51]. We hypothesized that delivery of an effective freeze dose (temperature and time) would result in complete C2 cell destruction. Further, we hypothesized that both apoptotic and necrotic pathways are activated following exposure to mild sub-freezing temperatures and that this molecular signaling plays a critical role in the extent of cell death.

Freezing protocol
Costar strips (Corning) with 75μl medium per well or 35mm Citation: Santucci  Falcon dishes with 1.5ml medium per dish were placed on a precooled block within a circulating temperature-controlled bath at a preset temperature. Samples were frozen to temperatures of -5, -10, -15, -20 or -25°C. Sample temperature was monitored with a type T thermocouple (Omega, Stamford, CT, USA) and ice nucleation was initiated by liquid nitrogen vapor (crystallized water vapor) when samples reached -2°C (±1°C).
Samples were held for 3, 5, or 10min and then allowed to thaw at room temperature (10min) before return to normothermic culture (37°C) to allow for system recovery and downstream assessment.

Cell viability
Sample viability was assessed post-thaw using the

Caspase inhibition assay
Pan caspase inhibitor VI (EMD Millipore, Billerica, MA, USA) was reconstituted in dimethyl sulfoxide at a concentration of 10mM and diluted to a fi nal concentration of 10μM in culture media prior to use. Inhibitor was applied in fresh medium to C2 subcultures in costar plates and kept at 37°C for 30minutes prior to freeze exposure.

Fluorescence microscopy
Samples were frozen as described and then fl uorescence

Impact of temperature and hold time on C2 survival
The C2 canine mastocytoma cell line was exposed to temperatures ranging from -5°C to -25°C and cell viability was assessed at 24h post-thaw (Figure 1). Regardless of exposure time (3, 5, or 10minutes), samples exposed to -5°C and -10°C demonstrated no signifi cant cell loss compared to controls (<5%; -5°C 5min p=0.62, -5°C 10min p=0.05, -10°C  5min p=0.37, -10°C 10min p=0.04). Exposure to -15°C resulted in a signifi cant increase in cell death, 24% (±1.8), 57% (±2.0), and 71% (±1.14) cell loss, when held for 3, 5, or 10minutes, respectively (Figure 1 (3min data not shown)) (p<0.001 for all samples). All samples, however, were found to recover to control levels by day 3 post-thaw. Exposure to -20°C resulted in a further increase in cell death in a time-dependent manner. Following exposure to -20°C cell survival was 12% (±1.4) (3min), 11.9% (±0.9) (5min), and 5.0% (±0.2) (10min) of untreated controls following 3, 5 or 10minute holds (respectively) at 24h post-freeze. While a low level of cell survival was noted following exposure to -20°C (5-12%), no re-growth was noted in any of the -20°C samples over the 3 day recovery period. Exposure to -25°C resulted in complete cell death regardless of the hold time. These data indicated that exposure time is a factor in C2 cell ablation at temperatures between -15°C and -20°C. However, when exposed to temperatures of ≤-25°C, complete ablation was achieved regardless of the exposure time.

Fluorescence microscopy correlates apoptosis and necrosis post-freeze
To visually assess the temporal regulation of cell death, fl uorescence microscopy analysis was conducted using probes specifi c for apoptosis (YO-Pro-1) or necrosis (Propidium Iodide (PI) at 1, 4, 8, and 24h post-thaw following exposure to -10°C, -15°C, and -20°C. The analysis confi rmed the relative level of cell death (apoptosis and necrosis) and viability at 24h within each population as was found in the viability studies ( Figure 2). Analysis at 1h following a -10°C freeze revealed an increase in the levels of apoptosis/necrosis over that of untreated controls. Continued presence of apoptotic and necrotic populations was observed 4h and 8h post-freeze. Yet by 24h post-freeze, -10°C samples did not differ signifi cantly from controls. Following freezing to -15°C a signifi cant population of apoptotic and necrotic cells were apparent as early as 1h post-freeze. A continued increase in both populations was observed at 4h and 8h post-freeze resulting in a signifi cant decline in viability at 24hours post-freeze. Samples exposed to -20°C also revealed an even greater level of positive staining for apoptosis and necrosis at 1, 4 and 8hours post-freeze resulting in <10% survival at 24hours, which was consistent with the metabolic activity assessment. Interestingly, the levels of cell death were found to peak at 8 hours post-thaw in both the -20°C and -15°C samples illustrating the involvement of a delayed cell death response.

Molecular analysis of relevant proteins
With the identifi cation of a delayed cell death response (apoptotic and necrotic activity) via fl uorescence microscopy, temporal (1, 4, 8 and 24hours) analysis of cell stress protein levels following freezing at -10°C and -15°C was conducted via western blotting (Figure 3). Protein analyses revealed reductions in the anti-apoptotic protein Bcl-2 levels following freezing when compared to controls. The greatest decrease in Bcl-2 levels was seen within 1hour following freezing to -10°C and -15°C (7 fold and 13 fold reductions, respectively; Table 1). Analysis of the pro-apoptotic Bax protein revealed no signifi cant changes following freezing (data not shown). This was not unexpected, as it is generally not the amount of Bax protein, but rather the translocation of Bax from the cytosol to mitochondrial pores that initiates mitochondrial mediated apoptosis. Analysis of the canonical pro-survival protein Akt (protein kinase B) revealed a decrease in phosphorylation and subsequent activation of Akt following freeze exposure. The greatest decrease in phosphorylated AKT (pAKT) was noted Figure 1: Assessment of C2 canine mastocytoma cell viability following freezing. C2 cultures were exposed to a 5 or 10 minute cooling regime yielding the nadir temperatures of -5°C, -10°C, -15°C, -20°C, or -25°C. Following thawing, sample viability (metabolic activity) was assessed at 24h, 48 and 72h post-freeze and compared to a pre-freeze controls. Results indicate that following freezing to -25°C there was complete C2 cell destruction.  Table 1).

Inhibition of caspases decreases the contribution of apoptotic cell death processes post-freeze
With the identifi cation of a delayed cell death and alterations in apoptosis related proteins following freezing, assessment of the impact of caspase inhibition on cell survival following freezing was conducted. This analysis was performed as a fi rst step in quantifying the level of apoptotic involvement in cell death following freezing. C2 cells were pre-treated with a pan caspase inhibitor for 30 minutes prior to freeze to -10°C, -15°C and -20°C. As with freezing only samples, exposure to -10°C in the presence of caspase inhibitors did not signifi cantly impact cell viability (Figure 4). Exposure to -15°C in the presence of caspase inhibition resulted in a 13.6% improvement in overall survival (26.6% increase) compared to non-inhibited -15°C samples (64.42% (±1.6) vs. 50.84% (±1.7), p<0.001 (Figure 4)). These results indicated that the apoptotic caspases signaling contributed to cell death following a -15°C freeze. When samples were exposed to -20°C in the presence of caspase inhibition no signifi cant change in cell survival was noted. Figure 3: Immunoblot analysis of cellular proteins following freezing to -10°C or -15°C. Samples were evaluated for alterations in phospho-Akt, Bcl-2, PARP and α-tubulin (loading control) temporally following freezing. A decrease in phosphorylated Akt (pAKT) was observed following freezing to -10°C and was found to be even greater following exposure to -15°C. Alterations in Bcl-2 and PARP (poly-ADP-ribose) protein levels were also observed at 4 and 8 hours post freeze suggesting the activation of a delayed mitochondrial cell death signaling response.  Figure 4: Assessment of the impact of caspase inhibition on C2 survival following freezing. Samples were treated for 30 mins with pan caspase inhibitor and then exposed to a 5 minute freeze to -10°C, -15°C, or -20°C. Caspase inhibition in samples exposed to -15°C resulted in an increase in cell survival compared to noninhibited -15°C samples, indicating apoptosis may play a signifi cant role in C2 death following freezing.

Primary mast cell tumor isolate response to low temperature exposure
In addition to the in vitro C2 studies, analysis of a primary grade 2, stage 2 canine MCT tumor sample was conducted.
The MCT sample was excised via fi ne needle aspirate biopsy procedure, sectioned, weighed and then exposed to -20°C ( Figure 5A). Following freezing, tissue sections were placed into ex vivo culture and analyzed for tissue viability at 1 and 24h post-thaw ( Figure 5B). The response of the tissue samples was found to be similar to the C2 in vitro results. Tissue sample viability at 1hour post-thaw was found to be 40% of nonfrozen controls. Following 24h of recovery, a further decrease in viability to 12% was observed. While a pilot ex vivo case study, the experimental results were found to correlate well with the in vitro freeze response data obtained with the C2 cell model following exposure to -20°C for 3 minutes.

Discussion
CA is an effective cancer treatment modality. However, the application of CA in veterinary medicine has been limited. In this study we investigated the effects of CA on MCT cells in an attempt to characterize MCT cell response to freezing, as well as identify the critical target temperature necessary to achieve total cell destruction. The data from this study demonstrated that CA was effective at destroying MCT cells when temperatures Citation: Santucci  These data correlate well with previous reports from our laboratory on CA in cell systems such as prostate and renal cancer [39,52], establishing that both the nadir temperature and time at temperature play a role in overall outcome.
When performing solid tumor cryosurgery, the temperature typically reached at the center of a cryogenic lesion ranges between -80°C and -180°C [11,47,50,51]. Extending radially from the center of the cryolesion, temperatures increase until the edge of the frozen mass, where temperatures are 0°C, nominally ( Figure 6). The corresponding isothermal gradient (temperature gradient profi le) within the frozen mass varies depending on application time, freeze repetition, and cryogen utilized [11,47,50,51]. As such, it is important to understand the characteristics of the cancer cell response to the range of temperatures associated with a cryogenic lesion. Our studies focused on the warmer sub-freezing temperatures associated with the periphery (outer edge) of the freeze lesion, as it is generally accepted that temperatures below -40°C result in complete cancer cell lysis through physical ice rupture [12,13].
The thermal range of 0°C to -40°C is characterized by a region of heterogenic cell responses which includes cell lysis, activation of necrotic and apoptotic pathways, as well as some cell survival [11,39,45]. Numerous reports by our group as well as others have demonstrated the transition from complete cell death to complete cell survival within this region [39,53,54,43].
Given these facts, it is important to identify the critical lethal temperature for MCT cells as well as understand the molecular responses of this cancer to CA to yield more effective treatment.
To this end, the data revealed the minimal lethal temperature for C2 MCT cells was -25°C in vitro whereas the -15°C to -20°C range resulted in a heterogeneous mix of necrotic, apoptotic and surviving cell populations. The data also demonstrated that regardless of the exposure temperature, necrosis was the primary mode of cell death. Apoptotic contribution to cell death following freezing has been reported primarily at warmer, sub-freezing temperatures [43,[55][56][57]. In this study, apoptotic involvement was found following exposure to -15°C and was   Citation: Santucci [58][59][60][61]. Many mutations in this gene cause the constitutively active form of the receptor, signaling the cell to proliferate even in the absence of its ligand, stem cell factor [58]. As such, c-kit specifi c small molecule inhibitors have been used with some success. Drug resistance, however, could become an issue in vivo [60]. Previous studies from our laboratory have shown the success of adjuvants used for cryosensitization [37,57,[62][63][64][65].
These and other studies have detailed the benefi t of adjunctive  [57,75,76,78]. Based on these reports coupled with the identifi cation of a delayed molecular cell death response activated following freezing of C2 cells, the use of adjuvant agents, including chemotherapy and immunotherapy agents as well as RTK inhibitors, in combination with CA is an area that may also provide benefi t for treatment of MCT's and as such should be explored in future studies.
In conclusion, the data from this study suggest that CA has the potential to be an effective therapeutic option for the The data presented herein suggest that CA may provide a minimally invasive, rapid therapeutic option for treating mast cell tumors. As such, additional studies in the application of CA for MCT treatment are warranted.