Q6

Characterization of Pancreatic Cancer Cell Thermal Response to Heat Ablation or Cryoablation

Introduction Pancreatic cancer (PaCa) is currently the fourth leading cause of cancer-related deaths in
the United States and the eighth worldwide. It is projected that over 48,000 individuals will be
diagnosed and over 40,000 will succumb to PaCa in 2015 [86]. The high lethality rates of PaCa are due to
a lack of effective treatment options [87]. The most common treatment of PaCa is a combination of
chemotherapy (e.g. gemcitabine, 5-FU, etc) and radiation [88-91]. This combinatorial regimen is
insufficient to provide a cure to PaCa and often is merely palliative for patients [87,92-93]. The reasons
why many cancer cells, including PaCa, are resistant to conventional therapies can be attributed to
“hallmarks of cancer,” as well as the presence of cancer stem cells [6]. Specifically, these hallmarks are:
resisting cell death, sustaining proliferative signaling, evading growth suppressors, activation of
metastasis and invading cells, inducing angiogenesis, and enabling immortalized replication and
proliferation [6]. One of these maxims, resisting cell death, directly relates to the normal cell death
functions of apoptosis and necrosis. Apoptosis, otherwise known as programmed cell death, is often
suppressed in most cancer types [6,13-14,94-95]. Necrosis, an inflammation-based cell death, results in
the circulation of intracellular contents around the site of cell death [6,13-14,96]. It is hypothesized that
in some cases, the activation of necrosis leads to further inflammation 23 and possibly the spread of
cancer-promoting chemokines [6,13-14]. With an improved understanding of cell death mechanisms in
solid cancers, researchers have become better able to treat these diseases by accounting for the
aforementioned hallmarks. Research has improved the survival rates of individuals treated for many
cancers. PaCa stands as a notable exception with 5-year survival rates remaining in the single digits and
an average lifespan of 6 months post-diagnosis [86]. As conventional options have not provided an
effective treatment for PaCa, there is a need for the development of new treatments to extend patient
lives and work towards a cure. Thermal therapies, such as cryotherapy and hyperthermia, represent
areas of continued growth as cancer treatments over the last decade [90]. In many cases, cancer cells
are found to be more susceptible to thermal exposure than drug-related therapies. This is due to a cell
being physically stressed to induce cell death, bypassing many other genetic based cancer defense
mechanisms. Freezing tissues causes cell rupture, hypoxia, ischemia, reperfusion, and extreme
hypothermia [43-44,97-100]. Heating cells results in protein denaturation, hyperthermia, cell membrane
failure, scarring, and destruction of tissue [54,101]. As the technologies used to apply a thermal injury
have improved, so has the effectiveness of the respective therapies. Thermal techniques possess a
unique ability in that, unlike surgical resection, they are less invasive and require shorter recovery times
[53,100-101]. This is a critical factor as the location of the pancreas is a major challenge in the treatment
of PaCa [102]. Cryotherapy is a minimally invasive technique that uses a cryogen, such as liquid nitrogen
(LN2) or argon gas, to freeze diseased tissue to ultracold temperatures [43-44,97- 100]. Cryoablation
subjects tissues to multiple cellular stresses beyond freezing, 24 including hypoxia, osmotic imbalance,
ischemia, and other forms of molecular stress [44,97,103-105]. One of the main uses of cryotherapy is
the treatment of prostate cancer. In 2008 the American Urological Association (AUA) published a best
practice statement on cryotherapy for treatment of prostate cancer [47]. The AUA and others also
recognize cryotherapy as an effective method of treatment for renal cancer [47,88,106]. Although
cryotherapy of the pancreas has been limited within the US and reserved primarily for pancreatitis,
cryotherapy offers great potential for the treatment of PaCa [49,107-109]. Heat ablation, such as
radiofrequency (RFA) and high-frequency ultrasound (HIFU), are also minimally invasive techniques that
induce a state of hyperthermia (>37°C) in cells and tissues. RFA has been utilized for the treatments of
liver, kidney and other solid tumors, but has not been used to a great extent in the treatment of PaCa
[54,57]. While this technique is relatively untested in PaCa, given the reported success in other cancers,
the use of heat ablation may also provide for an effective course of treatment for PaCa. Given the ability
of cancer to evade many molecular based therapeutic strategies coupled with the multi-faceted insult
(physical and molecular) provided by thermal therapies, we investigated the response of PaCa cells to
both cryoablation and hyperthermia. We hypothesized that understanding the characteristics of PaCa
cell response to thermal ablation would result in the identification of an improved therapeutic guidance
protocol for the treatment of PaCa. Given that the response of PaCa cells to thermal ablation has not
been characterized, we investigated PaCa cell response to freezing and heating in an effort to determine
the minimum thermal exposures necessary 25 to cause complete cell destruction. The results herein
suggest that thermal therapies may be a viable alternative option for the treatment of PaCa. Materials
and Methods Cell Culture: PANC-1 (CRL-1469) and BxPC-3 (CRL-1687) cells lines were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). Dubecco’s Modified Eagle’s Medium was used
to culture PANC-1 cells whereas RPMI-1640 medium was used for BxPC-3 (Caisson Laboratories, Inc,
Logan City, UT). Cell culture media was supplemented using 10% Fetal Bovine Serum (Atlanta Biologicals,
Lawrenceville, GA, USA) and 1% Penicillin/Streptomycin (Corning, Inc, Corning, NY). Cells were seeded
onto Costar 96 stripwell plates (Corning) at a cell density of approximately 3.75x104 cells/cm2 with
appropriate medium 24 hours prior to experimentation. Thermal Exposures: Culture medium was
exchanged with 75µl/well fresh media 30 minutes prior to experimentation. 8-well strips were placed
into pre-cooled aluminum blocks in a temperature controlled bath set to achieve and maintain target
temperatures. For freezing experiments, samples were exposed to -10, -15, -20 and -25°C for 5 minutes.
When sample temperature approached -2°C, ice nucleation was initiated with LN2 vapor to prevent
supercooling of samples. Following freezing, samples were placed at room temperature (RT) for 10
minutes to passively thaw before being returned to standard culture conditions. For heating
experiments, samples were exposed to 45, 48 and 50°C for 5 minutes. After the heat exposure, samples
were removed and placed at RT for 1 minute before returning to culture conditions. Thermal profiles of
samples under

Dual Thermal Ablation of Pancreatic Cancer Cells as an Improved Combinatorial Treatment Strategy
Introduction Pancreatic Cancer (PaCa) is one of the deadliest cancers in the world today. It is estimated
that 1 in 67 individuals will be diagnosed with PaCa in their lifetime [3,86]. Based on the latest statistics
from the National Cancer Institute, over 53,000 individuals are diagnosed with and over 43,000 die
annually from PaCa in the US alone [3,86]. Beyond the standard cancer treatment options,
chemotherapy and radiation, there has been minimal progress made in improving PaCa patient survival.
One of the latest attempts is the development of FOLFIRINOX, a combination of five chemotherapeutic
agents, which has shown some progress but is associated with toxic side effects if not managed properly
[117-122]. With the need to develop more effective treatment options, alternative approaches, such as
thermal therapy, may offer a viable strategy [123]. Cryotherapy is a minimally invasive technique that
utilizes a cryogen, such as argon gas or liquid nitrogen (LN2), to freeze a tissue, reaching temperatures
well below -100°C nearest the cryoprobe [104,124-125]. This ultra-cold environment causes multiple
physical and molecular stressors, such as ice rupture, osmotic imbalances, dehydration, oxygen
deprivation, and ischemia/reperfusion injury, among others [43,53,97,104,124- 125]. The primary
advantage of cryotherapy over radiation and chemotherapy is that cryoablation can be directly applied
to the diseased tissue. Further, unlike other treatment 53 strategies, molecular drug resistance can be
overcome with thermal injury [104]. Although cryoablation is not a mainstream thermal technique for
PaCa, it is a primary treatment option in other cancers [44,90,99,120,126-129]. In 2008, the American
Urological Association published a best practice statement on prostate cancer, recommending
cryosurgery as a primary treatment option [47]. Furthermore, the AUA has deemed it effective for the
treatment of renal cancer [47,128]. As our understanding of cryotherapy has improved, so has the
technology required to perform this technique, offering enhanced performance and improved patient
outcome. Another strategy for cancer ablation is that of heat based therapies, such as radio frequency
ablation (RFA) and high-frequency ultrasound (HIFU). Heat based treatments subject diseased tissue to
hyperthermia, inducing coagulative necrosis, protein denaturation, cytoskeletal breakdown, and severe
connective tissue damage [53-57,59]. Solid tumors in the liver and kidneys have been effectively treated
using hyperthermia. Yet, numerous side effects have been reported [54,57,59,128,130-131]. Despite
limited use of heat ablation therapy, we hypothesize that based on success in other tissues,
hyperthermia would provide for the effective ablation of PaCa [90,123,132-133]. Thermal therapies are
normally utilized in a context where one technique is applied to treat a given tumor based on the
patient’s specific needs. One advantage of thermal therapies over conventional treatments is that
thermal techniques overcome the “hallmarks of cancer” which challenges other treatment regimens,
such as chemotherapy efficacy [6,104,134]. Given that heat and cryoablation operate on opposite ends
of the thermal spectrum, it has been suggested that they may be utilized in combination as a single
treatment regime [114]. Similar to FOLFIRINOX as a combination chemotherapy 54 treatment regime,
we propose that a combinatorial thermal strategy may improve cancer ablation. It has been suggested
by Shafirstein et al that such a combination could result in improved tumor ablation [135]. In contrast to
the thermal model utilized by Shafirstein et al where breast cancer was frozen prior to a rapid heating,
we investigated first heating the target tissue followed by freezing to achieve cancer ablation. We
hypothesize that the combined exposure of heating and freezing on PaCa would act synergistically,
increasing the amount of cellular damage, resulting in rapid and complete PaCa destruction.
Furthermore, we hypothesize that by applying heat followed by freezing, the collateral damage
associated with heating would be reduced by providing a heat sink immediately following heat
exposure. Our results suggest that DTA may serve as a more effective means of targeting PaCa versus a
mono thermal therapy approach. Material and Methods Cell Culture: PANC-1 (CRL-1469) and BxPC-3
(CRL-1687) cells lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD).
Dubecco’s Modified Eagle’s Medium was used to culture PANC-1 cells and RPMI-1640 medium was used
for BxPC-3 (Caisson Laboratories, Inc, Logan City, UT). Cell culture media was supplemented using 10%
Fetal Bovine Serum (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% Penicillin/Streptomycin
(Corning, Inc, Corning, NY). Cells were seeded into Costar 96 stripwell plates (Corning) at a cell density of
approximately 3.75x104 cells/cm2 with appropriate medium 24 hours prior to experimentation. Thermal
Exposures: Culture medium was exchanged with 75µl/well fresh media 30 minutes prior to
experimentation. Eight-well strips were placed into pre-cooled or heated aluminum blocks in a
temperature-controlled bath set to reach target temperatures. For 55 freezing experiments, samples
underwent a 5-minute exposure in baths set to achieve temperatures of -10, -15 and -20°C. When
sample temperature approached -2°C, ice nucleation was initiated with LN2 vapor to prevent
supercooling of samples. Following freezing, samples were placed at room temperature (RT) for 10
minutes to passively thaw before being returned to standard culture conditions. For heating
experiments, samples underwent a 5-minute exposure in baths set to achieve temperatures of 45, 48
and 50°C. After the heat exposure, samples were removed and placed at RT for 1 minute before
returning to culture conditions. Thermal profiles of samples under each condition were recorded using a
T-Type Thermocouple (Omega Engineering, Stamford, CT) at one second intervals during exposure.
Double Exposure: Freezing exposure was performed using temperatures of -10, -15 and - 20°C. Samples
were subjected to a 5-minute exposure, passively thawed at RT for 5 minutes, and subsequently
refrozen at the same temperature for an additional 5 minutes. After the second exposure, samples were
passively thawed for 10 minutes at RT before returning to standard culture conditions. Heating exposure
used temperatures of 45, 48 and 50°C. Samples were heated for 5 minutes as described, placed at RT for
one

Molecular Basis of Treatment using Heat Ablation, Cryoablation or Dual Thermal Ablation in Pancreatic
Cancer Cells Introduction Pancreatic cancer (PaCa) remains one of the most lethal cancers in the world.
It is estimated that in 2018 over 53,000 individuals were diagnosed while over 43,000 died from PaCa
[86]. Traditional treatment options, such as chemotherapy and radiation, have provided limited success
in the treatment of PaCa and so a pressing need exists for the development of new strategies to improve
patient outcome [86]. One alternative treatment option is that of thermal therapies, including
cryotherapy and hyperthermal (heat) ablation. Cryoablation utilizes subzero temperatures that results in
ice crystal formation, changes in osmolarity and other physically damaging events which ablate the
cancer [43-44,97,99]. Heat ablation can be delivered via multiple avenues, including radiofrequency
ablation (RFA), microwave ablation, or high-intensity focused ultrasound (HiFU) [152]. Although none of
these thermal ablative therapies are yet a gold standard treatment option, there has been an increase in
utilization as more evidence in support of their effectiveness are reported [44,54,57,99,152]. We
previously identified the minimum lethal dose (time and temperature) required to achieve complete
destruction of several PaCa cell lines in vitro utilizing freezing and heat ablation [136,153-156]. While
these temperatures and times are achievable in the core of the lesion, tissue in the periphery often
don’t experience these temperatures and cells can survive. It is this region where concerns of
incomplete ablation and cancer reoccurrence are a concern. As such, there 79 remains a need to
improve cell death at moderate, sublethal temperatures experienced in the outer portion of an ablated
lesion. One such technique is the use of Dual Thermal Ablation (DTA). This method utilizes both heating
and freezing in tandem to deliver a destructive thermal load to the targeted area using temperatures
that would not be considered lethal when used as a single thermal modality. We have previously
described the impact of DTA on PaCa cells in support of its use for the treatment of PaCa and other
cancers [114,157]. To extend this previous study, we aimed to understand the molecular mechanisms by
which DTA is deemed more effective than either freezing or heating when applied as a monotherapy. By
observing any significant changes in protein expression of key cell death and stress pathways following
freezing, heating or DTA, a more thorough molecular understanding of how DTA exerts its ablation
effect will be better understood. Utilizing adjuvants or manipulating the tumor environment to increase
cancer susceptibility to thermal stress is another such path which has shown promise in increasing cell
death following exposure to mild sub-ablative temperatures [152,158]. One strategy to improving
cellular responsiveness to thermal ablation is to target molecular pathways which are activated within a
cell in response to treatment. One pathway that has garnered recent attention is autophagy. Autophagy,
a regulatory cellular process, normally acts to degrade unnecessary or damaged proteins, organelles and
intracellular contents so they can be utilized as energy resources for the cell [69,159- 160]. Several
studies have shown that autophagy plays a role in cell death and, as a result, autophagy has been
classified as programmed cell death type two; whereas apoptosis is type one [73,160-163]. Autophagy
functions by monitoring nutrient status in the cell via 80 mTOR, which when inhibited signals the ULK
complex to activate Beclin-1 to sequester ATG and LC3 proteins to generate a phagophore. The
phagophore then engulfs intracellular contents (i.e. ER, misfolded proteins, etc.) sealing these contents
within the fully formed autophagosome. The autophagosome then fuses with a lysosome which breaks
down the autophagosome and its contents, thereby providing energy and nutrients to the cell [68,70-
71,159]. The purpose and functioning of autophagy garnered recent attention as the 2016 Nobel Prize in
Physiology or Medicine was awarded to Yoshinori Ohsumi for his discoveries regarding autophagy’s
mechanisms of action and their connections to health and disease [72]. The role of autophagy in relation
to cancer has proven more difficult to decipher. It has been reported that autophagy contributes to
either cell survival or death processes (the autophagy paradox) depending on the type of cancer
[29,70,161]. Furthermore, cancer cells have been shown to suppress autophagy due to its anticancer
impact as well as up-regulate autophagy to provide an additional source of nutrients [161,164]. In the
case of PaCa, it has been reported that upregulated autophagy leads to poor patient prognosis [71,73-
76]. Studies have hypothesized this is due to the ability of autophagy to serve a protective role thereby
preventing cell death (apoptosis) [71,74-76,165]. It has been further postulated that the protective role
of autophagy in PaCa acts through repairing cellular damage following exposure to various external
stressors, resulting in cellular resistance to chemotherapy [75,164-165]. If correct, this hypothesis may
explain PaCa’s resilience to standard treatment options, further justifying exploration of therapeutic
strategies focused on modulating autophagy activity. To address this issue, we investigated the impact
of hyperthermal and cryoablation exposure on autophagic activity within PaCa cells. To hinder the ability
of 81 autophagy to rescue PaCa cells, we selected the autophagy inhibitor chloroquine (CQ) which acts
to neutralize endosomal pH, halting the acidification of the autophagosome by the lysosome in the later
stages of autophagy. It has been previously proposed that blocking autophagy in PaCa causes an
increase in cellular stress, therefore resulting in increased cell death through inhibition of the recovery
pathway needed for cancer cell proliferation [166-167]. We hypothesized that if autophagy is, in fact,
contributing to PaCa survival, inhibition of autophagy will result in enhanced PaCa cell susceptibility to
freezing or heat ablation, therefore increasing observed cell death. Methods Cell Culture: PANC-1 (CRL-
1469) and BxPC-3 (CRL-1687) cell lines were obtained from the American Type Culture Collection (ATCC,
Rockville, MD, USA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (PANC-1 cells) or
RPMI-1640 (BxPC-3 cells) (Caisson Laboratories, Inc, Logan City, UT, USA) and supplemented using 10%
Fetal Bovine Serum (Peak Serum Inc, Fort Collins, CO, USA) and 1% Penicillin/Streptomycin (Corning, Inc,
Corning, NY, USA). Cells were sub-cultured every four days and culture media replenished every two
days. Cells were utilized between passages 10 and 35 for all experiments. Cells were seeded onto Costar
96 stripwell plates (Corning) at a cell density of approximately 3.75x104 cells/cm2 in their respective
medium 24 hours prior to experimentation. Autophagy Inhibition: Samples were subjected to CQ
ranging from 25-200µM (Sigma, St. Louis, MO, USA) for 24 hours. Following exposure, CQ was removed
and cells were assessed using a metabolic activity assay (described below) to determine basel

Q7

Overview of Androgen Independent Prostate Cancer: Focus on Cryosurgical Applications and Promising
Treatment Options
Prevalence and Treatments According to the American Cancer Society, an estimated 600,920 Americans
will die from cancer in 2017 with 1,668,780 new cancer cases diagnosed overall [1]. Prostate cancer is
the most common cancer type diagnosed in men and accounts for as many as 1 in 5 new cancer
diagnoses. Though many treatment options for patients with cancer are successful in limiting
recurrence, there is a continued need for treatment modalities that will improve patient quality of life.
Improvements in early detection and screening have been instrumental to the decrease in cancer
mortality; detecting a cancer in its early stages generally lowers the risk that it will metastasize, which
makes a dramatic difference in treatment choice and eventual outcome. Additionally, these
improvements in early detection warrant research into minimally invasive therapies to facilitate
localized, rather than systemic, treatment options. Primary treatment options for prostate cancer often
include one or a combination of surgery (radical prostatectomy), chemotherapy, radiation, or androgen
deprivation therapy (ADT). Historically ADT has been widely utilized as early as the 1940’s when removal
of testosterone resulted in dramatic improvements in quality of life in patients with advanced prostate
cancers[2]. Since then both surgical and pharmacologic castration have been shown to greatly decrease
tumor volume in a short period of time as well as improve symptoms in patients with metastatic
disease. However, the overwhelming majority of men treated with ADT progress to androgen
independent prostate cancer (AIPC, also called castration resistant or hormone resistant prostate
cancer, CRPC/HRPC)[3, 4]. AIPC is extremely difficult to treat and across treatment modalities. 3 The 5-
year survival rate for CRPC metastatic prostate cancer is 25% with a median survival of 40 months [5, 6].
Principles of Cryotherapy/Cryosurgery Cryotherapy is one of several modes of thermal ablation
technology that is unique in that it offers a minimally invasive alternative to traditional methods that is
increasingly utilized for the treatment of prostate cancer. It may be used as a first line modality, often in
locally confined disease, or as a salvage therapy following other primary treatment. A 2008 retrospective
analysis of men given cryotherapy as a primary treatment reported 10 year disease free survival
comparable to that of traditional treatments [7, 8]. Compared to a radical prostatectomy, a cryosurgical
procedure is quicker, less invasive, and requires only local anesthetics or sedation, as the freezing itself
is painless. Insertion of a cryoprobe (or multiple probes) into the tumor mass is facilitated by ultrasound
imaging, and ice formation is closely monitored during the procedure to avoid overfreezing, both
visually on via ultrasound or MRI and also with multiple temperature monitoring sites throughout the
area [9, 10]. Cryoablation, as an energy deprivation therapy, is unique in that it initiates distinct forms of
cell death including physical damage due to ice formation, necrosis, activation of cellular stress
responses that lead to the induction of gene regulated apoptosis, vascular stasis and likely activation of
an ablative immune response. Each of these elements of the cell death cascade is separately influenced
by the physical parameters of the freeze-thaw process (i.e. cooling rate, duration at nadir temperature
and thawing rate, etc.). Additionally, distinct differences exist in a given cancer cell’s sensitivity to
freezing which is important to the ablative outcome. 4 The process of freezing tissue to ultra-low
temperatures results in extensive

Investigation of the Impact of Cell Cycle Stage on Freeze Response Sensitivity of Androgen Insensitive
Prostate Cancer 18 Abstract Cryoablation, an effective means of ablating cancer, is often used in
conjunction with adjuvants which target cancer cells in a specific cell cycle stage to increase treatment
efficacy. The objective of this study was to investigate the impact of cell cycle stage on cancer freeze
response as well as investigate the potential cellular kinetic effect of calcitriol, the active metabolic of
vitamin D3, when used as a cryosensitizing adjuvant in order to maximize prostate cancer cell death. Cell
cycle distribution of PC-3 cells was analyzed via flow cytometry to compare G1, S, and G2/M phase sub-
populations pre and post freeze as well as changes elicited by calcitriol pre-treatment. Distinct G1, S and
G2/M phase populations were obtained through FACS and S-phase thymidine synchronization. Post-
treatment viability was assessed using alamarBlue and fluorescence microscopy to assess live, apoptotic
and necrotic sub-populations. A small but statistically significant increase in S-phase and decrease in
G2/M phase populations was noted at 6h post freeze in asynchronous samples. Synchronization in S-
phase yielded an increase in cell death when combined with freezing to both -15°C and -20°C. Calcitriol
pre-treatment increased the G1 phase population by 20% and a synergistic decrease in viability
following freezing. However, G1 sorted populations combined with calcitriol treatment did not exhibit
this synergistic effect. Fluorescence microscopy of FACS sorted cells revealed necrosis as the
predominant form of cell death in all phases, though apoptosis did play a role. Though initial results
suggested a potential sensitivity, PC-3 cells exposed to freezing as sorted populations did not reveal
significant differences in cell death. As such, the data from this study suggest that there is no differential
in cell cycle stage sensitivity to freezing injury. 19 Introduction In 2015 an estimated 233,000 new cases
of prostate cancer were diagnosed in the United States [1]. As the most highly diagnosed non-skin
cancer in men, treatment for both early stage and advanced disease remains critical. Cryosurgery has
become a primary treatment option for prostate cancer and yields disease-free survival similar to other
therapies [2, 3]. One of the challenges of prostate cryosurgery is the determination of positive freeze
margins necessary to minimize recurrence risk while avoiding damage to the prostatic neurovascular
bundle and rectal wall. Advancements in cryosurgical technology have improved the overall efficacy of
treatments and minimized postoperative complications [4, 5]. However, often it is a challenge to achieve
the necessary temperature for total cellular destruction (>40°C) throughout the tumor; as such,
adjunctive treatments aim to increase the lethality of mild freezing by combining agent/drug exposure
with cryosurgery [6-8]. Previous studies have investigated conventional chemotherapeutic agents’
efficacy in conjunction with freezing [9-12]. The majority of chemotherapy drugs affect the DNA
synthesis or cell division processes, which acts as a cellular stress-inducing event and thus causes
activation of cell cycle checkpoints, DNA damage responses and the initiation of cell death cascades.
Similarly, ionizing radiation induces a reproducible effect whereby cells exhibit sensitivity based on cell
cycle phase; specifically, G2/M populations are the most radiosensitive, S the most radioresistant, and
G1 demonstrates intermediate sensitivity [13]. Similar cell cycle dependent responses have also been
reported with various chemotherapeutic agents wherein the drug targets cancer cells in specific cell
cycle stages [14, 15]. While the role cell cycle plays in therapeutic outcome 20 in radiation and
chemotherapy is understood, its role in cell response to cryoablation remains unknown. The literature
suggests that the multitude of stressors initiated during freezing, including ischemia, hypoxia, apoptotic
induction, and metabolic uncoupling all have a cell cycle related dependence when applied individually
[16-18]. The combinatorial action of these coupled with the physical and biochemical stressors occurring
during cryoablation may amplify or potentially negate the cell cycle dependence. As such, this study
examined post-freeze viability and cell cycle phase distribution as well as the responses of individual
phases to freezing injury as a first step in determining the role of cell cycle kinetics in cell death following
freezing. Pre
Dose Escalation of Vitamin D3 Yields Similar Cryosurgical Outcome to Single Dose Exposure in a Prostate
Cancer Model 43 Abstract Vitamin D3 (VD3) has been shown to be an effective adjunctive agent,
enhancing the destructive effects of freezing in both murine and human in vitro prostate cancer
cryoablation studies [1-3]. The sensitizing ability of calcitriol, the active VD3 metabolite, occurs after at
least 24h of exposure and is hypothesized to correspond to the completion of at least one cell division
cycle. Given the reported benefits of VD3 following short term exposure, we investigated if gradual dose
escalation of VD3 over several weeks would be as or more effective than a single moderately high dose
treatment one to two days prior to freezing. The objective of this study was to model the gradual
increase in VD3 levels within the body if an oral supplement dose regime were prescribed. Further, an
actively dividing (log phase) in vitro model was utilized to evaluate the effect of VD3 in combination with
cryoablation on aggressive, highly metabolically active (high Ki-67) prostate cancer. To this end, PC3
prostate cancer cells in log phase growth 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 then exposed to mild sub-lethal
freezing temperatures. The results demonstrate that both acute 24 hour exposure to 80 nM VD3 or dose
escalation resulted in enhanced cell death following freezing at -15°C or colder. However, no significant
differences were found between the two exposure regimes. Investigation into the involvement of
apoptosis in the initial 24 hour period post-freeze revealed that VD3 treatment induced both caspase 8
and 9 mediated cell death, with the largest increase seen in caspase 8 at 8 hours post-freeze. These
results indicate that both the intrinsic and extrinsic apoptotic pathways may be involved in the VD3
sensitization process resulting in increased cell death following a combinatorial freeze event. The
44 results of this study suggest that both acute and gradual dose escalation regimes of VD3 exposure
increases prostate cancer cell sensitivity to mild freezing. Importantly, this study expands upon previous
reports and suggests that VD3 sensitization in conjunction with freezing may offer an effective
treatment for both slow growth and highly aggressive prostate cancers. 45 Introduction The
development of adjunctive strategies for cryoablation is one of high interest due to the fact that simply
freezing a given tumor may not completely destroy all cancerous cells. Reports have demonstrated that
depending on the cancer type, cancer cells can survive temperatures as low as -40°C. For instance,
studies have shown that renal and liver cancer is destroyed at temperatures of -20°C or lower[4, 5]
whereas prostate cancer ranges from -25°C to -40°C depending on the molecular disposition of the
particular cancer (i.e. androgen sensitive vs. androgen insensitive prostate cancer, respectively[6-9]).
Given this differential cancer specific response, it is critical to attain a specific target temperature
(minimum lethal temperature) throughout a tumor to assure complete cancer destruction. Due to the
thermal gradients created within a frozen tissue mass, the necessity to freeze well beyond the edge of a
given tumor is often required. This positive freeze margin often results in the damage of non-targeted
tissues, thus creating unwanted comorbidities. For instance, in prostate cryoablation attainment of -
40°C at the edge of the prostate often requires a 1 cm positive freeze margin and, as such, often impacts
the neurovascular bundle. Further, with this positive freeze margin physicians must also be cognizant
not to freeze the rectal wall. Given the balance between attaining a targeted minimal lethal
temperature with a reduction of collateral damage associated with the positive freeze margin, the
development of adjunctive strategies which can increase prostate cancer sensitivity to milder sub
freezing temperatures is of great interest. The objective of these strategies is to elevate the minimum
lethal temperature through the use of low dose, minimally toxic, adjunctive anti cancer agents thereby
eliminating the need for a positive freeze margin while deploying a 46 highly effective focal treatment
strategy. Given the involvement of apoptotic cell death in freezing [7], agents that enhance apoptosis
are of great interest. Combinatorial agents that have been studied range from cytotoxic
chemotherapeutic agents[5, 10, 11] to neutraceuticals[1, 12], with the latter being the most attractive
option due to typically decreased side effects. One such agent is calcitriol, the active metabolite of
vitamin D3 (VD3). For the purposes of this paper, calcitriol and VD3 will be used synonymously. VD3 has
been reported to have positive anticancer properties in numerous in vitro and in vivo studies [1-4, 13-
16]. Clinical studies investigating the correlation between VD3 status and an individual’s cancer risk have
yielded conflicting results, particularly breast, colon, and prostate. Although some clinical studies have
found no significant differences between mortality rate and VD3 supplementation, many preclinical
studies have shown the benefit that supplementation has on cancer preve

Use of 1,25α Dihydroxyvitamin D3 as a Cryosensitizing Agent in a Murine Prostate Cancer Model

72 Abstract Cryotherapy has emerged as a primary treatment option for prostate cancer (CaP);
however, incomplete ablation in the periphery of the cryogenic lesion can lead to recurrence.
Accordingly, we investigated the use of a nontoxic adjunctive agent, Vitamin D3 (VD3) with cryotherapy
to sensitize CaP to low temperature induced, non-ice rupture related cell death. VD3 (active metabolite
calcitriol) has been identified as a possible adjunct in the treatment of cancer due to its anti-proliferative
and anti-tumorigenic properties. This study aimed to identify the cellular responses and molecular
pathways activated when VD3 is combined with cryotherapy in a murine prostate cancer model. Single
freeze-thaw events above -15°C had little effect on cancer cell viability; however, pre-treatment with
calcitriol in conjunction with freezing significantly increased cell death. The -15°C calcitriol combination
increased cell death to 55% following a single freeze, compared to negligible cell loss by freezing or
calcitriol alone. Repeat cryocombination yielded 90% cell death, compared to 65% in dual freeze-only
cycles. Western blot analysis following calcitriol cryosensitization regimes confirmed the activation of
apoptosis. Specifically, pro-apoptotic Bid and pro-caspase-3 were found to decrease at 1h following
combination treatment, indicating cleavage to the active forms. A parallel in vivo study confirmed the
increased cell death when combining cryotherapy with calcitriol pre-treatment. The development of an
adjunctive therapy combining calcitriol and cryotherapy represents a potentially highly effective, less
toxic, minimally invasive treatment option. These results suggest a role for calcitriol and cryoablation as
a combinatorial treatment for CaP with the potential for clinical translation. 73 Introduction In 2010 an
estimated 217,730 men will be diagnosed with prostate cancer (CaP) in the U.S [1]. While the current
treatment options available offer favorable cure rates, the development of new minimally invasive
treatments are vital to improving patient quality of life. In 2008 the AUA issued a Best Practices
Statement on cryosurgery for the treatment of CaP, indicating its emergence as a mainstream treatment
option [2]. A report by Cohen et al. on 10 year patient outcome has shown cryoablation to be equivalent
to that of other therapies used to treat CaP [3]. Advances in cryosurgery over the last 10-15 years have
greatly increased treatment efficacy and decreased negative side effects. These advances include
improvements in cryotechnology, the use of urethral warming devices to minimize urethral sloughing,
computer-aided ultrasound to guide the ablation process, as well as thermocouples to monitor
temperature [2, 4]. Cryotherapy, as with other thermal techniques, does not create a uniform treatment
zone; rather, there is a gradient of temperature, from the center where the temperature is at its lowest
with elevated temperatures toward the periphery [5]. In the center cells are typically destroyed due to
physical ice rupture and post-thaw necrosis. However, in the periphery it has been shown that other
forms of cell death are dominant, including apoptosis [6-8]. Local recurrence is thought to be a result of
incomplete cell death in the periphery of the freeze zone.[9] Chemotherapeutic drugs have been used as
combination treatments with cryotherapy to increase the efficacy of tumor cell death [6-9]. Many of
these drugs, such as Taxotere and 5-Fluorouracil, have been shown to sensitize CaP to freeze-induced
apoptosis [5-7]. In vivo, however, chemotherapeutic drugs present toxic side effects as well as concern
over 74 drug resistance. Accordingly, the need for the identification of novel, non-toxic treatments for
CaP is essential. For this reason, researchers continue to investigate possible adjuvants (cryosensitizers)
that when combined with low temperature ablation can increase the level of cell death in the periphery
of the cryolesion. One compound which has shown promise as a cryosensitizer is vitamin D3 (VD3)[10,
11]. Vitamin D circulates in the body bound to a vitamin D bin

Translational in vivo-like Modeling Studies 99 PART I: Preliminary Studies of 3D in vivo-like models In

2015 the National Institutes of Health (NIH) instituted a policy that requires the reasonable exploration
of alternative models prior to conducting animal studies. As such, we have focused on creating in vivo
like models that more closely mimic the microenvironment of a tumor to test the feasibility of various
treatment regimes. The research presented herein has progressed to further explore calcitirol
cryosensitization in 3D models of AIPC. In the ongoing studies presented in this chapter, we utilized a 3
dimensional tissue engineered model (TEM) of the prostate to test various freezing applications of
clinical significance. A complete TEM setup consists of layered 3mm individual TEMs to allow for optimal
nutrient exchange (avoiding a hypoxic microenvironment) as well as to provide a convenient method for
post-freeze assessment by stack disassembly [1] (Figure 1). Development of the TEM model has been
previously reported on by our laboratory [2]. Further, the utilization of TEM models has been
demonstrated in a number of settings including assessment of cryogurgical devices [3]. In the current
study, PC-3 cells were seeded into a collagen matrix and distributed within individual TEM layers of 3mm
depth. Multiple pTEM layers were stacked to create a 3D prostate surrogate to allow for the insertion of
a cryoprobe and iceball formation using a cryosurgical device. Methods Cell and 3D Cultures The human
prostate (PC-3) cancer cell line (ATCC, Rockville, Maryland) was maintained at 37°C, 5% CO2:95% air in
RPMI-1640 culture medium (Caisson Laboratories Inc, North Logan, Utah) fortified with 10% FBS (Atlanta
Biologics Inc, Atlanta, Georgia) and 1% penicillin/streptomycin (Corning, Inc, Corning, New York).
100 Cells were cultured in 75 cm2 t-flasks with media replenishment every 3 days. For generation of the
tissue-engineered models (TEM), rat tail type I collagen solution (BD Bioscience, Bedford,
Massachusetts) was used to form 0.2% wt/vol gel matrices as per standard protocol. Cells (1.5 x 106
cells/mL) were suspended in the collagen solution prior to solidification in 3 mm x 60 mm TEM ring
fixtures as per Robilotto et al. (2007) and Baust et al. (2017) and cultured for 24 hours prior to
utilization. Freezing Protocol Prior to freezing, individual cell-seeded TEMs were assembled into the 3D
stack configuration (60 mm x 60 mm) following SOP. The TEM stack was then submerged into a warm
circulating bath of culture media followed by placement onto a heat pad and stir table, and a cryoprobe
and thermocouple array were then inserted to monitor temperatures (Figure 1). Samples were held
until TEM and bath temperatures equilibrated at 32°C (± 2°C); TEM samples were frozen using the SCN
cryosystem (CPSI Biotech, Owego, NY) using a 1.8 mm cryoprobe with a 3-cm freeze zone length. TEM
models were frozen using a 5/5/5 or single 5 (in minutes) freeze/thaw/freeze protocol. Temperature of
the bath and within the TEM were monitored throughout the freezing process at fixed distances of 7.5,
10.5, 13, 16, and 19 mm extending radially from the surface of the cryoprobe at the center point of the
freeze zone using a type-T thermocouple array and recorded with an Omega TempScan at 10- second
intervals throughout the entire freeze cycle. At the completion of the freeze cycle, TEMs were allowed
to passively thaw in the warm circulating bath for 30 minutes prior to disassembly at which time the
individual TEM layers were returned to culture for recovery and assessment. 101 TEM Assessment
Following thawing and disassembly, individual TEM layers were measured via calipers to determine the
diameter of the iceball created following the freeze–thaw cycle (Figure 2). Iceball radii were also
measured at cardinal locations around the probe surface to determine symmetry of the freeze zone
created. Individual layers were then cut into half (bisection of the probe center) yielding 2 replicate
samples from each layer for analysis. One half of the layer sample was then placed into culture to assess
24 hours postfreezing recovery, whereas the other half was assessed for immediate (1 hour) postfreeze
viability. In situ sample viability assessment was performed using the fluorescent probes calceinAM and
propidium iodide (Cal/PI; Invitrogen, Carlsbad, California). Briefly, culture medium was decanted from
the TEM samples, and 2 mL of the working Cal/PI solution was added directly to each sample. Samples
were incubated in the dark at 37°C for 60 (+1) minutes. Fluorescent staining was visualized using a Zeiss
Axiovert 200 fluorescent microscope under a 10x magnification with the AxioVision 4 software (Carl
Zeiss, Germany). Panoramic digital images were recorded starting at the iceball center (probe surface)
and extended across the freeze region into the nonfrozen periphery. Following acquisition, a 5000 mm
(5 mm) scale bar and reference mark at a radius of 15 mm (represent a 3 cm diameter) were
autoimprinted onto each of the images using the AxioVision software to enable direct image
comparison. Data Analysis For ablation zone imaging studies, a minimum of 3 TEM repeats were
conducted. Following experimentation, data were combined and averaged (+standard deviation to
determine mean iceball size, temperature location, and ablation zone produced. Iceball, 102 thermal
zone, and ablation volumes were calculated based on freeze zone center point of TEM layer
measurements using an ellipsoid model to enable volumetric analysis of system performance. Freeze
zone size Following the freeze/thaw episode, iceball diameter and radii were measured using digital
calipers at the center point of the cryoprobe freeze zone. Ablation zone Fluorescent imaging of the TEM
began at the center point of the cryoprobe. Images were measured using the AxioVision software to
determine the size of the zone of ablation (PIpositive/Cal-negative [red] region). Real-time recordings of
the thermal profiles collected using the Omega TempScan system were converted to graphical format
using Microsoft Excel and analyzed to determine isotherm spread during the first and second freeze
intervals. Cryopellet Formation Cryopellets were formed by dropping a 50% ethanol, 50% water solution
into liquid nitrogen using a handheld P200 with a wide mouth tip. Pellets were manipulated with LN2
pre-cooled forceps (Figure 3a). Pellets were consistently ~3mm in diameter (Figure 3b). Cryopellet
Assessment Following thawing individual TEM layers were returned to standard 37C incubation for 24h.
Sample viability assessment was performed using the fluorescent probes calceinAM and propidium
iodide (Cal/PI; Invitrogen, Carlsbad, California). Briefly, culture medium was decanted from the TEM
samples and 2 mL of the working Cal/PI solution 103 (10 mL Cal þ 8 mL PI þ 2 mL phosphate-buffered
saline) was added directly to each sample. Samples were incubated in the dark at 37°C for 60 (+1)
minutes. Fluorescent staining was visualized using a Zeiss Axiovert 200 fluorescent microscope under a
10x magnification with the AxioVision 4 software (Carl Zeiss, Germany). Panoramic digital images were
recorded in a 7x7 region surrounding the center of the CP to allow for complete CP as well as nonfrozen
surrounding TEM. Results A single 5 minute freeze was conducted using a 1.8mm cryoprobe with a 3cm
freeze zone. The resultant cell death from this freeze exposure was measured using fluorescent probes
calcein AM and propidium iodide at both 1h and 24h post freeze via panoramic fluorescence
microscopy. Sensitized samples were exposed to Vitamin D3 [50 nM] for 24h prior to the freezing event.
TEMs pretreated with vitamin D3 [50 nM] for 24h had significantly reduced regrowth 1 day following
freeze exposure in comparison to non-treated samples (Figure 2). Non-vitamin D3 treat

Q888

DEVELOPMENT OF A TISSUE ENGINEERED HUMAN PROSTATE TUMOR EQUIVALENT FOR USE IN THE
EVALUATION OF CRYOABLATIVE TECHNIQUES

Abstract 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 (PC-3) 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 postthaw at low temperatures (< -40
°C) and moving toward the periphery (-20 °C) as recovery times reached 12 and 24 hr. Distal to the -10
°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 ~10 to 15 °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. 52 Introduction Cancer is the second leading cause of death in the US claiming
approximately 570,000 lives in 2005. Of those deaths, over 30,000 were from prostate cancers, making
it the second leading cancer killer among men [1]. Current treatment modalities include surgical
ablation, external beam radiation, brachytherapy, RF treatment, and chemotherapy. Cryosurgery, the
utilization of low temperatures to freeze and destroy unwanted tissue, has also emerged as a common
option in the treatment of cancerous lesions [2-4]. Like any therapy, improving cryosurgical efficacy
requires an understanding of both the physical parameters of the treatment itself, as well as the cellular
and biochemical responses of the targeted tissue (cells) to that treatment. Cryosurgery is performed
through the application of a heat sink (cryoprobe) to a desired area in order to generate a rapid cooling
rate and subsequent generation of a frozen mass of tissue, the cryolesion. This lesion is physically
characterized by a thermal gradient with the lowest temperature nearest the cryoprobe and -0.5 °C at
the freeze zone periphery with the formation of both intra- and extracellular ice [5]. Cells are initially
destroyed through either a freeze rupture, a physical destruction of the cells throughout much of the
lesion, or through a necrotic process resulting from chemical and osmotic stresses induced during
freezing [6]. Apoptosis, or programmed cell death, has also been shown to be a major contributor to cell
death following cryosurgery, typically associated with the freeze zone periphery and occurs in a time
frame ranging over hours to days post-thaw [2,7-9]. Additionally, the formation and propagation of this
freeze zone and its asso

TEMPERATURE DEPENDENT ACTIVATION OF DIFFERENTIAL APOPTOTIC PATHWAYS DURING

CRYOABLATION IN A HUMAN PROSTATE CANCER MODEL 76 Abstract Background: Critical to the
continual improvement of cryoablation efficacy is deciphering the biochemical responses of cells to low
temperature exposure. The identification of delayed-onset cell death has allowed for the manipulation
of cellular responses through the regulation of apoptosis. We hypothesized that in addition to delayed
apoptotic events associated with mild sub-freezing temperatures (10 to -25°C) cells exposed to ultra-low
temperatures (< -30°C) may undergo rapid, early onset apoptosis. Methods: Human prostate cancer
model and cells (PC3) were exposed to temperatures of - 60, -30, and -15°C to simulate a cryoablative
procedure. Using a combination of flow cytometry, fluorescent microscopy, and western blot analyses,
samples were assessed at various times post-thaw to identify the presence, levels, and pathways
involved in cell death. Results: Exposure to temperatures <-30°C yielded a significant apoptotic
population within 30 min of thawing, peaking at 90 min (~40%), and by 6 hr only necrosis was observed.
In samples only reaching temperatures > -30°C, apoptosis was not noted until 6 – 24 hr postthaw with
the levels of apoptosis reaching ~10% (-15°C) and ~25% (-30°C) at 6 hr postthaw. Further it was found
that early onset apoptosis progressed through a membrane mediated mechanism whereas delayed
apoptosis progressed through a mitochondrial path. Conclusions: These data demonstrate the impact of
apoptotic continuum whereby the more severe the cryogenic stress activated the extrinsic, membrane
regulated pathway while less severe freezing activated the intrinsic, mitochondrial mediated path. The
rapid induction and progression of apoptosis at ultra-low temperatures provides an explanation as to
why such results have not previously been identified following freezing. Ultimately, an 77 understanding
of the events and signaling pathways involved in triggering apoptosis following freezing may provide a
path for selective induction of rapid-onset and delayed programmed cell death pathways in an effort to
improve the overall cryoablation efficacy. 78 Introduction Cryosurgical ablation of prostate cancer is a
treatment modality that has demonstrated steady growth over the past ten years. This trend has been
reinforced by the publication of the first long term follow up study showing comparative results
between cryosurgery and more traditional treatments [1] and the Best Practice guidelines for prostate
cryosurgery issued by the American Urological Association (AUA) [2]. While clinical application continues
to grow, there remains a void in our understanding of the specific molecular based responses of cancer
cells following low temperature insults. This deficit, if not addressed, will likely hinder further
improvements in treatment efficacy. During a cryosurgical procedure, cells nearest the cryoprobe(s) (< -
60°C) are believed to predominantly die via rupture caused by the physical stresses of intra/extracellular
ice formation. Extending peripherally, as cooling rates decrease and temperatures remain more
elevated, mechanical destruction gradually gives way to biologically driven cell death. In this outer
region (> -40°C), cells undergo a combination of lessening physical rupture and increasing levels of
necrotic and apoptotic cell death [3, 4]. It is also in this region where incomplete tissue ablation can
occur, although some cancer cells can survive even at temperatures below -40°C [5]. Given the
incomplete initial destruction and the occurrence of cell mediated death, it is the cryolesion periphery
that has emerged as the primary focus of cellular/molecular research following freezing. In 1998
Hollister et al. identified the presence of apoptosis following freezing [6]. Since then, apoptosis, or
programmed cell death, has been shown to be a major contributor to cell death following cryosurgery
[7-10]. Occurring over a period of hours to days post-thaw, apoptosis is typically associated with the
freeze zone periphery and is largely modulated 79 by the caspase family of proteases [11] and the two
distinct paths of caspases- dependent apoptosis, intrinsic and extrinsic. The intrinsic, or mitochondrial-
mediated pathway, is characterized by the “opening” of the mitochondrial permeability transition pore
[12-14], release of cytochrome c [15, 16], and activation of caspase-9 [17, 18]. The intrinsic pathway has
been shown to be the predominant form of cryo-induced delayed-onset cell death [19]. The second
caspase dependent pathway is the extrinsic, or membranemediated, pathway. Induced by death
receptor ligands [20, 21], loss of growth factor signaling [22, 23], or alterations in cell attachment
signaling [24], the extrinsic pathway progresses through caspase-8. The role of membrane modulated
cell death following cryosurgery, while reported, is less understood. Recently, early-onset programmed
cell death occurring within hours post-thaw was observed in cells exposed to temperatures below -20°C
[25]. These observations prompted further investigation into the role of early onset apoptosis in cell
death f

ASSESSING THE THERMAL PERFORMANCE OF A NOVEL CRYOSURGICAL DEVICE USING A 3-DIMENSIONAL

TISSUE ENGINEERED CULTURE MODEL 107 Abstract With the growing adoption of cryotherapeutic
procedures to treat a wide array of disease states, comes an increased need for a deeper understanding
of the responses of cells and tissues to freezing insults, as well as the technologies and clinical
techniques underpinning the freeze procedure. Freezing parameters, such as the extent of ice growth
and the distribution of isotherms within the freeze volume, have typically been analyzed with phantom
gel models (acellular hydrogels), while the response of cells and tissues to freezing have been studied in
animal models or in vitro cell monolayers. In an effort to combine these test platforms, we have
developed a 3-dimensional, tissue engineered culture model (TEM) that provides a freeze medium with
clinically relevant volumes and thermal characteristics, while also allowing for the post-thaw assessment
of cellular ablation. In this study, we employed the TEM model to assess the freeze zone and ablative
performance of a novel cryosurgical device employing supercritical nitrogen (SCN) as the cryogen. Using
the SCN device and a 2.0 mm x 40.0 mm or a 1.8 mm x 30.0 mm cryoneedle, a 5/5/5 min
freeze/thaw/freeze procedure was performed in our TEM models seeded with either prostate cancer
(pTEM) or renal cancer cells (rTEM). Measurements of the freeze zone diameters revealed iceballs 3.86
cm (± 0.25) cm for pTEMs and 3.94 cm (± 0.25 cm) for rTEMs following the freeze procedure, which
calculate to freeze volumes of 28.87 cm3 and 33.33 cm3 respectively. The distribution of the -20 °C and -
40 °C isotherm, as determined by temperature recordings, were found to be 2.53 cm (± 0.25 cm) and
2.02 cm (± 0.25 cm) for pTEMs and 2.65 cm (± 0.25 cm) and 2.10 cm (± 0.25 cm) for rTEMs. The volume
of the -20 °C and -40 °C isotherms comprised an average 45.3 % and 28.7 % of 108 the total frozen
volumes. In addition to the thermal profile, the ablative potential of the SCN system was assessed with
fluorescent microscopy of both the pTEM and rTEM models. Results show an ablative zone with a
diameter of 3.00 cm and volume of 18.85 cm3 in the pTEM samples, and 3.50 cm and 25.65 cm3 in the
rTEM samples. These volumes correlate to 60.4 % (pTEM) and 78.9 % (rTEM) of the total frozen volume
and indicate a critical temperature of -30 °C for the prostate cancer cells and -20 °C for the renal cancer
cells, results supported by clinical reports. The ablative data also indicates the ring of incomplete cell
death in the freeze zone periphery was 0.43 cm and 0.22 cm wide for the pTEMs and rTEMs
respectively. The data presented in this study demonstrate that the TEM culture model is able to
provide a reliable, cost effective, and repeatable test platform for the assessment of cryosurgical
technologies and techniques. Furthermore, the SCN cryosurgical system demonstrated the ability to
generate large ablative volumes under physiological heat loads in half the typical freeze duration (5/5/5
min vs. 10/5/10 min) of current technologies. Together, the TEM model and SCN system provide useful
tools in the study and application of cryotherapeutic procedures. 109 Introduction Cryoablative
therapies rely on the precise placement of a cryoprobe and the circulation of a cryogen to extract
thermal energy and generate freezing temperatures within the targeted tissue. As heat is removed, a
thermal gradient is generated in which the distance from the cryoprobe surface is predictive of the
relative cooling rate and nadir temperature experienced by the tissue: as the distance from the
cryoprobe increases, cooling rates decrease, and temperatures increase. The destruction of the cells and
tissues targeted during a cryoablative procedure is a result of numerous mechanisms including the
formation of intracellular and extracellular ice, the accumulation of osmotic and oxidative stress, the
initiation of apoptotic and other pro-death biochemical signaling cascades, and through the vascular
stasis and immunological responses that occur in situ [1-5]. The growth in the use of cryoablative
therapies has been driven by several factors. Technological advancements such as planning software
and intraoperative ultrasound that guide cryoprobe placement and track the advancing ice front; fine
gauge thermocouple needles that accurately monitor tissue temperatures; devices that can warm and
protect adjacent, non-targeted tissues; and multi-probe cryosurgical devices able to sculpt the
cryolesion, have made cryoablative therapies more consistent and effective [6]. In addition to
technological improvements, a growing understanding of the molecular responses of the cells and
tissues to freezing insults has made the field more translational and evidence based [7-10], and the
publication of long term follow-up studies showing

Q99

IMPLEMENTATION OF AN EDUCATIONAL INTERVENTION TO IMPROVE THE CORRECT USE OF ASTHMA

INHALERS

Metered-dose inhalers (MDI) are routinely prescribed for patients with asthma. Healthcare professionals
are encouraged to check the patient’s ability to use an inhaler as inhalers are most effective when used
correctly. However, many healthcare professionals are unable to use an inhaler correctly. This pilot
study tested the ability of nurse practitioner students to correctly use an MDI. Findings indicated that
nurse practitioner students are unable to use an MDI correctly. These students then participated in a
“teach to goal” educational intervention. Four weeks later, students were significantly more likely to be
able to use an MDI correctly (p=0.000). All nurse practitioner students completed the Healthcare
Professional Asthma Knowledge Questionnaire before and after the intervention. Although scores
increased, from 10.67 to 11.33, this was not statistically significant (p=0.252). There was no relationship
between ability to use an inhaler correctly at pre or post-test and years of experience as an RN, previous
education on inhalers, or personal use of an MDI. Age was only significant in the pretest as the younger
students were more likely to be able to use an MDI correctly; there was no significant difference at the
post-test. These findings indicate that all nurse practitioner students should be taught how to use an
MDI during their course of study
ed as Intal is no longer available in the U.S., and question 12 was eliminated as it discussed an Australian
specific program. In question 18, Eformoterol was changed to long-acting beta 2 agonist to be more
general as students may not know about specific medications. Therefore, the questionnaire contained a
total of 14 questions (see Appendix D). A convenience sample was used. All participants were nurse
practitioner students at Binghamton University. Participants were contacted via the Decker School of
Nursing graduate email listserv. Participants were at least18 years old and enrolled as adult/geriatric,
family, or community-health nurse practitioner students at Binghamton University. Exclusion criteria
was current certification as a nurse practitioner. The implementation of the educational program was
done in a classroom at the Decker School of Nursing. There was no need for funding for this project.
Participants were entered into a raffle to win a $20 gift card. Minimal expenses were encountered and
were paid for by the researcher. 19 Table 2 Operationalization of Variables Operational definition Type
of measurement Question Choices Demonstration of inhaler use Pre-test inhaler use Categoricalnominal
Demonstration of inhaler use according to NHLBI’s Steps 0=incorrect 1=correct Post-test inhaler use
Categoricalnominal Demonstration of inhaler use according to NHLBI’s Steps 0=incorrect 1=correct
Demographic information Age Categoricalordinal Respondent’s age 110 Nurse Practitioner program
Categoricalnominal Respondent’s Nurse Practitioner Program 1=Family 2=Community Formal education
received on metereddose inhaler Categoricalnominal Respondent’s reception of formal education 0=No
1=Yes Personal use of metered-dose inhaler Categoricalnominal Respondent’s personal use of metered-
dose inhaler 0=No 1=Yes Healthcare Professional Asthma Knowledge Questionnaire True of false
responses to 14 item questionnaire Categoricalnominal Questions 1 to 14 0=Incorrect 1=Correct 20
Research Question 1 What is the relationship between “teach to goal” educational intervention and pre
and post inhaler use? Null hypothesis: There is no relationship between “teach to goal” educational
intervention and pre and post inhaler use. Independent variable: “Teach to goal” educational
intervention Dependent variable: Percentage of participants with post-test proper inhaler use This will
be evaluated using t-test for dependent samples. Research Question 2 What is the relationship between
participation in an educational program designed to increase ability to use an MDI and participant’s
pretest to posttest knowledge scores on the Healthcare Professional Asthma Knowledge Questionnaire?
Null hypothesis: There is no relationship between participation in an educational program designed to
increase ability to use an MDI and participant’s pre and post knowledge score on the Healthcare
Professional Asthma Knowledge Questionnaire. Independent variable: “Teach to goal” educational
intervention Dependent variable: Score on Healthcare Professional Asthma Knowledge Questionnaire
This will be evaluated using a t-test for dependent measures. Research Question 3 What is the
relationship between demographic variables (age, gender, years of RN experience, previous education
on MDI, and personal use of MDI) and proper use of an MDI? 21 Null hypothesis: There is no
relationship between demographic variables and proper use of an MDI. Independent variable:
Demographic variables Dependent variable: Proper use of MDI This will be evaluated using Pearson’s
chi-square test. 22 Chapter 4 Outcomes Twenty nurse practitioner students participated with 18 females
and 2 males. All participants were final semester master’s level students. All were in the family nurse
practitioner program except one who was in the community health nurse practitioner program. In the
first session, only 2 participants (10%) were able to use an MDI correctly. The most commonly missed
steps were not breathing out fully before inhalation and holding breath and counting to 10 after
inhalation. No participants required more than one round of “teach to goal”. Only 15 participants
returned for the second session. Of those, 12 were able to use the MDI correctly (80%). What is the
relationship between “teach to goal” educational intervention and pre and post inhaler use? The ability
of participants to use an MDI correctly was compared before and after the “teach to goal” intervention.
2 out of 20 participants were able to correctly use the inhaler before the intervention. After the
intervention, 12 out of 15 participants were able to use the MDI correctly. Using a paired t-test, t=7.483,
p=0.000. Therefore, there is a statistically significant difference in the ability to use an MDI between
before and after the “teach to goal” intervention. What is the relationship between participation in an
educational program designed to increase ability to use an MDI and participant’s pretest to posttest
knowledge scores on the Healthcare Professional Asthma Knowledge Questionnaire? 23 Scores on the
Healthcare Professional Asthma Knowledge Questionnaire were compared before and after the “teach
to goal” intervention. The questionnaires were scored out of 14 possible points, with 1 point for each
correct answer. The mean score before the intervention was 10.67 and 11.33 after the intervention.
Using a paired t-test, t=1.195, p=0.252. Although the scores increased, the increase was not statistically
significant. The pretest mean of 10.67 indicates that these students started with knowledge about
asthma and the use of MDIs. What is the relationship between demographic variables (age, gender,
nurse practitioner program, years of RN experience, previous education on MDI, and personal use of
MDI) and proper use of an MDI? Age. Of the two participants who were able to demonstrate proper use,
one was in the age group less than 25, and one was in the age group of 25 to 29. Using Pearson’s chi-
square, x 2=10.123 and p=0.038. However, since the majority of the population is in the 25 to 29 age
group, this would be expected. In the post-test, participants in all age groups were able to correctly
demonstrate use of an MDI. Using Pearson’s chi-square, x 2=2.5 and p=.475, which is not statistically
significant. Therefore, age was only associated with correct inhaler use in the pre-test. Gender. Only 2
males did the pretest and neither male completed the post-test. Therefore, it cannot be determined if
there is a relationship between gender and proper use of an MDI. Nurse Practitioner program. Only one
participant was a community nurse practitioner program student, with the remaining students from the
family nurse 24 practitioner program. Therefore, it cannot be determined if there is a relationship
between nurse practitioner programs and proper use of an MDI. Years as RN. The two participants who
correctly demonstrated use of an MDI had less than 5 years of experience as an RN. However, it is not
statistically significant, x 2=1.818, p=.403. After the intervention, participants from all years of
experience categories were able to correctly demonstrate use of an MDI; x2=.625, p=.732 which is not
statistically significant. Therefore, years of experience as an RN was not found to be associated with
ability to use an MDI. Previous education on MDI. Participants were asked if they had previously
received education on how to use an MDI. For the pre-test, of the two who were able to correctly use
the MDI, one had previously received education and one had not. Using Pearson’s chi-square and
Fisher’s exact test, there is no statistical significance, x2=0, p=1. For the post-test, 6 who had received
education before the intervention and 6 who had not were able to correctly use the MDI. There is no
statistical significance, x2=.268 and p=1. Therefore, there is no relationship between previous education
on MDI use and ability to use an MDI. Personal use of MDI. At the pretest, all 4 participants who
responded that they had personally used an MDI were unable to correctly demonstrate use of the MDI.
Using Pearson’s chi-square and Fisher’s exact test, x2=.556, p=1.Therefore, it is not statistically
significant. For the post-test, all 4 participants who personally used an MDI were able to correctly
demonstrate use. Thus, no statistical significance was found (x2=1.364 and p= .516). No statistically
significant relationship between personal use of an M
q10

ARE CHILDREN WITH AUTISM SPECTRUM DISORDER LESS SUSCEPTIBLE TO VISUAL ILLUSIONS? AN
INVESTIGATION OF WEAK CENTRAL COHERENCE AND ENHANCED PERCEPTUAL FUNCTIONING THEORIES

Perceptual abnormalities have long been observed in individuals with Autism Spectrum Disorder (ASD).
Research suggests that superior visual processing on a variety of tasks evidenced in individuals with ASD
may be related to enhanced local processing. Two theories (Weak Central Coherence, Frith 1989;
Enhanced Perceptual Functioning, Mottron & Burack, 2001) have been proposed to account for this
superior local processing. Visual illusions are one measure that has been used to test these theories,
producing mixed results. The purpose of the present study was to address the discrepancy in results
across studies by conducting a direct replication of the initial study investigating susceptibility to visual
illusions in ASD (Happe, 1996). The current study also extended the scope of previous research by
including eye-tracking data. 36 children (17 with ASD, 19 typically developing) completed a visual illusion
task and an existing measure of central coherence. Results indicated no group differences in illusion
susceptibility; however, individual differences in illusion susceptibility were related to increased local
processing at the start of viewing the illusions. Implications of these findings, limitations, and future
directions are discussed.

hat children with ASD would display a longer proportion of fixation duration on the separate elements,
as compared to typical children, and that this would 10 predict lowered susceptibility to the illusions.
Each illusion was also partitioned into the first three seconds of viewing time. It was hypothesized that
children with ASD would display local precedence by exhibiting a longer duration of fixations on the
elements at the start of the illusion and that this would predict lowered susceptibility to the illusions 11
Method Measures To assess the presence and severity of ASD symptoms, the Childhood Autism Rating
Scale-Second Edition (CARS-2; Schopler, Van Bourgondien, Wellman, & Love, 2010), the Social
Communication Questionnaire (SCQ; Rutter, Bailey, & Lord, 2003), and the DSM-IV-TR Checklist for
Autism Spectrum Disorders were administered. The CARS-2 is an observational rating scale that was
developed to assist in identifying individuals with ASD and distinguishing the severity of the disorder.
The measure consists of 15 items that are rated on a scale from 1 (behavior is within normal limits
compared to typically developing individuals of the same age) to 4 (behavior is severely abnormal
compared to same age peers). The CARS-2 has been demonstrated to have moderate to excellent
psychometric properties. The SCQ, originally the Autism Screening Questionnaire (Berument, Rutter,
Lord, Pickles, & Bailey, 1999), is a parent rating scale with 40 yes/no items that is designed to screen for
ASD in children over 4 years of age with a mental age of at least 2 years. The SCQ evaluates reciprocal
social interaction, language, and communication as well as repetitive and stereotyped behaviors. The
scale can be completed in ten minutes and generates a total score with a cutoff of 15 or greater
indicating the possibility of an ASD. The SCQ is not meant to be used as a diagnostic measure in and of
itself, but can be useful for determining children that require further evaluation. The SCQ demonstrates
12 high rates of sensitivity and specificity and is highly correlated with the Autism Diagnostic Interview-
Revised, which is considered a gold standard in the diagnosis of autism (Berument et al., 1999; Witwer &
Lecavalier, 2007). Additionally, a DSM-IV-TR diagnostic checklist was used for the purposes of obtaining
a research diagnosis. This checklist was completed following behavioral observations of the child and
parent completed measures. The checklist assesses social, communication, and repetitive behavior and
can be used to differentiate between Autistic disorder, Asperger’s syndrome, and Pervasive
Developmental Disorder Not Otherwise Specified. Legal guardians of participants also completed a
demographic questionnaire in order to obtain information regarding child age, ethnicity, and
socioeconomic background. In order to evaluate cognitive ability, children were administered either the
Wechsler Preschool and Primary Scale of Intelligence-Fourth Edition (WPPSI-IV) or the Wechsler
Intelligence Scale for Children-Fourth Edition (WISC-IV), depending on the child’s age. Children ages 4 to
5 years 11 months were administered the WPPSI-IV. Children ages 6 years and older were administered
the WISC-IV. For both the WPPSI-IV and WISC-IV, the standard battery was administered to obtain a
verbal, performance, and full scale IQ (FSIQ) score. The performance IQ score was derived from the
perceptual reasoning index for the WISC-IV and from the fluid reasoning index for the WPPSI-IV. The
WPPSI-IV takes approximately 45-60 minutes to complete the core subtests for children within the 4-7
year-old age range. The standardization sample consisted of 1,700 children ages 2-7 years old that was
matched demographically to current U.S. Census data. Reliability and validity ranges from good to
excellent. Reliability coefficients for 13 the primary index scores range from .86 to .94, with the FSIQ
demonstrating a mean internal consistency of .96. The WPPSI-IV correlates highly with the WPPSI-III and
WISC-IV and demonstrates expected convergent and discriminant validity with other achievement tests
(i.e., The Wechsler Individual Achievement Test, WIAT-III). The WISC-IV takes approximately 65-80
minutes to administer and demonstrates similarly good to excellent psychometric properties. The
standardization sample consisted of 2,200 children in the United States ages 6-16 years old that were
divided into 11 age groups with 200 children per age group. The sample was diverse and representati

q555

THE EFFECT OF CERVICAL INTRAEPITHELIAL NEOPLASIA AND TREATMENT SURGERIES ON

FECUNDABILITY ALEXANDRA KLANN ABSTRACT Introduction: Approximately 6 million couples in the
United States experience infertility. Because few risk factors for infertility are known, identification of
modifiable determinants is an important public health goal. Cervical intraepithelial neoplasia, CIN,
occurs when the surface cells of the cervical tissue begin to change, and is caused by infection with a
high-risk type of human Papillomavirus (HPV). CIN may affect the cervix’s immunological function,
resulting in changes in mucus production, reduced protection against infections, and alterations in
sperm transport through the cervical canal. CIN can also progress to invasive cervical cancer. There are
four main CIN treatment procedures that aim to remove pre-cancerous cells from the cervix; loop
excisions, commonly known as electrosurgical excision procedure (LEEP) or large loop excision of the
transformative zone (LLETZ); cryosurgery; conization; and laser ablation. Because the goal of these
procedures is to remove abnormal cells, healthy cervical cells may inadvertently be removed as well,
leading to further changes in cervical mucus production, sperm motility, and reduced protection against
infection. Because of the changes to the cervical tissue and its function, CIN and its surgical treatments
may affect fecundability. vi Methods: We analyzed data from Pregnancy Study Online (PRESTO), a
preconception cohort of 5,594 North American pregnancy planners enrolled and followed between 2013
and 2018. At baseline, participants reported whether they had abnormal Pap tests and their age at their
first abnormal Pap test, as well as cervical procedures and their age at the procedure. We estimated
fecundability ratios (FR) and 95% confidence intervals (CI) using proportional probabilities models
adjusted for sociodemographics, smoking, number of sexual partners, history of sexually transmitted
infections/ pelvic inflammatory disease, and HPV vaccination. Results: A history of abnormal Pap test,
which we used as a proxy for cervical dysplasia, was positively associated with current and past smoking,
gravidity, parity, irregular menses, number of sexual partners, history of chlamydia, genital warts and
herpes, as well as a history of pelvic inflammatory disease. Of the women with an abnormal Pap test,
the average age at first abnormal Pap test was 23.0 (std=4.5) years and the average number of abnormal
Pap tests was 2.1 (std=1.7). We found little association overall between a history of abnormal Pap test
and fecundability (FR=1.03, 95% CI: 0.96, 1.11). The results did not differ when the data were examined
by number of abnormal Pap tests, or type of procedure. There was also little association between time
since the diagnosis or procedure and pregnancy attempt and fecundability. There was however a slight
decrease in fecundability within the first 2 years of diagnosis/ procedure, with FRs that tended to
increase with increasing time since diagnosis/procedure. vii Discussion: We found little association
overall between a history of abnormal Pap test or cervical dysplasia, including excisional surgeries, and
fecundability. These results are consistent with most other studies demonstrating no clear adverse
effects of CIN and treatments. Recency of diagnosis or procedure did not appreciably affect these
findings. Although we found a very slight decrease in fecundability within the first two years since
diagnosis or procedure, fecundability became similar to that of undiagnosed/untreated women after 2
years, and then increased slightly. Conclusion: We found little association between a history of
abnormal Pap and CIN treatments and fecundability. A major limitation of our study is that the data
were selfreported, which may have resulted in non-differential exposure misclassification.

dy examines the association of cervical dysplasia and its treatment with TTP among the Pregnancy Study
Online (PRESTO).45 The three main hypotheses are as follows: Hypothesis 1: Women with a history of
cervical dysplasia (e.g. abnormal Pap tests) will have a longer TTP than women without a history of
cervical dysplasia, indicating a role for HPV infection in subfertility. Hypothesis 2: Women with persistent
or recurrent cervical dysplasia (e.q. >2 abnormal Pap tests) will have a longer TTP than women whose
cervical dysplasia resolves (e.g. 1 abnormal Pap test). Hypothesis 3: Women with a surgery for cervical
dysplasia (LEEP/cone/laser/cryosurgery) will have a longer TTP than women with dysplasia who did not
undergo surgery, indicating possible effects of both dysplasia and surgery. Hypothesis 4: With increasing
time since most recent procedure or cervical dysplasia diagnosis, TTP will return to that of
untreated/undiagnosed women. 16 METHODS Study population Pregnancy Study Online (PRESTO) is an
ongoing web-based prospective cohort study of women who are planning their pregnancy in the U.S.
and Canada.45 The study began enrollment in June 2013 and participants are recruited mainly through
advertising on social media and pregnancy related websites. Women are eligible for this study if they are
between the ages of 21 and 45, not using contraception or fertility treatments, in a stable relationship
with a male partner, and not currently pregnant. The participants complete a baseline questionnaire and
bimonthly follow-up questionnaires online for up to 12 months or until pregnancy, whichever occurs
first. Over
q444

Vision-based Flap Planning Software Tool for Reconstructive Facial Plastic Surgery

The success of reconstructive facial plastic surgery is determined not only by the treatment of the given
pathology but also the restoration or maintenance of normal morphological features and the reduction
of scarring. Failure to consider these aesthetic components can have significant negative psychological
effects for the patient. A vision-based approach to automatic lesion segmentation and incision planning
is proposed. Optical camera images of the face will be analyzed for lesions and estimated lines of
relaxed skin tension using feature-based registration. Simulation of stresses within wound closures will
consider skin mechanical properties and craniofacial structures to implement a constitutive model for
finite element stress analysis. The objective of this research is to develop a consistent and effective
approach to flap planning for reconstructive facial plastic surgery. This system has applications for
surgical training, surgical automation and control, and the improvement of patient care through the
reduction of treatment variati

majority of the algorithm processing was developed using Matlab R2016b. The use of Matlab allowed
for an easy-to-use framework for image processing, GUI development, access to peripherals such as the
webcam, and general computations. Matlab allowed for rapid deployment of ideas as the intricacies of
low-level software development could be abstracted. 3.1.2 COMSOL Multiphysics Finite element
analysis was completed using COMSOL Multiphysics. While the developed algorithm does not depend
on any one specific FEA software suite, COMSOL Multiphysics was chosen for its extensive Matlab
interface. This allowed the entire FEA solution to be initiated and controlled by Matlab. Results from the
COMSOL solver could be easily integrated within the user interface developed with Matlab’s GUIDE
framework. 3.1.3 SolidWorks SolidWorks 2016 was used to perform some processing of the patient face
template geometry to facilitate FEA. 3.2 Graphical user interface (GUI) A graphical user interface (GUI)
was developed to allow for easy use of the tool by the user. The Matlab GUIDE system was used to
develop this GUI, and development was guided by several design criteria: • Interface and interactive
elements, such as buttons, must be easy to use and understand. 14 Chapter 3. Methods 15 • Interface
should require minimal interaction from user to obtain desired results. • Results must be easily
understood and interpreted by user. • Interface should be somewhat aesthetically pleasing. It was
important that the GUI be developed for modern computing interfaces, and this included touchscreen
devices. As a result, GUI elements of appropriate sizes were chosen to allow for direct touchscreen
interaction. This was chosen to be as unobtrusive as poss

q33

TARGETING SYSTEM XC - DIRECTLY BY PHARMACOLOGICAL INHIBITION Page | 115 3.1 Introduction

Recently, the anti-inflammatory agent and system xc - inhibitor SSZ was demonstrated to reduce
behavioral manifestations of CIBP in nude mice [45]. While reduction in tumor cell glutamate release in
vivo was the proposed mechanism for its efficacy, this assertion was made without experimental
verification. We hypothesized that system xc - could be targeted with SSZ in an immunocompetent
mouse model of CIBP to reduce glutamate in the bone-tumor microenvironment and assuage CIBP. 3.2
66.1 cells release glutamate via system xc - Several tumor cell lines, including those of mouse, rat and
human origin, release glutamate in vitro via the cystine/glutamate antiporter system xc - [152]. We
investigated the expression and function of this transporter in the spontaneously occurring, murine
(BALB/cfC3H) mammary adenocarcinoma cell line 66.1. This cell line is highly tumorigenic and has
previously been used by our group and others [25, 357, 363] for in vitro and in vivo investigations of
metastatic bone disease [364]. To demonstrate expression of system xc - in 66.1 cells, we used
immunocytochemistry to visualize the antiporter. System xc - , like other members of the heteromeric
amino acid transporter family, has both a shared heavy subunit (4f2hc) and a light subunit (xCT). xCT is
unique to system xc - and is required for the transporter’s amino acid exchange function.
Immunofluorescent xCT staining was evident in untreated 66.1 cells (Figure 5). Page | 116 Figure 5 Page
| 117 Figure 5. The spontaneously occurring murine mammary adenocarcinoma cell line 66.1 expresses
xCT. 66.1 cells were plated on glass coverslips, fixed, probed with an anti-xCT antibody and stained with
DAPI nuclear counterstain. Representative images are shown of xCT and DAPI immunofluorescence, as
indicated. Page | 118 To assess whether 66.1 system xc - is functional, 66.1 glutamate release into
Lglutamine-free medium was measured over time (Figure 6). Medium was collected for determination
of glutamate concentration 0, 6, 18 and 24 hr post application of Lglutamine-free media. Medium
glutamate concentration increased significantly from 10 nM / 10,000 cells at time 0 to 400 nM / 10,000
cells over 24 hr. System xc - is the only glutamate transporter implicated in breast tumor cell glutamate
release to date [152]; we verified its role in 66.1 glutamate release using two established system xc -
inhibitors: (S)-4-carboxyphenylglycine [356] (CPG-4, Figure 7A) and SSZ [356] (Figure 7B). Both CPG-4 (1,
3 and 10 µM) and SSZ (3, 10 and 30 µM) reduced 66.1-derived glutamate release in a concentration
dependent manner after an 18 hr incubation period. These concentration ranges were selected based
on previous work [45] and the desire to minimally effect cell viability. Medium glutamate con

TARGETING SYSTEM XC - INDIRECTLY BY MODULATION OF REACTIVE OXYGEN AND NITROGEN SPECIES

Page | 137 4.1 Introduction As an alternative to direct inhibition of system xc - with SSZ, we next
determined whether 66.1 system xc - expression could be reduced with a redox modulator. Since
system xc - is involved in mobilizing antioxidant defenses in cells, it is not surprising that up-regulation
occurs in response to oxidative challenge [154]. System xc - -mediated glutamate transport can be
induced by peroxynitrite, a potent oxidant that is significantly elevated in invasive breast carcinomas
[161]. We hypothesized that peroxynitrite may drive system xc - expression, tumor cell glutamate
release and CIBP. 4.2 Peroxynitrite regulates 66.1 system xc - functional expression To test this
hypothesis in vitro, we applied the superoxide, nitric oxide-donating compound, SIN-1, to 66.1 cells. SIN-
1 is an effective pharmacological agent for administering peroxynitrite (the product of the spontaneous
reaction of superoxide and nitric oxide) at the level of the cell in a relatively stable form, as authentic
peroxynitrite is highly volatile and may decompose into inactive metabolites before reaching the cell.
66.1 cells were treated with SIN-1 (50 nM to 5 µM) for 18 hours: a concentration range and time point
selected based on those known to increase system xc - mediated transport in other cell types [366]. SIN-
1 significantly increased xCT levels in whole cell lysates at both 500 nM and 5 µM (2.6 fold and 3.7 fold,
respectively), as compared to those of vehicle (0.01% DMSO)-treated cells (Figure 12). Lower SIN-1
concentrations did not affect system xc - expression. To determine the functional relevance of
peroxynitrite driven increases in system xc - protein expression, we investigated glutamate transport
from 66.1 cells in the presence Page | 138 of SIN-1. Incubation with SIN- increased tumor cell glutamate
release in a concentration dependent manner as compared to vehicle-treated cells (Figure 13). At
concentrations of 5 and 50 µM, SIN-1 significantly increased 66.1-mediated cell glutamate release up to
1.5 fold over an 18 hour period. Inclusion of the established system xc - inhibitor SSZ (3, 10 and 30 µM)
significantly reduced SIN-1-induced glutamate release (Figure 13). Together, these data suggest that
peroxynitrite (donated by SIN-1) increases system xc - - mediated glutamate expulsion from 66.1 cells.
Page | 139 A B Control 50 nM SIN-1 500 nM SIN-1 5 µM SIN-1 0 1 2 3 4 5 SIN-1 - 50 nM 500 nM 5 µM
*** * Fold Change xCT Expression Over Control 57 kDa 43 kDa 0 nM 50 nM 500 nM SIN-1 xCT β-Actin 5
µM A B Control 50 nM SIN-1 500 nM SIN-1 5 µM SIN-1 0 1 2 3 4 5 SIN-1 - 50 nM 500 nM 5 µM *** * Fold
Change xCT Expression Over Control 57 kDa 43 kDa 0 nM 50 nM 500 nM SIN-1 xCT β-Actin 5 µM Figure
12 Page | 140 Figure 12. The superoxide/nitric oxide donor SIN-1 induces xCT expression. The
spontaneously occurring murine mammary adenocarcinoma cell line 66.1 was treated with SIN-1 (50 nm
– 5 µM) or vehicle (media). After 18 hr cells were harvested and prepared for Western blot analysis.
Samples were analyzed for expression of the functional subunit of the system xc - transporter, xCT (A).
Relative levels of xCT expression, standardized to actin in each lane, were determined by densitometric
analysis (B). Results are expressed as mean ± S.E.M. of four separate experiments. Asterisks represent
data points that are significantly different from vehicle

FURTHER CHARACTERIZATION OF CANCER PAIN MODELS Page | 196 5.1 Introduction As plainly stated
by 20th century statistician George E.P. Box, “all models are wrong, but some are useful." While Box was
referring to mathematical modeling, the same sentiment holds true for disease modeling in animals.
Animal models of human diseases are only helpful when we fully understand in what ways they reliable
recapitulate human pathophysiology and in what ways they deviant from the human presentation. In
our studies of the role of system xc - in CIBP, we have gained a significant amount of knowledge about
the neurochemical and behavioral signature of a murine model of CIBP. In addition to we have identified
novel biochemical and behavioral changes that result when CIBP is produced by the inoculation of 66.1
cells into the adult, female Balb/c mouse femur. 5.2 Elevated spinal nitration in CIBP A number of pain
states (e.g., inflammatory pain, chemotherapy-induced peripheral neuropathy) as well as opiate-induced
hyperalgesia and antinociceptive tolerance are associated pre-clinically with altered spinal cord nitration
[229, 378]. This nitration occurs as a result of the overproduction of reactive nitrogen species, including
peroxynitrite. Spinal cord nitration can result in a dysregulation of central glutamatergic
neurotransmission by altering both synaptic availability of and neuronal response to glutamate [378]. To
determine whether spinal cord nitration contributes to central sensitization in our murine model of
CIBP, whole lumbar spinal cord was collected 14 days post-surgery from sham and tumor-bearing
animals. In collaboration with Dr. Daniela Salvemini’s group at St. Louis University School of Medicine,
total and nitrated Page | 197 proteins levels of manganese superoxide dismutase (MnSOD), the
glutamate transporter GLT-1 and glutamine synthetase (GS) were assessed by immunoprecipitation with
an anti-nitrotyrosine antibody followed by Western blot analysis. When compared to Sham animals,
mice with CIBP showed a significant increase in the nitration of MnSOD and GS (Figure 34A, E). No
significant changes were observed in the total MnSOD or GS fractions (Figure 34B, F). Likewise, spinal
levels of nitrated or total GLT-1 protein were not changed during CIBP (Figure 34C, D). This is the first
report of altered nitration status in the spinal cord in preclinical CIBP. This alteration in nitration status
may contribute to enhanced glutamatergic neurotransmission and central sensitization in CIBP. Page |
198 A C E B D F A C E B D F A C E B D F A C E B D F A C E A B C D E F B D F Figure 34 Page | 199 Figure 34.
MnSOD and GS Nitration is elevated in CIBP. Lumbar spinal cords from tumor-bearing animals had
higher expression of nitrated MnSOD and GS, as compared to non-tumor-bearing (Sham) mice (n=9-12)
(A, E). Neither nitrated nor total protein levels for GLT-1 (C), MnSOD (B), GS (F) or GLT-1 (D) were
changed. All results are represented as the mean optical density standardized to α-tubulin per lane ±
S.E.M. To determine statistical significance Student’s t-test for unpaired data was used. *p < 0.0

Q222

Introduction: Fractures represent a common orthopaedic injury and create a large financial burden for
the health care system. Non-union fractures often require surgery to assist the healing process.
Understanding the origin of the postnatal skeletal stem cells will allow for locally delivered therapeutic
treatments to be more exactly spatially targeted for fracture healing. Two forms of post-natal bone
formation were studied, callus formation after fracture and ectopic bone growth. Both forms of bone
formation closely follow the mechanisms of endochondral ossification. The Prx1 gene that is known to
be expressed by skeletal stem/progenitor cells within the periosteal tissues was used to spatially follow
this cell population during ectopic and fracture induced bone formation. Objectives: The purpose of this
study was to define the cell lineages that arise from Prx1 expressing cells in fracture and ectopic bone
models. Methods: Prx1 expressing cells were tracked using Prx1CreER-GFP x RosaAi14 (dTomato
indicator) and Prx1CreER-GFP x Ai14i (dTomato indicator) in the Rag1tm strain of transgenic mice. These
mice strains respectively received a closed stabilized fracture or human demineralized bone matrix
(DBM) that was surgically implanted onto the periosteal surface of the femur to initiate the
development of ectopic bone. Tissue was collected at either day 10 or 14 post-fracture surgery or day 8
post-DBM implantation. Prx1CreER-GFP expression was induced by tamoxifen or control animals
received no vi injection or corn oil. Three different tamoxifen induction protocols (30 days prior to
surgery to allow for washout, three days prior to, or continuously after either fracture or ectopic bone
induction were used. Fluorescent microscopy of the histological images were performed to assess the
cell populations that expressed Prx1 and cell counting was used to quantify the percentages of Prx1
positive cells in specific regions of interest. qRT-PCR of Prx-1 mRNA expression was used to provide the
relative gene expression of Prx1 in a variety of different tissues. Results: Control animals that received
corn oil or no Tamoxifen showed low levels (~5- 15%) labeled cells, however there was a ~2 fol

As discussed earlier, Prx1 cells are known to be a contributor to fracture healing as it has been
discovered within both osteogenic and chondrogenic lineages in the callus (Murao et al., 2013). The
fracture portion of this study permitted for investigation into how differing Tamoxifen schedules and
harvest dates affect the recruitment of Prx1 cells. Two harvest time points, POD 10 and 14, and three
different Tamoxifen procedures: prefracture injections, washout, and continuous injections were used.
As suspected, all samples showed some presence of Prx1 cells in and around the callus. Post-operative
harvest dates of 10 and 14 days were chosen to analyze calluses at different stages of healing. At
POD10, most of the callus surrounding the fracture consists of cartilage, whereas POD14 has a callus
that contains the beginnings of ossified bone. Separate Tamoxifen injection schedules leads to labeling
Prx1 cells at specific time points. The washout method tends to follow the lineages of stem cells that
were labeled one month before surgery or harvest. Continuous injections of Tamoxifen attempts to
identify expressing Prx1 cells through the process. Lastly, Tamoxifen injections prior to the fracture
surgery target cells and their lineages that are recruited to the callus site during fracture repair, but not
necessarily through the process. Comparing the POD10 samples, the experimental mice that received
continuous Tamoxifen injections displayed approximately half of their total nucleated cells to be 49 Prx1
positive within the callus. The sample given only pre-fracture injections of Tamoxifen possessed about
one third of their total nucleated cells as Prx1 positive. The POD14 specimen represented a larger
percentage of Prx1 positive cells, but this could be due to mainly two factors. First, the harvest date was
four days later than the POD10 samples and therefore could potentially have allowed for more
expansion and differentiation. Also, it should be noted that the POD 14 received Tamoxifen washout.
When establishing a baseline response, the washout method did lead to a higher percentage of labeling
than the continuous injections. Thus either a greater number of initial stem cells became labeled that
expanded into their differentiated lineages or some fraction of committed cells derived from the initial
stem cell expansion contributed to the bone formation of the callus. Effects of Ectopic Bone on Prx1
Recruitment The recruitment of Prx1 derived cells to ectopic bone development was investigated. All
periosteal implants were harvested on POD 8, but either received continuous Tamoxifen, Tamoxifen
washout, or a single Tamoxifen injection 3 days prior to the DBM surgery. Similarly to the fracture
model, these different Tamoxifen methods allowed for close tracking of the Prx1 positive cells during
recruitment for ectopic bone growth. When comparing the percentage of Prx1 positive cells in each
scenario, it was interesting to see that the fracture callus had about 50% derived from Prx1, while the
baseline samples possessed approximately 14% and the only about 25% contributed to ectopic bone
formation. Given that the ectopic bone method was in the absence

Q11

TCDD Disrupts Cranial Cartilage and Dermal Bone Development in Zebrafish Lar

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) disrupts craniofacial development in zebrafish

larvae in part by decreasing sox9b (SRY [sex determining region Y]-box 9b) expression in craniofacial
cartilage. However, the identification of associated chondrocytic changes responsible for the decreased
jaw size and timing of TCDD-induced repression of sox9b remain poorly understood. We show that
smaller jaw size is due to a decrease in size and number of chondrocytes in craniofacial cartilage of
zebrafish. By immunostaining a mitotic marker in the background of a transgenic zebrafish expressing
EGFP under control of the sox9b promoter, we discovered that TCDD exposure beginning after the
period of pharyngeal arch restriction decreases perichondrial cell proliferation. When TCDD exposure
occurs at 4 hours post fertilization (hpf), it downregulates sox9b expression in the whole embryo, but
only after pharyngeal arches are populated by cranial neural crest cells. TCDD was found to decrease
ossification of osteoblasts in the perichondrium of cartilage that previously expressed sox9b. TCDD is
shown in this study to cause clefting of the parasphenoid for the first time in fish, reminiscent of TCDD-
induced cleft palate in mice. Thus, dermal and perichondrial bone development of the craniofacial
skeleton are clearly disrupted by TCDD exposure in the zebrafish larvae. This dysmorphic response of the
zebrafish craniofacial skeleton after exposure to TCDD is consistent with findings demonstrating
disruption of axial bone development in medaka and repression of sox9b
The transcription factor SOX9 is a member of the SRY-related high-mobilitygroup box (SOX) superfamily
of genes. In mammals, Sox9 plays important roles in many developmental processes including
craniofacial, skeletal and heart morphogenesis, retinal and brain development, and gonad
differentiation. Human mutations in SOX9 or the SOX9 promoter result in campomelic dysplasia, a
severe genetic disorder, which disrupts skeletal, craniofacial, cardiac, neural and reproductive
development. Due to the teleost fish genome duplication, zebrafish (Danio rerio) have two Sox9 genes:
sox9a and sox9b. Loss of sox9b in zebrafish results in loss of function phenotypes that are similar to
those observed in humans and mice. In order to generate a transgenic sox9b:EGFP reporter line, we
cloned a 2450 bp fragment of the sox9b promoter and fused it to an EGFP reporter. Consistent with
reported sox9b expression and function, we observed sox9b:EGFP in the developing heart, skeletal and
craniofacial structures, brain, retina, and ovaries. Our resulting transgenic line is a useful tool for
identifying and studying sox9b function in development and visualizing a number of zebrafish organs
and tissues in which sox9b is normally expressed

Enhancers of the SRY-related high-mobility box 9 (SOX9) gene integrate a myriad of signaling pathways.
To delineate the elements that recapitulate sox9b expression in zebrafish, we cloned and sequenced
multiple lengths of the sox9b promoter and characterized their expression by transgenic and transient
analysis with an enhanced green fluorescent protein (EGFP) reporter. The regions of the sox9b promoter
used to drive EGFP caused organ-specific expression as follows: notochord (- 902/-1386 bp), heart (-
562/+29 bp), brain (-902/-1816 bp), and jaw (-902/-1386 bp). With respect to heart, a minimal promoter
containing a conserved TATA box and an EGR2 element recapitulated sox9b expression transiently, and
a 591 base pair sox9b:EGFP transgenic fish also reported heart expression. This suggests that a smaller
region of the sox9b promoter (-74/+29 bp) may be sufficient to drive EGFP expression in the heart. In
addition, a 2450 base pair region 5’ of the sox9b contained eight conserved non-coding elements that
included putative HIF1α, CCAAT, EGR2, and core promoter elements. One CCAAT box was conserved
with sox9a suggesting the presence of a non-partitioned regulatory element. Finally, three sequences in
the longest cloned sox9b promoter contained stable second