cryosensitizers: ablation at the edge ablation at the edge john g. baust ... control fre (-15)...
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CRYOSENSITIZERS: Ablation at the Edge
John G. BaustInstitute of Biomedical Technology
State University of New YorkBinghamton, NY USA
Precision targeting
ChallengeUncertain margin
Responsiveness gradient
0 oC
-15 oC
Cryolesion
Thermal variability within lesion
Cooling rates
Temperature
Multiple Modes of Cell Death
Freeze Rupture
Necrosis
Apoptosis
Freeze Rupture
Necrosis
Apoptosis
- - - - - - - - - - -
Effect of Freezing on Viability of Prostate Cancer Cells In Vitro
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100 110
Temperature (C)
% V
iabi
lity
Clarke (2000)
Hollister (1998)Bischoff (1997)
Znati (1998)
Freezing Response (15 min) of Human Prostate Cancer Cells (PC-3) Treated with Double Freeze Cycle
0
25
50
75
100
Control -5°C -10°C -15°C -20°C -25°C -40°CCondition
Perc
ent V
iabi
lity Pre-Freeze
Day 1Day 3Day 5Day 7Day 9
Freezing Response (15 min) of Human Prostate Cancer Cells (LNCaP) Treated with Double Freeze Cycle
0
25
50
75
100
Control -5°C -10°C -15°C -20°C -25°C -40°CCondition
Perc
ent V
iabi
lity Pre-Freeze
Day 1Day 3Day 5Day 7Day 9
*
*
**
*
*
p< 0.005
p< 0.005
Tissue-Engineered Prostate Model
PC-3
PC-3
LNCaP
LNCaP
1-Day Post Freeze
PC-3 Cell Recovery After Cryo, Chemo, and Combination
0
10
20
30
40
50
60
70
80
90
100
110
Control 5-FU 2day 5-FU 4day -20 12hr prior 1 dayprior
2 dayprior
4 dayprior
Perc
ent V
iabl
e
2 Days
4 Days
6 Days
*
* *
P < 0.001
PC-3 Cell Recovery Following Exposure to Freezing (-150C) Alone or in Combination with Cytotoxic Agents
0
10
20
30
40
50
60
70
80
90
100
110
Control Fre (-15) 5FU/Fre Tax/Fre Etop/Fre Cis/Fre FA/Fre
% V
iabl
e
2 Days
5 Days
8 Days
11 Days
14 Days
Double Freeze
Double Freeze/TRAIL
Double Freeze/Taxotere
-105 C -5 C-20 C-40 C-60 C
-105 C -1 C-20 C
Single Freeze
-40 C-60 C
Comparison of Combinatorial Approaches to Cryosurgery
Factors Underlying Ablative Response
HypothermiaMetabolic UncouplingEnergy DeprivationWaste AccumulationIonic ImbalancepH Shifts (acidosis)Membrane Phase TransitionsCytoskeletal Disassembly
FreezingExtracellular IceHyperosmolalityCell Volume DisruptionProtein DenaturationIntracellular IceShearing Membrane DisruptionVascular Stasis
Induction of ApoptosisIschemiaHypoxiaNutrient deprivationIonic imbalanceIncreased free radicalsEnergy deprivationMembrane alterationsBiochemical alterations
Induction of Necrosis
All Events
Hallmarks of Cancer
Proliferative SignalingEvade Growth SuppressorsResist ApoptosisInduce AngiogenesisAvoid Immune System DestructionReprogram Cellular EnergeticsEnable Replicative Immortality (CSC)Recruit Non-cancerous Stromal Cell to Create MicroenvironmentMobilization = Metastasis
Effects of VD3 and Freezing at -15°C (15 min) on Human Prostate Cancer Cell Lines LNCaP LP and PC-3 AR
0
20
40
60
80
100
120
Control 50nM FR171 -15°C Only -15°C with50nM FR171
Control 50nM FR171 -15°C Only -15°C with50nM FR171
LNCaP LP PC-3 AR
Condition
Perc
ent V
iabi
lity
Pre-FreezeDay 1Day 3Day 5Day 7Day 9
BJU Intl 2011
Effects of VD3 and Freezing at -15°C (15 min) on Human Prostate Cancer Cell Lines LNCaP HP and PC-3
0
20
40
60
80
100
120
Control 50nM FR171
-15°C Only -15°C with50nM FR
171
Control 50nM FR171
-15°C Only -15°C with50nM FR
171
LNCaP HP PC-3
Condition
Perc
ent V
iabi
lity
Pre-FreezeDay 1Day 3Day 5Day 7Day 9
BJU Intl 2011
BJU Intl. 2011
Hallmarks of Cancer
Proliferative Signaling – loss of cell cycle controlEvade Growth Suppressors - -p53Resist Apoptosis - +Bcl-2, -p53Induce Angiogenesis - +VEGFAvoid Immune System Destruction – surface markersReprogram Cellular Energetics - glycolysisEnable Replicative Immortality - CSCRecruit Non-cancerous Stromal Cells Create to Microenvironment – tumor associated-endotheliumMobilization = Metastasis - - cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors -Resist Apoptosis - +bcl2, -p53Induce Angiogenesis - +VEGFAvoid Immune System Destruction -Reprogram Cellular Energetics -Enable Replicative Immortality - CSCRecruit Non-cancerous Stromal Cell to Create Microenvironment -Mobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis - +bcl2, -p53Induce Angiogenesis - +VEGFAvoid Immune System Destruction -Reprogram Cellular Energetics -Enable Replicative Immortality - CSCRecruit Non-cancerous Stromal Cell to Create Microenvironment -Mobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis – inhibits Bcl-2Induce Angiogenesis - +VEGFAvoid Immune System Destruction -Reprogram Cellular Energetics -Enable Replicative Immortality - CSCRecruit Non-cancerous Stromal Cell to Create Microenvironment -Mobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis – inhibits Bcl-2Induce Angiogenesis – inhibits VEGF transcriptionAvoid Immune System Destruction -Reprogram Cellular Energetics -Enable Replicative Immortality - CSCRecruit Non-cancerous Stromal Cell to Create Microenvironment -Mobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis – inhibits Bcl-2Induce Angiogenesis – inhibits VEGF transcriptionAvoid Immune System Destruction - ?Reprogram Cellular Energetics – uncouples metabolismEnable Replicative Immortality - CSCRecruit Non-cancerous Stromal Cell to Create Microenvironment -Mobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis – inhibits Bcl-2Induce Angiogenesis – inhibits VEGF transcriptionAvoid Immune System Destruction - ?Reprogram Cellular Energetics – uncouples metabolismEnable Replicative Immortality - ablates CSC (?)Recruit Non-cancerous Stromal Cell to Create Microenvironment -Mobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis – inhibits Bcl-2Induce Angiogenesis – inhibits VEGF transcriptionAvoid Immune System Destruction - ?Reprogram Cellular Energetics – uncouples metabolismEnable Replicative Immortality - ablates CSC (?)Recruit Non-cancerous Stromal Cell to Create Microenvironment – ablates stromal cellsMobilization = Metastasis - -cell adhesion molecules
Hallmarks of CancerVD3 + Cryo Induced Changes
Proliferative Signaling – restores of cell cycle controlEvade Growth Suppressors – restores p27, p53Resist Apoptosis – inhibits Bcl-2Induce Angiogenesis – inhibits VEGF transcriptionAvoid Immune System Destruction - ?Reprogram Cellular Energetics – uncouples metabolismEnable Replicative Immortality - ablates CSC (?)Recruit Non-cancerous Stromal Cell to Create Microenvironment – ablates stromal cellsMobilization = Metastasis - ++cell adhesion molecules
Key Findings
VD3 pre-treatment pulse (2 days) followed by freezing (in vitro and in vivo) resulted in
Total cell ablation without of re-growth for both androgen-sensitive (AS) and androgen-insensitive (AI) prostate cancers.
Increased levels of both necrotic and apoptotic cell death cascades.
Increased caspase activity and duration in AI cells more significantly than was observed for AS cells.
Elevated caspase-9 activity in the AI cells indicates that vitamin D3 treatment prior to freezing was able to increase mitochondrial mediated apoptotic cascades to levels comparable with freeze-sensitive AS cells.
Single Pulse Vitamin D3 exposure offers a promising neo-adjunctive cryosensitization strategy.
Vitamin D3 Increases Cryoablation Efficacy
Cryosurgical Technology (boat)
Cryoablation: Self Taught