8/2/2012
Vascular and Tumor Response to Microbeam
Radiation Therapy In Vivo using a Murine
Window Chamber Tumor Model
M.K. Boss, A.N. Fontanella, J. Zhang,
G.M. Palmer, S. Chang, M.W.
Dewhirst
Outline
• Effects of ionizing radiation on tumor and
stromal cell biology
• Rationale for microbeam irradiation
• Methods
• Results
– Angiogenic response
– Tumor cellular response
• Conclusions and Applicability to marginal miss
Dual reporter cell lines:
HIF-1-eGFP, CMV-RFP
HIFHIF-1 reporter:
5 copies of VEGF HRE + minimal CMV
HCT116 and 4T1 tumor lines
Hoechst
Perfusion
HIFHIF-1
Reporter
RFP
HIFHIF-1
Reporter Microvessels
Overlay
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Hypoxia-inducible Factor 1α (HIF-1α)
Angiogenesis
Apoptosis
pH Regulation
Glycolysis
Mutagenesis
Metastasis
Therapeutic
Resistance
Reoxygenation post RT increases free
radicals
H2DCFA
or
DCFA
PBS
2 x 5Gy
24hr after 2nd
RT dose
+
+
N
N
N
N
Mn+
N+
N
N
N+
Moeller et al, Cancer Cell, 2004
bar = 300µm
Free radicals post RT
increase HIF-1α
α and protect vasculature
RT
+
PBS
+
+
N
N
N
N
M n+
N
N
N+
RT
+
SOD
mimet
N+
RT = 5Gy x 2
bar = 300µm
Moeller et al, Cancer Cell, 2004
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HIF-1 upregulation post XRT (5 x 3Gy) increases
angiogenesis: knockdown sensitizes tumor endothelial
cells to RT
HCT 116
HIF-1 (+)
HIF-1 (-)
PC3
HIF-1 (+)
HIF-1 (-)
Moeller et al, Cancer Cell, 2005
High doses per fraction may kill
endothelial cells directly
Garcia Barros et al, Science, 2001
Background:
GRID, microbeam therapy
• High dose
• Spatially fractionated
• Reduced normal tissue
toxicity
• Ideal dose/spatial
geometry?
• Long-term tumor control?
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Enabling technology for compact MRT device :
Carbon nanotubes (CNT) field emission for high dose rate and
high spatial and temporal resolution x-ray source array.
eeee3 µm
Microscope image of CNTs.
Each CNT is a tiny electron gun
controlled by electric field.
Chang (UNC)
Methods and Preliminary Data:
Specific Aim 2 – The microbeam irradiator
•
•
•
•
Photon energy = 160 keV
Dose rate = 1-2 Gy/min
Total Dose = 50 Gy
Beam Width = 300 μm
Tumor
Radiation path
Study Design:
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8/2/2012
Hypotheses for 30-50 Gy single dose
• Partial tumor irradiation with microbeam will
increase HIF-1, stimulate an angiogenic
response and promote tumor regrowth
• Whole tumor irradiation will shut down
perfusion and eliminate tumor completely
Wide-field, 30Gy
Microbeam, 30Gy
Preliminary Results –
Effects post 30Gy single dose
Hb sat
Day 0, no treatment
Day 2, 48h post-treatment
Serial images post 30 Gy microbeam
Angiogenesis
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Serial images post 50Gy microbeam
2 hr post
Pre
1 day
Hyperemia - angiogenesis
3 days
5 days
6 days
Central necrosisangiogenesis
Central necrosis
Hyperemia - angiogenesis
Serial images post post 50Gy widefield
HIF-1-GFP Expression: Microbeam vs.
Widefield – 50Gy
Normalized Mean GFP (HIF-1) Expression
Mean GFP (relative to pre-treatment)
3
2.5
2
1.5
Partial
Whole
1
0.5
0
Pre
Post
D1
D2
D3
Time Point
D4
D5
D6
D7
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Vascular length density changes over
time: microbeam vs. widefield- 50Gy
Normalized Vascular Length Density
1.2
VLD (relative to pre-treatment)
1.1
1
0.9
Partial
0.8
Whole
0.7
0.6
0.5
Pre
Post
D1
D2
D3
Time Point
D4
D5
D6
D7
Result does not reflect peritumoral angiogenesis – still being analyzed
Hemoglobin Saturation of Tumor:
Microbeam vs. Widefield – 50Gy
Normalized Hemoglobin Saturation
8
%HbSat (relative to pre-treatment)
7
6
5
4
Partial
Whole
3
2
1
0
Pre
Post
D1
D2
D3
Time Point
D4
D5
D6
D7
Result does not reflect peritumoral hyperemia – still being analyzed
Pre-angiogenic tumor cell and vessel behavior
Day 0
Day 2
Day 4
Proliferation / Chemotaxis
Bar = 300µm
Day 8
Angiogenesis
C.Y. Li et al., JNCI, 2000
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8/2/2012
Radiation exposure stimulates
epithelial-mesenchymal transition
Garcia Blanco – Dewhirst, Unpublished
Evidence for epithelial – mesenchymal transition
post 50Gy microbeam
We speculate that partial-tumor irradiation may
induce EMT-facilitated cell migration.
Cell motion observed in bridge area –
2hr confocal time lapse
Bridging tumor cells do
not remain stationary
over 90 minutes
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Second example of tumor cell
migration – 50Gy microbeam
M5-Pre
Primary Tumor
Satellite tumors
Day 7
Primary Tumor
Conclusions
• 30Gy microbeam irradiation creates strong but
transient angiogenic response
• 50Gy microbeam
– Exhibits overall decline in microvessel density in
tumor, similar to wide field vs. peritumoral increase
– Very little evidence for reoxygenation, compared with
wide field
– Less upregulation of HIF-1 vs. widefield
– Appears to stimulate epithelial – mesenchymal
transition, promoting tumor cell migration and
colonization of relatively unirradiated areas.
Implications for marginal miss
following IMRT
• Irradiation with IMRT doses are likely to stimulate
at least a transient hyperemic and angiogenic
response, that extends beyond the irradiated
zone.
• Stimulation of tumor cell migration away from
irradiated site and potential for metastasis
promotion exists
• Further work is required at relevant IMRT doses
and with other tumor models to investigate these
hypotheses
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8/2/2012
Macrophage infiltration post RT
contributes to tissue response
Acknowledgements
•
•
•
•
•
•
Andrew Fontanella
Greg Palmer
Keara Boss
Mariano Garcia Blanco
Sha Chang
J. Zhang
CNT-based compact MRT system has produced
high peak-to-valley dose ratio (~1000).
Red Channel
EBT-2 Film Dosimetry of CNT MRT profile
Green Channel
10
1
Dose (Gy)
-3000
-2000
-1000
0
1000
2000
3000
1300
0.1
0.01
0.001
Position (microns)
Dosimetry film
(EBT-2)
Chang (UNC)
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8/2/2012
CNT-based compact MRT
Synchrotron-based MRT
Micro-CT
MRT
mouse
UNC has developed nanotechnology-based compact MRT system for research to
enable widespread MRT research.
Chang (UNC)
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