Zaver M. Bhujwalla
Director, Division of Cancer Imaging Research
The Russell H. Morgan Department of Radiology
and Radiological ScienceThe Johns Hopkins University
School of MedicineBaltimore, MD 21205, USA
&Past President, WMIS
Molecular Imaging –A New Field For a New
World
Molecular Imaging Society of IndiaMarch 9, 2014Mumbai, India
• The field of molecular imaging is an exciting fusion of many differentscientific disciplines including imaging technologies, molecular biology,and chemistry that is providing major new insights and advances intodifferent diseases and their treatment.
• Reflecting the society’s integrated multimodal approach that spansdiscovery to preclinical and clinical applications, the membership consistsof scientists, clinicians, young researchers and students from verydifferent disciplines with the common goals of better understandingpathological processes noninvasively, developing interventions to obtainearly diagnostic and prognostic information, and developing image-guidedtherapy.
• It is a transformative new field for a new world where many differentdisciplines work together in understanding and effectively treatingdiseases that globally extract a heavy social and economic burden.
WMIS VISIONTo encompass and globally promote preclinical and clinical
multi-modal imaging applications to understand and effectively treat diseases
Basic Preclinical First-in-human
Transformative
At the Transformative Edge of Molecular Imaging
Meiyappan Solaiyappan
‘New cancer cases will risefrom an estimated 14 millionannually in 2012 to 22 millionwithin two decades. Over thesame period, cancer deaths arepredicted to rise from 8.2million a year to 13 million.’
‘In 2010, the economic cost of the disease worldwide wasestimated at $1.16 trillion.’
‘The cancer burden in developingcountries is reaching pandemicproportions. Globally, cancer kills morepeople each year than AIDS,tuberculosis and malaria combined, andthe number of deaths is growing rapidly.’
Annals of Oncology 21: 680–682, 2010
‘India is facing a cancer epidemic.’
The Lancet, 379: 992 - 993, 2012
Cancer is the second leading causeof death after heart disease in theUS.
PhysiologicalStromal
Vascular and lymphatic networks
Substrates
H+
H+ H+
pH
Macrophages
Fibroblasts
HRE Reporter Gene (EGFP)
mRNAHIF-1
Hypoxia
Tumor
ECM
Collagen fibers
GluGlu
Stasinopoulos et al., Exploiting the tumor microenvironment for theranostic imaging, NMR in Biomedicine, 2011.
Diffusion Imagingb=500
Low ADC
High ADC
T2WI
Normal
Tumor
Dynamic Contrast MRI
T1WI
Jacobs MA, Stearns V, Wolff AC, Macura KJ, Blumeke DA, Et. al.,
Journey through the Tumor Microenvironment
• Multi-modal imaging (MRI-optical) to understand therelationship between vasculature and hypoxia
• Inflammation – COX-2 – and the multifaceted impact of asingle enzyme on the tumor microenvironment – an exampleof how a Gordian knot can also be an Achilles heel
• Theranostic imaging
O2 Concentration
Well OxygenatedModerate Hypoxia
HighlyHypoxic
Radioresistance
Drug resistance
< 2.5mm Hg
Hypoxia in Tumors
• Hypoxia is a major cause of radiation and chemo-resistance
HRE Reporter Gene (EGFP)
mRNAHIF
Green fluorescencereporter protein
hypoxia
HIF stabilized
oxygenation
HIF proteolysed
Cancer cells stably transfected with HRE-EGFP/RFP
Peripheral oxygenated region
pO2 ~ 42 mmHgHypoxic region
pO2 ~ 0 mmHg
Raman et al., Cancer Research, 2006
HRE-RFP MDA-MB-231 tumor. Mouse injected with Hoechst 33342
pO2 = 0.0 mmHg
Normoxia Hypoxia
Combined MRI and Optical Imaging
VascularVolume
0
344 ul/gm
Permeability Surface Area Product
0
24 ul/gm-min
MRI
low vascular volume
highpermeability
VASCULARIZATION - HYPOXIA
1 mm thick fresh sectionfrom MR imaged slice
OPTICAL
high fluorescence(high HRE activity)
Raman et al., Cancer Research, 2006
Albumin-GdDTPA contrast agent
MDA-MB-435 MDA-MB-231 MCF-7 MatLyLu PC-3 DU-145
Bhujwalla et al., Neoplasia 2001.
Meiyappan Solaiyappan
Vascular volume displayed in redPermeability for albumin-GdDTPA (~ 90 kD) displayed in green•few yellow areas
Vector transfectedPC-3 tumor
VEGF overexpressingPC-3 tumor
Journey through the Tumor Microenvironment
• Multi-modal imaging (MRI-optical) to understand the relationship betweenvasculature and hypoxia and the extracellular matrix
- Poor vascularization leads to hypoxia- Leaky vessels occur in poorly vascularized hypoxic regions
• Inflammation – COX-2 – and the multifaceted impact of a single enzyme onthe tumor microenvironment – an example of how a Gordian knot can alsobe an Achilles heel - if COX-2 addicted tumors can be identified
Stasinopoulos et al., Frontiers in Pharmacology, 2013
Inflammation (Latin, īnflammō, "I ignite, set alight") is part of the complexbiological response of vascular tissues to harmful stimuli, such aspathogens, damaged cells, or irritants. Inflammation is a protective attemptby the organism to remove the injurious stimuli and to initiate the healingprocess.
Cancers have wound-like environments such as hypoxia and acidicextracellular pH – ‘tumors are wounds that do not heal’ (Dvorak, NEJM, 1986)
Silencing of COX-2 in MDA-MB-231 cells
COX-2
GAPDH
COX-2
GAPDH
Clone 2: Clone stably transfected with a plasmid coding for a COX-2
shRNA
Pooled: Pool of four clones stably transfected with the COX-2
shRNA plasmid
IL-1β (10 ng/ml) - - - - + +
+ +
Stasinopoulos et. al. Mol Cancer Res. 5, 435 (2007)
TPA (50 nM) - - - - + +
+ +
Silencing of COX-2 delays tumor onset in SCID mice T
um
or
volu
me (
mm
3)
0 20 40 60 800
200
400
600
800
1000
MDA-MB-231Empty vectorClone 2Pooled
700 mm3
548 mm3
3142 mm3
time (days)
2 mm0
2x105
4x105
6x105
24 48 72
Cell
num
ber
Incubation time (h)
Pooled
Clone 2
Empty vector
MDA-MB-231
4/10
1/10
n/a
n/a
measurable tumors
(90 days)
0/10
1*/11
n/a
n/a
measurable tumors
(60 days)
0/10
0/11
9*/9
10*/10
measurable tumors
(30 days)
10
11
9
10
number
of mice
Stasinopoulos et al., Mol. Cancer Res.
2007
Arachidonic acid metabolism-related genes
Oncogenes
MMP1CXCR4 – qPCR verified IL11SMAD1 - qPCR verified SPDEFNOVHAS2KRT19NGFRJAG1 - qPCR verified CUGBP2S100P
-60 -50 -40 -30 -20 -10 0
Tumor suppressor genes
0 1 2 3 4 5
THBS1LUMEBI3CLU
ROBO4GBP1
SCARA3TPM1
6
214 856 424
231 vs Clone 2 231 vs Pooled
381 1483 679
Total down-regulated genes
Total up-regulated genes
Fold loss Fold induction
231 vs Clone 2 231 vs Pooled
PTGS2
PTGS2*PLA2G4APTGES
-40 -30 -10-20 0
231-Pooled231-Clone2
231-Pooled231-Clone2
231-Pooled231-Clone2
COX-2 silencing alters the transcriptome of MDA-MB-231
cells
Stasinopoulos et al., Mol.
Cancer Res. 2007
High COX-2
COX-2 silenced
Proton Spectroscopy
Shah et al., NMR in Biomed., 2010
Shah et al., NMR in Biomed. 2010
COX-2-knocked down Clone 2
(35 days post-injection)
COX-2-knocked down Pooled
(35 days post-injection)
MDA-MB-231
(34 days post-injection)
Stasinopoulos et al.,
Mol. Cancer Res. 2007
COX-2 silencing inhibits invasion and metastasis
Cells are plated on polyacrylamide gel containing fluorescent beads. Cells exert traction forces on these beads following their attachment.Cells are trypsinized and the beads return to their unstrained position.Deformation field is measured and quantified.
Fourier Transform Traction Microscopy
20 µµµµm
(Phase Contrast) (Fluorescence)
(Tolić-Nørrelykke, Butler, Chen, Fredberg, Wang, Am J Physiol Cell, 2002)
Traction map of human airway smooth muscle cell
COX-2
GAPDH 0
500
1000
1500
2000
PG
E2
(ng/µ
l/200,0
00 c
ells
)
COX-2 expression and PGE2 production are reduced, but not silenced in Clone 13 cells
*
* p< 0.05
Clone 13MDA-MB-231
Permeability maps acquired over 30 min. following i.v.
injection of the macromolecular contrast agent albumin-GdDTPA
0
30
(µl/g.m
in)
COX-2-LowCOX-2-High
Pooling(influx rates)0
93
(µl/g.m
in)
-44
0
(µl/g.m
in)
COX-2-LowCOX-2-High
Draining(efflux rates)
Macromolecular transportPermeability
COX-2 changes permeability and macromolecular transport
From: Stasinopoulous, Kakkad et al.
Second harmonic generation microscopy of collagen 1 fibers
Clone 13
COX-2 changes collagen fiber content and density
MDA-MB-231
From: Stasinopoulous, Kakkad et
al.,
perfused
cancer
cells
human tumor xenografts
COX-2
Pain
Immunity
Development Labor
Arthritis
Neurode-
generation
Asthma
Cancer
Collagen-1 fibers
ECM
High
COX-2
Low COX-
2
Macromolecular transport
Draining
(efflux rates)
Pooling
(influx rates)
High
COX-2
Low COX-
2
Permeability
High
COX-2
Low COX-
2
High
COX-2
COX-2
silenced
Invasion
High
COX-2
COX-2
silenced
Metastasis
High COX-2
COX-2
silenced
Proton Spectroscopy
Mechano-molecular imaging Gordian Knot or Achilles Heel?
Target
pHe
Vascular/ lymphatic
endothelial cell
receptors
Cancer cell
receptors
Stroma/stromal cell receptors
Hypoxia
MRS/ I
MRI
PET/
SPECTUS
Optical
Imaging
cDNA
siRNA
Prodrug/
enzyme
Photo-
dynamic
therapy
Radiation
Chemo-
therapy
Therapy
• Liposomes
• Nanoparticles• Micelles• Viral vectors
Carrier
Stasinopoulos et al., Exploiting the tumor microenvironment for theranostic imaging, NMR in Biomedicine, 2011.
‘THERANOSTIC IMAGING’
– combining detection with
treatment
Exploiting the TME for
Theranostics
Minimize damage to
normal tissue
THERANOSTIC IMAGING
PSMA
Chen, Penet et al., ACS Nano 2012
Cho et al., J. Nucl. Med. 2012
Aboagye and Bhujwalla, Cancer Research, 1999 (breast cancer)Ackerstaff et al., Cancer Research, 2001 (prostate cancer)Iorio, Podo et al., Cancer Research, 2005 (ovarian cancer)Shah et al., 2013 (pancreatic cancer)Glunde, Bhujwalla, Ronen, Nature Reviews Cancer, 2011
SPECT imaging of SCID mouse bearing PIP (PSMA +ve, red arrow) and FLU (PSMA –ve, green arrow) tumor. Mouse was injected i.v. with 1.4 mCi 111In labeled PSMA-targeted nanoplex (150 mg/kg in 0.2 ml). Decay corrected SPECT/CT images at 48 hand 72 h demonstrate increased accumulation in PSMA expressing PIP tumors.
GAPDH
PSMA
PC3 PipPC3 Flu
Chen, Penet et al., ACS Nano 2012
Theranostic Imaging
• bCD active
Chen, Penet et al., ACS Nano 2012
Theranostic Imaging - siRNA
Pre-treatment 48 h post-treatment
Tota
l ch
oli
ne
de
nsi
ty m
ap
s
6 4 2 6 4 2ppm ppm
tCho tCho
• Reduction of total choline
PSMA
• Personalized ‘omics’-based probes for the TME• Multiple siRNA (metabolism/vascularization)• Multiple targets
• Understand and target the TME
U87-stb-CXCR4
U87-stb-CXCR4U87 U87
24 h 48 h
H&E
Ki67
Ki67
U87
U87-C
XC
R4
H&E
Nimmagadda et al., unpublished data
Challenges• Cost
• Immunogenicity
• Toxicity studies in mice
• cGMP synthesis
• FDA/IRB approval
• Phase 0/I trial
Johns Hopkins Center for Translational Molecular Imaging (CTMI)
Acknowledgements
Support from NIH P50 CA103175, P30 CA006973, R01 CA73850, R01 CA82337, R01 CA136576 and R01 CA138515 is gratefully acknowledged.