Molecular imaging – a new field for a new world By Zaver M. Bhujwalla

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



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


MDA-MB-231


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


Theranostic Imaging

• bCD active


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.

Health & Medicine

Molecular imaging – a new field for a new world By Zaver M. Bhujwalla