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STOCHASTIC MODELING OF BIOLOGICAL PROCESSES: QUANTITATIVE
OPTICAL IMAGING AND
TUMOR-INDUCED ANGIOGENESIS
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Amir
H. Gandjbakhche, PhD, Head,
Section on Biomedical Stochastic Physics Victor
Chernomordik, PhD, Staff Scientist Franck
Amyot, PhD, Visiting Fellow Moinuddin
Hassan, PhD, Visiting Fellow David
Hattery, PhD, Adjunct Scientist Asmaneh
Siavosh, BS, Postbaccalaureate Fellow Alexander
Sviridov, PhD, Guest Researcherb Abby |
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With
our main focus on quantitative theories applicable to in vivo quantitative
optical spectroscopy and tomographic imaging of tissues, we develop
quantitative theories, methodologies, and instrumentation to study biological
phenomena whose properties are characterized by elements of randomness in
both time and space. Our research requires analysis of different optical
sources of contrast such as endogenous or exogenous fluorescent labels,
absorption, and/or scattering. We design and conduct experiments and computer
simulations to validate theoretical findings. In addition, we conduct
collaborative research nationally, internationally, and within the Intramural
Research Program to investigate physiological sites for which optical
techniques might be clinically practical and might offer new diagnostic
knowledge and/or less morbidity than existing diagnostic methods. We also
study tumor-induced angiogenesis, which plays an essential role in the
establishment of tumor malignancy. To gain a better understanding of the
mechanisms underlying angiogenesis, we are developing stochastic models for
qualitative analysis of the biological events that constitute angiogenesis.
We develop methodologies applicable to in vivo quantitative tissue
spectroscopy and tomographic imaging, conduct experiments on tissue-like
phantoms, and run computer simulations to validate our findings. With
successes at the bench, we are bringing our imaging and spectroscopic devices
to the bedside. Time-resolved
tomography of thick tissue Chernomordik,
Sviridov, Hassan, Hattery, Gandjbakhche; in collaboration with Cubeddu,
Hebden, Rinneberg, Russo, Smith, Weiss, Zaccanti Using
time-domain scanning mammographs and based on the random walk (RW) model of
time-resolved contrast functions, we continued to analyze multispectral in
vivo data provided by our collaborators for the quantification of optical
properties of breast abnormalities (e.g., tumors). The additional data
confirm the tumors’ higher blood volume and higher hypoxicity than
surrounding tissues. A new time-resolved optical mammograph, built by the Canadian
company Advanced Research Technology (ART), is scheduled for installation at
the NIH for clinical protocols to evaluate new therapies for breast cancer.
Our main goal is to assess the role of optical mammography in therapy
response evaluation by correlating the optical signal acquired before,
during, and after neo-adjuvant therapy in breast cancer lesions. The
assessment will allow us to determine how optically derivable parameters of
breast cancers correlate with biological measurements of the tumor specimens
and how such correlation compares with other imaging modalities (MRI and
PET). We
have found differences in the scattering coefficients at two projections,
suggesting that tumors exhibit spatial structures. To obtain closed-form
analytic solutions, we have generalized the RW formulas for transmission and
reflection geometries to take into account possible differences in scattering
probabilities in different directions (i.e., parallel and perpendicular to
fibers). We recently initiated a joint project with University College London
to substantiate experimental RW theory (RWT) formulas with experimental data
from anisotropic phantoms. Preliminary results show that the RWT expressions
well describe experimental time-of-flight intensity distributions for different
orientations of the fiber-like structures. We plan to continue the studies
for different clinically relevant geometries and combine the RWT approach
with polarization analysis of reflected light to substantiate our findings on
anisotropic structures of tissues and tissue-like phantoms. RWT can also be used for fiber orientation. Many
biological tissue components such as collagen, muscle fibers, keratin,
retina, and glucose possess polarization properties. Depolarization of the
polarized light is strongly dependent on the bulk optical properties of the
tissue (absorption and scattering coefficients) and on optical anisotropy.
Mapping the degree of polarization may carry valuable information about the
superficial and subsurface structures of the skin and other tissues and may
contain information that cannot be observed visually or photographically. In
many cases, it is useful to enhance the subsurface structures, especially if
distorted in the procedure of polarization degree patterning. For this purpose,
a statistical analysis and methodology of noise filtering can help enhance
the hidden structures and estimate the characteristic sizes and
directionality of possible structures. We plan to map the degree of
polarization to visualize structural information about the skin and study the
propagation of polarized light in tissue and tissue-like phantoms and the
possible application of polarized light for tissue diagnostics. In
animal experiments, we took digital photographs of X-ray–irradiated
mouse skin by illuminating the skin with linearly polarized light (650 nm
wavelength). The patterning of degree of polarization allowed us to detect
earlier skin fibrosis structures that were not observable visually. Data
processing of degree-of-polarization images based on Fourier filtering of
high-frequency noise improved subjective perception of the revealed
structure. We developed a Pearce correlation analysis that provided
information about skin structure, size, and directionality. In a related
project, we investigated structures of mouse and human skin in vivo by
using polarized videoreflectometry. We focused an incident beam (linearly
polarized, wavelength 650 nm) on the sample surface and used two types of
tissue-like media as controls to verify the technique: isotropic delrin and
highly anisotropic demineralized bone with a priori knowledge of the
preferential orientation of collagen fibers. Equi-intensity profiles of
light, backscattered from the samples, were fitted with ellipses that
appeared to follow the orientation of the collagen fibers. The ratio of the
ellipses’ semiaxes correlated well with the ratio of reduced scattering
coefficients. We analyzed variation of equi-intensity profiles with distance
from the incident beam for different initial polarization states of the light
and the relative orientation of polarization filters for incident and
backscattered light. For the anisotropic media (demineralized bone and human
and mouse skin), we observed a qualitative difference between intensity
distributions for cross- and copolarized orientations of the polarization
analyzer up to 2.5 mm from the entry point. We expect that polarized
videoreflectometry of the skin may be a useful tool for assessing skin
fibrosis resulting from radiation treatment. Dagdug L, Weiss GH, Gandjbakhche A. Effects of anisotropic
optical properties on photon migration in structured tissues. Phys Med
Biol 2003;48:1361. Dudko OK, Weiss GH, Chernomordik V, Gandjbakhche A. Photon
migration in turbid media with anisotropic optical properties. Phys Med
Biol 2004;49:3979-3989. Hebden J, Garcia Guerrero JJ, Chernomordik V, Gandjbakhche AH.
Experimental evaluation of an anisotropic scattering model for a slab
geometry. Opt Lett 2004;29:2518-2520. Sviridov A, Chernomordik V, Hassan M, Russo A, Eidsath A, Smith
P, Gandjbakhche A. Intensity profiles of linearly polarized light
backscattered from skin and tissue-like phantoms. J Biomed Opt 2004, in
press. Three-dimensional
reconstruction of localized in vivo fluorescence Chernomordik, Hattery,
Gannot I, Gandjbakhche; in collaboration with Chen, Gannot G, Russo, Smith To
improve our reconstruction method further and make it a general tool for
optical biopsy, we have used an animal model: mice with squamous cell
carcinoma in their oral cavity. The model is easily repeatable and useful for
further investigations and refinement. The aim is to follow the immune
response to tumor cells. In our experiments, we observed formation and
development of a single fluorescent object localized in the tumor area. We observed
good correlation between the detected intensity profiles and the profiles
reconstructed from the theoretical model. We
have also initiated a collaboration with Wei Chen. Using BSA-coated CdMnTe/Hg
quantum dots, we have demonstrated that nanocrystals are a useful
angiographic contrast agent in mice. We administered the nanocrystals via
jugular injection; the excitation source was a 633 nm He-Ne laser, and we
captured the fluorescence, which peaked around 775 nm, with a sensitive CCD
camera with a bandpass filter. To make a preliminary assessment of the depth
of penetration for excitation and emission, we imaged a beating mouse heart,
both through an intact thorax and after a thoracotomy. We addressed the
temporal resolution associated with imaging the nanocrystals in circulation
and measured the blood clearance for the contrast agent. In addition, we
directly imaged the vessels surrounding and penetrating a murine squamous
cell carcinoma in a C3H mouse. In an in vitro study of a
blood/nanocrystal mixture, we observed no significant photobleaching or
degradation of the nanocrystals over the course of an hour under continuous
excitation. The preliminary results suggest that the NIR-fluorescent quantum
dots may be ideally suited for pharmacokinetic studies. In
the future, it will be important to characterize more fully the elimination
of the nanocrystal from the body in our or other animal models. Along with
gaining an understanding of the pharmacology of the nanocrystal, we would
like to target it and other nanocrystals to specific tissues. The use of
proper shell functional groups as well as of proper cross-linking agents and
the establishment of noncovalent bonding, such as electrostatic interactions
with other agents, could aid in nanocrystal trafficking. In addition, we
would like to explore the use of upconverting quantum dots. In this system,
luminescence arises from a two-photon process at a higher energy than the
excitation, thereby minimizing the effects of autofluorescence on the
measurements. We will attempt to develop quantum dots (i.e., Mn-doped CdTe)
that are suited for both optical and magnetic (ESR) measurements. Eidsath A, Chernomordik V, Gandjbakhche A, Smith P, Russo A.
Three-dimensional localization of fluorescent masses deeply embedded in tissue.
Phys Med Biol 2002;47:4079-4092. Gandjbakhche A, Chernomordik V, Hattery D, Hassan M, Gannot I.
Tissue characterization by quantitative optical imaging methods. Technol
Cancer Res Treat 2003;2:537-552. Gannot I, Garashi A, Gannot G, Chernomordik V, Gandjbakhche A.
Quantitative 3-D imaging of tumor labeled with exogenous specific
fluorescence markers. Applied Optics 2003;42:3073-3080. Morgan N, English S, Chen W, Chernomordik V, Gandjbakhche A,
Smith PD, Russo A. Real time in vivo non-invasive optical imaging using
near-infrared quantum dots. Acad Radiol 2004, in press. Monitoring
blood circulation in Kaposi’s sarcoma and complex regional pain
syndrome type I Hassan, Hattery,
Vogel, Gandjbakhche; in collaboration with Dionne, Yarchoan For
anti-angiogenic or pain therapies, factors associated with blood flow are of
particular interest. We have developed and are using several noninvasive
imaging techniques such as near infrared multispectral imaging, thermography,
and laser Doppler imaging (LDI). We use the multispectral imager to
reconstruct local variations in skin analyte concentrations such as oxy- and
deoxy-hemoglobin and blood volume. LDI measures the blood velocity of small
blood vessels, which generally increases as blood supply demand increases.
Combining multispectral imaging and LDI allows us to determine changes in
blood volume, oxygenation state, and blood velocity of the microvasculature
and location of the abnormality. Thermography, the third imaging technique,
provides the temperature as a means of assessing blood flow. Thermographic
patterns in medical diagnostic applications may be related to increased blood
flow associated with increased metabolic activity. The use of the three
imaging modalities provides a detailed analysis of tissue function as a
predictive tool for the outcome and individualization of therapeutic
strategies. In addition, the combined use of the techniques is important in
order to avoid spurious results. It is
now well recognized that new blood vessel formation is an essential component
in tumorigenesis. Currently, there is substantial interest in developing
therapies to treat cancer through inhibition of vessel formation.
AIDS-associated Kaposi’s sarcoma (KS) is a useful model for both
studying anti-angiogenesis approaches and imaging the results of newly
developed therapies. Three ongoing NCI clinical trials have so far
investigated 30 KS patients and have used thermography, LDI, and
multispectral images to record over the lesion for comparison with normal
skin either adjacent to the lesion or on the contralateral side. We obtained
measurements before therapy and after a regimen of liposomal doxorubicin and
interleukin-12 for 18 and 42 weeks. Before treatment, KS lesions generally
show higher blood volume, deoxy-hemoglobin, temperature, and blood velocity
than normal skin. We found a strong correlation between temperature and blood
velocity as well as with blood volume. After 18 weeks of anti-angiogenesis
drug treatment, the blood volume, deoxy-hemoglobin, temperature, and blood
velocity of the lesions were significantly lower than at baseline. Further
analysis of the data promises to deepen our understanding of tumor
angiogenesis and the effects of specific anti-angiogenic treatment. Complex
regional pain syndrome type I (CRPS-I) has long been clinically recognized.
Although the pathogenesis of CRPS-I is not clear, the syndrome appears as a
regional disorder of a limb characterized by burning pain; edema; autonomic
dysfunction such as altered skin color, temperature; sudomotor activity, a
movement disorder; or tropic changes such as muscle or skin atrophy. We are
exploring the applicability of thermography and LDI as techniques to
investigate and track physiological parameters associated with chronic pain
before and after treatment. In an
ongoing double-blind clinical trial conducted by the NIDCR, all subjects with
CRPS receive an individualized regime of physical therapy and standard
treatment to control their pain. In addition, they receive an experimental
drug or placebo tablets for five weeks. Patients return for an additional
five weeks of treatment in which the regimen is switched (i.e., those
preciously receiving the drug are given placebo tablets and placebo patients
receive the experimental drug. Infrared cameras record thermal patterns, with
thermograms of the painful areas compared with adjacent pain-free areas to
determine the differential temperature pattern. We measure response to cold
by placing a noninvolved hand or leg in cold water (2 to 3°C) for 10 seconds
and recording for five minutes. LDI assesses blood perfusion in the pain and
pain-free sites. The thermal pattern of the painful site before entry into
the drug or placebo treatment is warmer or cooler by at least 1°C and spread
over a large area. The results have not been unblinded, and the effect of the
drug relative to the placebo cannot be assessed yet. Hassan M, Hattery D, Chernomordik V, Toda K, Fukuhara K, Mittal
D, Rowan J, Shah J, Gerber L, Dionne R, Kopin I, Gandjbakhche A. Infrared
thermographic imaging for the assessment of temperature asymmetries in reflex
sympathetic dystrophy. In: Diakides N, ed. Proceedings of the
International IEEE EMBS Conference, Cancun, Mexico. IEEE
2003;1102-1104. Hassan M, Hattery D, Vogel A, Chernomordik V, Demos S, Aleman K,
Little R, Yarchoan R, Gandjbakhche A. Multi-modality imaging techniques to
assess angiogenesis associated with Kaposi’s sarcoma. In: Bigio I, ed. Proceedings
of the OSA Biomedical Optics Topical Meeting, Miami, Florida, OSA 2004;
CDROM (FG5). Hassan M, Hattery D, Vogel A, Chernomordik V, Demos S, Aleman S,
Little R, Yarchoan R, Gandjbakhche A. Noninvasive infrared imaging for
quantitative assessment of tumor vasculature and response to therapy. In:
Romani GL, ed. Proceedings of the International IEEE EMBS Conference,
San Francisco, California. IEEE 2004;1200-1202. Hassan M, Little R, Vogel A, Aleman K, Wyvill K, Yarchoan R,
Gandjbakhche A. Quantitative assessment of tumor vasculature and response to
therapy in Kaposi’s sarcoma using functional noninvasive imaging. Technol
Cancer Res Treat 2004;3:451-458. Stochastic
modeling of tumor-induced angiogenesis Amyot, Siavosh, Gandjbakhche; in collaboration with
Camphausen Angiogenesis
morphogenesis is a complex process that results in the formation of a fractal
vascular network. During the early stage of tumor-induced angiogenesis,
endothelial cells (ECs) are released into the extracellular matrix (ECM) and
adhere to each other, thus forming a special pattern. Thereafter, small
finger-like capillary sprouts form from an accumulation of ECs that are
recruited from the parent vessel. The sprouts grow in length with the
migration and further recruitment of ECs. After rearrangement, the created
net resembles a mature vessel system. The tumor, now well fed, resumes its
rapid growth. Moreover, the connectivity of the tumor to the vasculature
causes metastasis. Our
research project focuses on the multistep process of tumor-induced angiogenesis.
The process starts with the release from the tumor of tumor angiogenesis
factors (TAFs) such as EGF, VEGF, or FGF. The TAFs degrade the ECM and
promote the migration of endothelial cells from existing blood vessels to
form a network of new blood vessels feeding the tumor. Once the process is
complete, tumor cells can migrate (metastasize) to other organs. Although
extensive work has been done at the molecular level, the details of
macroscopic network formation are not well understood or quantified. The
complexity of the paths that ECs undertake during angiogenesis is such that
only stochastic models can quantitatively describe the process, which
includes interactions of several parameters (concentration of different TAF
molecules, the components of ECM such as collagen or fibronectin, and the
migration, proliferation, and differentiation of ECs). Our objective is to
study the role of each factor described above in in vitro cell
cultures and, if successful, in animal models. We
have developed a stochastic model that begins with the secretion of several
TAFs from cancerous cells of a solid tumor. The goal of the mechanism of
tumor-induced angiogenesis is to perfuse the tumor with vessels. In this
project, we measure the perfusion threshold value for different ECM material
densities. In other words, in what is a typical percolation threshold
problem, we measure the probability for a sprout to reach the tumor by
following a disordered system, i.e., the repartition of macromolecules in the
ECM. The goal is to determine a threshold concentration that allows
percolation from one edge to the other. Currently,
in vitro experiments are under way to explore EC migration through a
basement membrane gel. It is during this migration stage that vascular
morphogenesis occurs. Cells migrate over distances that are an order of
magnitude larger than their radius and then aggregate when they come in
contact with a neighboring cell. The aggregates form clusters that themselves
aggregate, creating a network of cord-like structures. Several experiments
involving concentrations of cells and types of growth factors allowed us to
record the aggregation of ECs with respect to time. We have developed a model
based on the diffusion-limited colloidal aggregation (DLCA), which allows us
to quantify the network's cord-like structure kinetics. Based on experiments
and simulations, we have shown that the DLCA model is well adapted to
reproduce the in vivo first stage of angiogenesis. The work provides
an explanation, by quantification, of the influence of different growth
factors (i.e., FGF, VEGF, and EGF) on the morphology and growth kinetics of
the vascular network induced by a tumor. aAssociate
Professor, bRussian
Academy of Sciences cUniversity
of Maryland at COLLABORATORS Kevin Camphausen, MD, Radiation Oncology
Branch, NCI, Wei Chen, PhD, Nomadics, Rinaldo Cubeddu, PhD, Raymond Dionne, DDS, PhD, Pain and
Neurosensory Mechanisms Branch, NIDCR, Gallya Gannot, DDS, PhD, Laboratory of
Pathology, Center for Cancer Research, NCI, Bethesda, MD Jeremy Hebden, PhD, University
College James Mulshine, MD, Cell
and Cancer Biology Branch, NCI, Herbert Rinneberg, PhD, Physikalisch-Technische
Angelo Russo, MD,
PhD, Radiation Biology Branch, NCI, Paul Smith, PhD,
Division of Bioengineering and Physical Science, ORS, NIH, George H. Weiss, PhD, Center
for Information Technology, NIH, Robert Yarchoan, MD, HIV
and AIDS Malignancy Branch, NCI,
Giovanni Zaccanti, PhD,
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