National Science Foundation     |     Directorate for Engineering  (ENG)
Division of Chemical, Bioengineering, Environmental, & Transport Systems  (CBET)
CBET Award Achievements  (Formerly "CBET Nuggets")
Notable Accomplishments from CBET Awards
Cancer Drug Fingerprinting Inside Tumor Tissue
David Nolte  -  Purdue UniversityWest Lafayette, IN
John Turek

This NSF-funded research, led by Dr. David Nolte at Purdue University in Indiana, has developed an extension to spectral fingerprinting, which adds specificity to Nolte's technique for particular mechanisms, and therefore the ability to monitor specific modes of action of drug candidates. On a previous NSF grant, Nolte pioneered the capture of intracellular motion from deep inside living tissue - - which was a unique aspect of his motility-contrast imaging technique.

Background:  Cells live in three dimensions (3D).  They move, communicate, and divide in 3D.  On the other hand, cell cultures, in which the vast majority of cellular studies are performed, are intrinsically two-dimensional (2D).  Cell shape on culture plates can be far from the natural cell shape in tissue, affecting both intra- and inter-cellular signalling.  Most importantly, the efficacy of drug testing may be misrepresented by their effect on 2D culture.  For these reasons, the NSF-funded research, led by Prof. David Nolte of Purdue University, explored the effect of drugs on cells in their natural 3D setting inside living tissue far from surface-induced gradients.  They use laser ranging and digital holography to capture intracellular motion inside small tumors.  The motion is captured as dynamic 'shimmering' laser speckle (random intensity patterns produced by optical wavefront interference).  Previously, the Nolte group had shown that dose-response studies of anti-cancer drugs could be performed using the measured speckle from as deep as 1 millimeter inside tissue.  In their recent work, they have demonstrated that one can now identify spectral fingerprints associated with different biomechanical contributions to the dynamic speckle and its response to drugs.  This is a crucial step forward to establish the specificity of these tissue-based drug assays to particular drug action.  Motility contrast will provide a new direct-detection imaging approach in the relevant 3D environment for high-throughput screening to find new and more specific mitotic drug candidates to aid cancer drug research.

Results:  Dynamic speckle has been captured from inside living tissue using coherence-gated digital holography.  The time-variation of the speckle has statistical properties that are extracted to quantify the degree of intracellular motion at depth.  The simplest measure, that was established previously, is a motility (ability to move spontaneously) metric based on the normalized standard deviation (NSD) of the speckle light intensity.  This approach has good sensitivity to various types of different drugs, but it lacked overall specificity.  Drugs can act on cells and tissues through many different mechanisms, each of which can affect intracellular motion.  For instance, membrane fluctuations are thermally driven, but depend on the viscosity of the cytoplasm and on the actin-dependent bending stiffness of the membrane.  As another example, organelle motility depends on ATP synthesis as well as on actin filament and microtubule density.  While drugs can target one or several of these, the NSD motility metric by itself cannot distinguish among these mechanisms.
In the recent research, a differential power spectral analysis moves beyond the single-metric approach.  By looking at the temporal spectrum of the fluctuations, frequency bands that relate to different cellular mechanisms have been identified.  For instance, a low frequency band (1/20 sec-1) has a response that is sensitive to actin-affecting drugs (cytochalasin D).  A mid-frequency band (1/5 sec-1) is sensitive to the mechanics of the cell, including temerature sensitivity and sensitivity to microtubule-affecting drugs.  A high-frequency band (1 sec-1) has a more complicated response that can be sensitive to drugs that inhibit anaerobic glycolysis, but that also can be affected by changes in cytoplasm viscosity.
The differental spectra are presented as spectrograms plotting frequency along one axis and time along the other.  The spectrogram of a drug acts as a specific signature by considering the drug action on the three different frequency bands.  An example is shown in Figure 1 for the actin drug cytochalasin D compared with the microtubule drug nocodazole captured 400 microns deep in a tumor.  Note that the low-frequency band shows strong enhancement in the case of cytochalasin, reflecting the decreased membrane bending stiffness and a decrease in the cellular viscosity because of actin depolymerization.  Nocodazole, on the other hand, shows a strong suppression of both the mid- and high-range frequency bands.
David Nolte 1
    Figure 1.
Comparison of the spectrograms (fingerprints) of [Figure 1a] cytochalasin D (an actin depolymerization drug) with [Figure 1b] nocodazole (a microtubule depolymerization drug).  The low-frequency band in the case of cytochalasin shows enhanced power density, reflecting the decrease in the membrane binding stiffness because of the depolymerization of the actin cortex, which gives the membrane much of its mechanical stiffness.  These data were captured 400 microns deep inside living tissue responding to the applied drugs.
David Nolte 2
    Figure 2.
Comparison of the spectrograms (fingerprints) of [Figure 1a] cytochalasin D (an actin depolymerization drug) with [Figure 1b] nocodazole (a microtubule depolymerization drug).
Credit for both Images:  David Nolte, Purdue University

Scientific Uniqueness:  Capturing intracellular motion from deep inside living tissue is a unique aspect of the motility-contrast imaging technique developed under the previous NSF grant.  The extension to spectral fingerprinting adds specificity to this technique for particular mechanisms, and therefore the ability to monitor specific modes of action of drug candidates.

This project addresses the NSF Strategic Outcome Goals, as described in the NSF Strategic Plan 2006-2011, as follows:
Primary Strategic Outcome Goal:      (1) Discovery:  This project applies physics to the problem of quantifying health and disease.  The pariticipation of the physical sciences in biological research, and in particular in the study of the physics of cancer, is widely acknowledged as a critical need in the scientific R&D community of the US.  While biological science has made great strides in understanding the qualitative features of health, physics and engineering provide the basis for developing new paradigms.  The discovery of freqency bands that have specificity to different cellular mechanisms is a major advance in understanding information that can be extracted from three-dimensional optical imaging of living tissue.
                                                                   (1) Discovery Categories:
                                                                           -  Biology
                                                                           -  Engineering

Secondary Strategic Outcome Goal:  (2) Learning:  The Nolte lab at Purdue University is one of the most highly sought-after labs by both undergraduate and graduate students.  The research is featured prominently on the Purdue campus with many local talks presented on campus, as well as international invited talks.  The Nolte lab over the past year has had 4 graduate students (one from an underrepresented group) participating in a number of biophotonic projects, including this one, a post-doc, and two undergraduate students (now graduate students at Berkeley and Harvard, one from an underrepresented group, and one who has received an NSF graduate fellowship partially based on his work in the Nolte lab).

                                                                   (2) Learning Categories:
                                                                           -  Undergraduate Education and Undergraduate Student Research
                                                                           -  Graduate Education and Graduate Student Research

This Award Achievement represents Transformative Research.  The approach is unique, establishing a new paradigm for high-throughput screening in 3D tissue.  In this research highlight, the new paradigm is the recognition that motion and heterogeneity, rather than being parasitic to biomedical imaging, becomes the crucial contrast agent.  This new view opens many avenues for science and commercialization.

The Intellectual Merit of this research:  The technique developed in this research is the first to use the actual dynamics of the cells as an imaging contrast agent.  With the extension to spectral fingerprinting of the dynamic speckle, the technique now has high specificity to particular mechanisms of action of drug candidates.  This research cuts across several research fields, including biomedical imaging, cellular motility, biomechanics, cancer research, and pharmacological drug screening.  The identification of specific modes of drug action and cellular response inside of tissue can contribute to each of these fields.

The Broader Impacts of this research include:
(1Benefits of this research to society:  This research opens the way to high throughput tissue-based screening (HTS) with high specificity.  HTS now uses fluorescent optical endpoints in 2D culture, which has not been effective in identifying new drug candidates for the pharma pipelines.  With the advance made here, this old paradigm can be replaced by more representative 3D screens, using the actual dynamics of the cells as the contrast agent.  Motility contrast will provide a new direct-detection imaging approach in the relevant 3D environment for high-throughput screening to find new and more specific mitotic drug candidates to aid cancer drug research.
(2Advancing discovery and understanding while promoting teaching, training, and learning:  The research group works with science education majors and develops hands-on intracellular motility imaging experiments for grades 7-12.
(3Enhancing the infrastructure for research and education::  The research group integrates the experiments with the outreach program that reaches students in grades 7-12, as well as parents and teachers throughout Indiana.

Area of Emphasis (Themes) for FY 2010 Highlights included in this research project:
(1Interdisciplinary, high-risk, and potentially transformative
(2Speeds translation of promising fundamental research into innovations that can be commercialized
(3Enhances health and quality of life

Program Director:
Leon Esterowitz
CBET Program Director - Biophotonics
NSF Award Number:   0756005
Award Title:   Motility Contrast for Endogenous Multi-functional Imaging of Tissue
PI Name: 
  David Nolte and
John Turek
Institution Name:   Purdue University;  West Lafayette, IN/FONT>
Program Element Code:   7236
CBET Award Achievement:

  FY 2010

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This Award Achievement was Updated on 23 August 2010.