The Section on Medical Biophysics develops new biophysical and optical methodologies for biomedical research and clinical applications. Recently, the section has been focusing on the refinement of technological approaches to characterizing early stages of disease and to developing strategies for prevention of progression and monitoring responses to therapy in cancer and age-related macular degeneration (AMD). Further development of Laser Capture Microdissection (LCM) and related laser technologies has coupled the isolation of small populations of cells from sections of complex tissues with global analysis of gene expression. Statistical analysis of gene expression has been applied to identify candidate lists for stage-specific molecular markers within microdissected cell populations, focusing on improvements necessary to identify critical but less abundantly expressed genes.

Laser Microdissection Technology Development
Cantoni, Obiakor, Parlato, Malekafzali, Bonner
As the list of expressed human genes is completed, a major scientific and medical challenge will be to track the complex molecular events that drive normal tissue differentiation and progression of pathologic lesions. With refinements in PCR, hybridization microarrays, and mutation screening, both DNA and mRNA can be extracted from tissue biopsies or cytology specimens and analyzed with a parallel panel of hundreds or even thousands of genetic markers. Because cells in complex tissue are biochemically and physically affected by surrounding cells and by remote stimuli from greater distances, the task of analyzing critical gene expression patterns in development, normal function, and disease progression depends on the extraction of specific cells from their complex tissue milieu. Our laboratory, in collaboration with NCI, has invented and developed Laser Capture Microdissection (LCM) (Arcturus Engineering, Inc., has commercialized the technology). LCM provides a rapid, reliable method to procure pure populations of specified cells from microscopic regions of tissue sections in a form useful for subsequent quantitative, multiplex molecular analysis. Routine clinical use of this approach will require (1) reliable preservation of the macromolecules of interest within a sample; (2) ensuring that critical clonal populations are included and can be identified within an ex vivo sample; and (3) quantitative extraction and analysis of critical subsets of marker macromolecules.

We are studying the polymer physics and chemistry of the LCM processes and competitive ablative laser microdissection techniques in order to lead technology integration with biological and clinical research. Recent improvements include "noncontact LCM" prototypes that use automated beam steering and pulse sequences. The automated beam steering and pulse sequences allow reliable activation of flexible transfer films in order to inject picoliter volumes of polymer into individually targeted cells, thereby permitting capture and retraction of the cells over precisely controlled air gaps of 10 to 20 microns. A new method for simultaneous complete release of targeted cells from the microscope slide is based on simultaneous ablation of 100nm-thick polymer coatings. Quantitative recovery of DNA, mRNA, and proteins from the injected polymer matrix has been demonstrated with standard aqueous tissue extraction buffers. The LCM process, which loads picoliter cell volumes onto equivalent volumes of polymer matrix, is being applied to integrated purification and analysis of specific macromolecular constituents. In parallel efforts, we have been investigating the biophysics of UV laser ablation and have demonstrated the role played by polymer breakdown and ionization in electrostatic interactions determining target collection efficiency. Polymer multilayer coatings with charge neutralization appear to improve ablative laser microdissection, which has advantages particularly for collecting larger populations of cells required for automated cytometry and many proteomics discovery efforts. In collaboration with NIAID, we have demonstrated reliable molecular analysis in LM-isolated immunolabeled single cells at the single-molecule level, using nested PCR and DNA sequencing. As we proceed to single-cell isolation and greater knowledge of molecular changes in pathology, specific molecular targeting will become increasingly important to ensure rapid, precise collection of subpopulations that are not discernible by morphology alone. We are incorporating fluorescence microscopy and real-time image analysis methods, including remote image-guided sample targeting.

Gene Expression Complexity and Statistics of Gene Expression
Kuznetsov, Stepanov, Bonner
If microdissection and molecular analysis can be made clinically practical, the expression levels of sets of approximately 20 to 100 critical, stage-specific disease markers within a selected cell population might provide reliable diagnosis and intermediate endpoints of response to molecular therapies in individual patients. We have been developing robust statistical tools to facilitate identification of critical genes and pathways that characterize both normal and aberrant cellular function, particularly those suitable for "multiplex" analysis of a small number of specific cells. Analysis of large gene expression databases (SAGE and LCM cDNA libraries) suggests that a large fraction of all genes are expressed in any specific cell type and that the genes' expression levels universally exhibit a highly skewed power-law distribution similar to those characterizing many other complex systems. We are developing mathematical models for the evolution of such distributions that evidence increasing biological complexity.

In collaboration with NCI, we have developed standard procedures for the isolation of normal and pathological cells from clinical specimens in order to follow macromolecular changes during cancer progression within prostate, breast, lung, and ovary tissues. Using statistical multivariate analysis, we have developed mathematical algorithms for the selection of "candidate genes" from such cDNA libraries. The candidate genes' expression frequencies have a strong correlation with disease progression. We use our models of the statistics of expression levels in cell populations to identify genes differentially expressed in cancer progression. To date, our analysis points to a critical role for many less abundantly expressed genes at a critical stage of ovarian cancer progression and suggests that, for most cancers, critical diagnostic marker sets should include such low-abundance transcripts. This notion is guiding our research in statistics of low-level gene expression and suitable detection methods.

Gene Expression during Normal Development and Pathology Progression
Parlato, Malekafzali, Bonner
We have applied single-cell LCM capture technology to studies of gene expression during normal development (spermatogenesis and thyroid bud development) and in stage-specific pathological cells (e.g., cancer precursors). In collaboration with Roberto DiLauro's laboratory in Naples, Italy, we examined gene expression patterns associated with the primordial thyroid and the adjacent cells from which it derives in wt and ko mice. In collaboration with Fred Mushinski (NCI), we are applying the same LCM methodology to study early changes in the well-established plasmacytoma model in BALB/c mice. Jointly, we have developed robust LCM techniques for isolation of specific cells from mouse cryosections and recovered good yields of high-quality mRNA. In collaboration with Life Technologies, Inc., we have made cDNA libraries of high diversity and message length from 100 ng of microdissected mRNA (~5000 cells). cDNA microarray hybridization allows characterization of the global gene expression profiles in these libraries while preserving the libraries for isolation of full-length message of identified stage-specific genes. We are planning to apply these methods validated in reproducible animal models to clinical specimens of early stages of disease progression in which microdissection of critical rare cell populations is required. We foresee an evolution of molecular diagnosis from one based on the qualitative or quantitative analysis of a few key macromolecules to one in which complex multivariate databases will be analyzed by special clustering algorithms. These should allow us to better identify highly correlated clinical cases and then characterizing their response to molecular therapies specifically designed to prevent progression. To this end, we have refined the laser microdissection of rare cell populations, particularly those that might be accessible by serial minimally invasive cell harvesting from patients. We have continued to organize and hold an annual conference at NIH on Laser Capture Microdissection and Macromolecular Analysis of Normal Development and Pathology, bringing together about 500 researchers to discuss research and methodological advances made possible by LCM.

Prevention of Progression of AMD through Photoprotection
Meyers, Ostrovsky, Behr, McConnell, Bonner
Age-related macular degeneration (AMD) is the most common cause of severe visual loss in the elderly. Early stages of the disease are ophthalmoscopically detected with increasing frequency with age (particularly over 60 years). Lipofuscin accumulates with age in the retinal pigment epithelium (RPE). It is the dominant fluorphore in the retina, and, in Stargardt's disease, its increased rate of accumulation is associated with early macular degeneration. This led us to hypothesize in the early 1980s that lipofuscin was photochemically active and responsible for acute retinal damage at high blue light levels and caused chronic photochemical (free radical production) stress that would increase with age (lipofuscin accumulation). If lipofuscin photochemistry played a critical role in initiation and progression of early age-related macular degeneration, then simple optical filters (yellow-colored lens) might be expected to reduce dramatically the rate of photochemical free radical production. We designed (Bonner: 500nm-long pass sunglasses; Ostrovsky: 430nm-long pass intraocular lens) such filters, which have been used clinically. Recent discovery of a photochemical active product (A2E) created by rod photoreception at high light levels and our experimental determination of A2E photochemical action spectrum in RPE cell culture led us to reinvestigate optimal designs for optical filters to reduce or prevent progression of early AMD. We have generated models of spectral irradiance in the human macular RPE following cataract surgery and IOL implantation and as the lens ages. The models suggest the importance of both the reduction of the maximal bleaching of rod rhodopsin and the rate of photodegradation of A2E and its toxic photoproducts by visible light. We have proposed new long wave-length cutoffs for AMD protective optical filters and are currently seeking to characterize spectrally different RPE photoproducts to evaluate the potential for noninvasive monitoring of the molecular effects of such filters in patients.