Edward B. Brown

In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy

Intravital microscopy coupled with chronic animal window models has provided stunning insight into tumor pathophysiology, including gene expression, angiogenesis, cell adhesion and migration, vascular, interstitial and lymphatic transport, metabolic microenvironment and drug delivery. However, the findings to date have been limited to the tumor surface (< 150 μm). Here, we show that the multiphoton laser-scanning microscope can provide high three-dimensional resolution of gene expression and function in deeper regions of tumors. These insights could be critical to the development of novel therapeutics that target not only the tumor surface, but also internal regions.

Role of tumor–host interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor–host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.

Mitochondrial membrane potential probes and the proton gradient: a practical usage guide

Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.

Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot–labeled bone marrow–derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

Diffusion and convection in collagen gels: implications for transport in the tumor interstitium.

Diffusion coefficients of tracer molecules in collagen type I gels prepared from 0-4.5% w/v solutions were measured by fluorescence recovery after photobleaching. When adjusted to account for in vivo tortuosity, diffusion coefficients in gels matched previous measurements in four human tumor xenografts with equivalent collagen concentrations. In contrast, hyaluronan solutions hindered diffusion to a lesser extent when prepared at concentrations equivalent to those reported in these tumors. Collagen permeability, determined from flow through gels under hydrostatic pressure, was compared with predictions obtained from application of the Brinkman effective medium model to diffusion data. Permeability predictions matched experimental results at low concentrations, but underestimated measured values at high concentrations. Permeability measurements in gels did not match previous measurements in tumors. Visualization of gels by transmission electron microscopy and light microscopy revealed networks of long collagen fibers at lower concentrations along with shorter fibers at high concentrations. Negligible assembly was detected in collagen solutions pregelation. However, diffusion was similarly hindered in pre and postgelation samples. Comparison of diffusion and convection data in these gels and tumors suggests that collagen may obstruct diffusion more than convection in tumors. These findings have significant implications for drug delivery in tumors and for tissue engineering applications.

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.

PHOTOLYSIS OF CAGED CALCIUM IN FEMTOLITER VOLUMES USING TWO-PHOTON EXCITATION

A new technique for the determination of the two-photon uncaging action cross section (deltau) of photolyzable calcium cages is described. This technique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. The two-photon action cross sections of three calcium cages, DM-nitrophen, NP-EGTA, and azid-1, are studied in the range of excitation wavelengths between 700 and 800 nm. Azid-1 has a maximum deltau of approximately 1.4 GM at 700 nm, DM-nitrophen has a maximum deltau of approximately 0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to predict the amount of cage photolyzed and the temporal behavior of the liberated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focused with a 1.3-NA objective, essentially all of the azid-1 within the two-photon focal volume would be photolyzed with a 10-micros pulse train of approximately 7 mW average power. The initially localized distributions of free calcium will dissipate rapidly because of diffusion of free calcium and uptake by buffers. In buffer-free cytoplasm, the elevation of the calcium concentration at the center of the focal volume is expected to last for approximately 165 micros.

Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors

Transport parameters determine the access of drugs to tumors. However, technical difficulties preclude the measurement of these parameters deep inside living tissues. To this end, we adapted and further optimized two-photon fluorescence correlation microscopy (TPFCM) for in vivo measurement of transport parameters in tumors. TPFCM extends the detectable range of diffusion coefficients in tumors by one order of magnitude, and reveals both a fast and a slow component of diffusion. The ratio of these two components depends on molecular size and can be altered in vivo with hyaluronidase and collagenase. These studies indicate that TPFCM is a promising tool to dissect the barriers to drug delivery in tumors.

In Vivo Imaging of Tumors

INTRODUCTION Light microscopy of tumors, as for other thick, scattering tissues such as the brain or the developing embryo, is limited by light penetration and optical access. because of these problems, epifluorescence and confocal microscopy are typically limited to the outer 50-100 microm of the accessible tumor tissue. Most mouse tumors must be exteriorized for examination under the light microscope, a procedure that limits the duration and repeatability of imaging. this protocol describes the generation of chronic window preparations in the mouse. These preparations allow an implanted tumor to grow for several weeks in an optically accessible location in vivo, making it possible to examine the living tumor with high-resolution light microscopy in a repetitive manner. Two chronic window preparations are described: (1) the dorsal skinfold chamber, which allows in vivo imaging of tumors growing in the subcutaneous space, and (2) the cranial window, which allows in vivo imaging of tumors growing on the brain surface.

Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research

Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.