Ralph S. DaCosta

New optical technologies for earlier endoscopic diagnosis of premalignant gastrointestinal lesions.

Gastrointestinal malignancies continue to be the second leading cause of cancer‐related deaths in the developed world. The early detection and treatment of gastrointestinal preneoplasms has been demonstrated to significantly improve patient survival. Conventional screening tools include standard white light endoscopy (WLE) and frequent surveillance with biopsy. Well‐defined endoscopic surveillance biopsy protocols aimed at early detection of dysplasia and malignancy have been undertaken for groups at high risk. Unfortunately, the poor sensitivity associated with WLE is a significant limitation. In this regard, major efforts continue in the development and evaluation of alternative diagnostic techniques. This review will focus on notable developments made at the forefront of research in modern gastrointestinal endoscopy based on novel optical endoscopic modalities, which rely on the interactions of light with tissues. Here we present the ‘state‐of‐the‐art’ in fluorescence endoscopic imaging and spectroscopy, Raman spectroscopy, optical coherence tomography, light scattering spectroscopy, chromoendoscopy, confocal fluorescence endoscopy, and immunofluorescence endoscopy. These new developments may offer significant improvements in the diagnosis of early lesions by allowing for targeted mucosal excisional biopsies, and perhaps may even provide ‘optical biopsies’ of equivalent histological accuracy. This enhancement of the endoscopist’s ability to detect subtle preneoplastic changes in the gastrointestional mucosa in real time and improved staging of lesions could lead to curative endoscopic ablation of these lesions and, in the long term, improve patient survival and quality of life.

Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy.

Abstract In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation–emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250–520 nm, with fluorescence emission spectra collected in the range 260–750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal.com/).

Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa.

Background/Aims: In vivo autofluorescence endoscopic imaging and spectroscopy have been used to detect and differentiate benign (hyperplastic) and preneoplastic (adenomatous) colonic lesions. This fluorescence is composed of contributions from the epithelium, lamina propria, and submucosa. Because epithelial autofluorescence in normal and diseased tissues is poorly understood, this was the focus of the present study. Methods: Whole colonic crypts were isolated, and short term primary cultures of epithelial cells were established from biopsies of normal, hyperplastic, and adenomatous colon. Autofluorescence (488 nm excitation) was examined by confocal fluorescence microscopy. Fluorescently labelled organelle probes and transmission electron microscopy were used to identify subcellular sources of fluorescence. Results: Mitochondria and lysosomes were identified as the main intracellular fluorescent components in all cell types. Normal and hyperplastic epithelial cells were weakly autofluorescent and had similar numbers of mitochondria and lysosomes, whereas adenomatous (dysplastic) epithelial cells showed much higher autofluorescence, and numerous highly autofluorescent lysosomal (lipofuscin) granules. Conclusions: Short term primary cell cultures from endoscopic biopsies provide a novel model to understand differences in colonic tissue autofluorescence at the glandular (crypt) and cellular levels. The differences between normal, hyperplastic, and adenomatous epithelial cells are attributed in part to differences in the intrinsic numbers of mitochondria and lysosomes. This suggests that the detection of colonic epithelial fluorescence alone, if possible, may be sufficient to differentiate benign (hyperplastic) from preneoplastic and neoplastic (adenomatous) colonic intramucosal lesions during in vivo fluorescence endoscopy. Furthermore, highly orange/red autofluorescent intracellular granules found only in dysplastic epithelial cells may serve as a potential biomarker.

A multifunctional polymeric nanotheranostic system delivers doxorubicin and imaging agents across the blood-brain barrier targeting brain metastases of breast cancer.

Metastatic brain cancers, in particular cancers with multiple lesions, are one of the most difficult malignancies to treat owing to their location and aggressiveness. Chemotherapy for brain metastases offers some hope. However, its efficacy is severely limited as most chemotherapeutic agents are incapable of crossing the blood-brain barrier (BBB) efficiently. Thus, a multifunctional nanotheranostic system based on poly(methacrylic acid)-polysorbate 80-grafted-starch was designed herein for the delivery of BBB-impermeable imaging and therapeutic agents to brain metastases of breast cancer. In vivo magnetic resonance imaging and confocal fluorescence microscopy were used to confirm extravasation of gadolinium and dye-loaded nanoparticles from intact brain microvessels in healthy mice. The targetability of doxorubicin (Dox)-loaded nanoparticles to intracranially established brain metastases of breast cancer was evaluated using whole body and ex vivo fluorescence imaging of the brain. Coexistence of nanoparticles and Dox in brain metastatic lesions was further confirmed by histological and microscopic examination of dissected brain tissue. Immuno-histochemical staining for caspase-3 and terminal-deoxynucleotidyl transferase dUTP nick end labeling for DNA fragmentation in tumor-bearing brain sections revealed that Dox-loaded nanoparticles selectively induced cancer cell apoptosis 24 h post-injection, while sparing normal brain cells from harm. Such effects were not observed in the mice treated with free Dox. Treatment with Dox-loaded nanoparticles significantly inhibited brain tumor growth compared to free Dox at the same dose as assessed by in vivo bioluminescence imaging of the brain metastases. These findings suggest that the multifunctional nanoparticles are promising for the treatment of brain metastases.

Porphyrin-Cross-Linked Hydrogel for Fluorescence-Guided Monitoring and Surgical Resection

We demonstrate that porphyrins can be used as efficient cross-linkers to generate a new class of hydrogels with enabling optical properties. Tetracarboxylic acid porphyrins reacted with PEG diamines to form a condensation polyamide in a range of appropriate conditions, with respect to reaction time, diisopropylethylamine initiator concentration, porphyrin-to-PEG ratio, porphyrin concentration, and PEG size. The network structure of the hydrogel maintained a porphyrin spacing that prevented excessive fluorescence self-quenching despite high porphyrin density. The near-infrared properties readily enabled low background, noninvasive fluorescence monitoring of the implanted hydrogel in vivo, as well as its image-guided surgical removal in real time using a low-cost fluorescence camera prototype. Emission could be tuned by incorporating copper metalloporphyrins into the network. The approach of creating hydrogels using cross-linking porphyrin comonomers creates opportunities for new polymer designs with strong optical character.

Fluorescence and Spectral Imaging

Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barretts esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots). This is an area of great promise, but still in its infancy, and preclinical studies are currently under way.

Autofluorescence-Based Detection of Early Neoplasia in Patients with Barrett’s Esophagus

Barrett’s esophagus (BE) is a condition in which the normal stratified squamous lining of the distal esophagus is replaced by metaplastic columnar epithelium. Its importance arises from it being a precancerous condition with the potential to progress to esophageal adenocarcinoma, the incidence of which is dramatically rising in Western countries [1, 2]. Malignant transformation in BE is believed to follow the metaplasia-dysplasia-carcinoma sequence [3], so there is a possibility to detect and treat neoplastic lesions at an early and curable stage. In this article, we will discuss fluorescence-based imaging used adjunctively with conventional endoscopy for the detection of early neoplastic lesions in BE. We will use the term ‘early neoplasia’ to denote lesions with low-grade dysplasia (LGD), high-grade dysplasia (HGD) or early cancer (EC) in BE. Current Surveillance Strategies

Quantifying tissue microvasculature with speckle variance optical coherence tomography

In this Letter, we demonstrate high resolution, three-dimensional optical imaging of in vivo blood vessel networks using speckle variance optical coherence tomography, and the quantification of these images through the development of biologically relevant metrics using image processing and segmentation techniques. Extracted three-dimensional metrics include vascular density, vessel tortuosity, vascular network fractal dimension, and tissue vascularity. We demonstrate the ability of this quantitative imaging approach to characterize normal and tumor vascular networks in a preclinical animal model and the potential for quantitative, longitudinal vascular treatment response monitoring.

Directed Differentiation of Skin-Derived Precursors Into Functional Vascular Smooth Muscle Cells

Objective—The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)–directed differentiation of Sox2+ multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. Methods and Results—We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-&bgr;1 or -&bgr;3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription–polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle &ggr;-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. Conclusion—SKPs are Sox2+ progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization.

Photodiagnostic techniques for the endoscopic detection of premalignant gastrointestinal lesions

Considerable attention is given to the clinical diagnosis of gastrointestinal (GI) malignancies as they remain the second leading cause of cancer‐associated deaths in developed countries. Detection and intervention at an early stage of preneoplastic development significantly improve patient survival. High‐risk assessment of asymptomatic patients is currently performed by strict endoscopic surveillance biopsy protocols aimed at early detection of dysplasia and malignancy. However, poor sensitivity associated with frequent surveillance programs incorporating conventional screening tools, such as white light endoscopy and multiple random biopsy, is a significant limitation. Recent advances in biomedical optics are illuminating new ways to detect premalignant lesions of the GI tract with endoscopy. The present review presents a summary report on the newest developments in modern GI endoscopy, which are based on novel optical endoscopic techniques: fluorescence endoscopic imaging and spectroscopy, Raman spectroscopy, light scattering spectroscopy, optical coherence tomography, chromoendoscopy, confocal fluorescence endoscopy and immunofluorescence endoscopy. Relying on the interaction of light with tissue, these ‘state‐of‐the‐art’ techniques potentially offer an improved strategy for diagnosis of early mucosal lesions by facilitating targeted excisional biopsies. Furthermore, the prospects of real‐time ‘optical biopsy’ and improved staging of lesions may significantly enhance the endoscopists ability to detect subtle preneoplastic mucosal changes and lead to curative endoscopic ablation of these lesions. Such advancements within this specialty will be rewarded in the long term with improved patient survival and quality of life.