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• W2224995654 abstract "Proc Amer Assoc Cancer Res, Volume 46, 20055665 PURPOSE In this study we demonstrate the use of multiphoton imaging to study human carcinoma development and invasion in an in vitro human tumor model that recapitulates a normal tissue microenvironment (NTM). The human tumor model is based on an organotypic skin equivalent that consists of a fully stratified epidermis resting on a fibroblast- embedded collagen matrix. The keratinocyte and fibroblast cells exhibit endogenous two-photon fluorescence (TPF) when examined using multiphoton microscopy; while the fibrillar collagen matrix exhibits second harmonic generated light (SHG), a high-contrast, nonlinear scattering mechanism that has recently been applied to biological tissues. INVASION AND MMPS The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are overexpressed in tumor tissue, and are associated with the creation of a permissive environment for tumor invasion. We plan to examine collagenase MMP-1 and gelatinase MMP-2 in part to mimic the well-known proMMP-2 activation mechanism involving a ternary complex formed by membrane-bound MT1-MMP, tissue inhibitor of MMP-2, and proMMP-2. Once activated, MMP-2 participates in pericellular proteolysis by degrading primary structural proteins of the basement membrane. EXPERIMENTAL PROCEDURES Multiphoton imaging was used to assess the effect of proteolysis on the surrounding NTM by (1) characterizing intact engineered models of normal human skin, and (2) assessing the response of human skin models to proteolysis by purified MMP-1 and -2. MMP-treated human skin models were imaged over time, and proteolytic processing of type I collagen was quantified. RESULTS TPF+SHG images indicate that both the epidermal and dermal compartments of the mature skin equivalent tissue are clearly visible. The pattern of keratinocyte differentiation is evident: cellular morphology changes with depth as expected. Preliminary experiments using bacterial collagenase enzyme treatment were successful in demonstrating visualization and quantification of collagen degradation over time. Approximately 20 minutes after the addition of collagenase, a precipitous decrease (70-90%) in the collagen signal was observed. CONCLUSIONS Based on our findings using endogenous sources of contrast, we conclude that changes in the collagen organization, as measured by a decrease in SHG intensity, is a reliable and sensitive indicator of the onset and advancement of tumor invasion. Spectral-based quantification of collagen organization may be a novel and highly sensitive screen for anti-metastatic therapeutic agents. FUTURE To form the human tumor model, GFP-tagged squamous cell carcinoma cells will be incorporated into the epidermal compartment of the skin equivalent, and in vitro tumor development will be analyzed using multiphoton microscopy." @default.
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• W2224995654 date "2005-05-01" @default.
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• W2224995654 title "Assessment of an in vitro human model of stromal invasion using second harmonic generation and multiphoton imaging" @default.
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