Get PDF Email Share Share with Facebook Tweet This Post on reddit Share with LinkedIn Add to CiteULike Add to Mendeley Add to BibSonomy Get Citation Copy Citation Text V. Leautaud, V. Mack, J. N. Wright, J. Lu, D. Yu, and R. Richards-Kortum, "Optical Imaging of Transformed Breast Epithelial Cells and Breast Tumor Microenvironment," in Biomedical Optics and 3-D Imaging, OSA Technical Digest (CD) (Optica Publishing Group, 2010), paper BSuD76. Export Citation BibTex Endnote (RIS) HTML Plain Text Citation alert Save article

Objective. Structured illumination microscopy is an inexpensive alternative to confocal microscopy that allows optical sectioning at a sub-cellular resolution. However, its application in imaging biological tissue has been limited by inadequate contrast present in them especially in reflectance imaging. Novel, optically active contrast agents like gold nanoparticles and quantum dots targeted against biomarkers of cancer can be integrated with structured illumination to image both the morphological and biochemical changes associated with epithelial pre-cancers.Methods. We modified the optical path of a widefield microscope to implement structured illumination both in reflectance and fluorescence modes. For imaging, we used 25-nm-diameter gold nanoparticles and CdSe quantum dots for reflectance and fluorescence imaging, respectively, to label three-dimensional tissue constructs of SiHa cervical cancer cells. Contrast agents were targeted against the epidermal growth factor receptor (EGFR) using an anti-EGFR monoclonal antibody. Agents targeted with a non-specific IgG antibody served as a control to monitor non-specific labeling.Results. Our result shows that optically sectioned images taken with structured illumination are very comparable to those obtained using confocal microscopy. Moreover, images of three-dimensional cultures stained with the anti-EGFR agents show significantly more image intensity than those stained with the IgG targeted control.Conclusion. Our findings suggest that the combination of novel optical contrast agents and structured illumination can differentiate neoplastic cells which overexpress EGFR from non-nal cells in intact tissue. Combining structured illumination microscopy with novel contrast agents can potentially provide a powerful and inexpensive tool to aid in the detection of cervical pre-cancers. (C) 2005 Elsevier Inc. All rights reserved.

A ball lens coupled fiber-optic probe design is described for depth-resolved measurements of the fluorescence and reflectance properties of epithelial tissue. A reflectance target, fluorescence targets, and a two-layer tissue phantom consisting of fluorescent microspheres suspended in collagen are used to characterize the performance of the probe. Localization of the signal to within 300 microm of the probe tip is observed by use of reflectance and fluorescence targets in air. Differential enhancement of the fluorescence signal from the top layer of the two-layer tissue phantom is observed.

We present continuing developments of immuno-targeted gold nanoparticles as reflectance-based optical contrast agents for cancer-detection in-vivo-. We demonstrate the ability to label EGFR (a hallmark for epithelial cancer) in several models of increasing biological relevance.

Structured-illumination microscopy delivers confocal-imaging capabilities and may be used for optical sectioning in bio-imaging applications. However, previous structured-illumination implementations are not capable of imaging molecular changes within highly scattering, biological samples in reflectance mode. Here, we present two advances which enable successful structured illumination reflectance microscopy to image molecular changes in epithelial tissue phantoms. First, we present the sine approximation algorithm to improve the ability to reconstruct the in-focus plane when the out-of-focus light is much greater in magnitude. We characterize the dependencies of this algorithm on phase step error, random noise and backscattered out-of-focus contributions. Second, we utilize a molecular-specific reflectance contrast agent based on gold nanoparticles to label disease-related biomarkers and increase the signal and signal-to-noise ratio (SNR) in structured illumination microscopy of biological tissue. Imaging results for multi-layer epithelial cell phantoms with optical properties characteristic of normal and cancerous tissue labeled with nanoparticles targeted against the epidermal growth factor receptor (EGFR) are presented. Structured illumination images reconstructed with the sine approximation algorithm compare favorably to those obtained with a standard confocal microscope; this new technique can be implemented in simple and small imaging platforms for future clinical studies.

Molecular characterization of cancer could have important clinical benefits such as earlier cancer detection based on molecular characterization, the ability to predict the risk of cancer progression, real time margin detection, the ability to rationally select molecular therapy and to monitor response to the therapy. We present a new class of molecular specific contrast agents for optical imaging of carcinogenesis in vivo-gold nanoparticles conjugated with monoclonal antibodies specific for cancer biomarkers.

We present a new class of molecular specific contrast agents for optical imaging of carcinogenesis in vivo - gold nanoparticles conjugated with monoclonal antibodies specific for cancer biomarkers. The nanoparticles can resonantly scatter visible light that provides an opportunity for optical interrogation using reflectance spectroscopic and imaging modalities.

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.

Several studies have correlated escape from TGF-beta-mediated cell cycle arrest with the tumorigenic phenotype. Most often, this escape from growth control has been linked to dysfunctional TGF-beta receptors or defects in the TGF-beta-mediated SMAD signaling pathway. In this report, we found that highly metastatic 4T1 mammary carcinoma cells express functional TGF-beta receptors capable of initiating SMAD-mediated transcription, yet are not growth inhibited by TGF-beta1. We further observed that TGF-beta directly contributes to the metastatic behavior of this cell line. Exposure to TGF-beta caused 4T1 cells to undergo morphological changes associated with the metastatic phenotype and invade more readily through collagen coated matrices. Furthermore, expression of a dominant negative truncated type II receptor diminished TGF-beta signaling and significantly restricted the ability of 4T1 cells to establish distant metastases. Our results suggest that regardless of 4T1 resistance to TGF-beta-mediated growth inhibition, TGF-beta signaling is required for tumor invasion and metastases formation.