Search

Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

Products of the COX reaction are frequently elevated in solid tumors and their roles in the malignant phenotype have been extensively investigated. We have shown that COX-2 is essential for the growth of MDA-MB-231 cells in the fat pad of SCID mice and for their extrapulmonary colonization following injection in the tail vein of SCID mice. The molecular changes that follow shRNA-mediated silencing of COX-2 include a significant downregulation of LEF-1, a transcription factor normally activated during development following the Wnt-induced nuclear translocation of β-catenin. We also report that COX-2-silenced cells have reduced nuclear accumulation of LEF-1 protein and that the COX-2 product PGE2 partially restored nuclear LEF-1 expression in COX-2-silenced cells. Further, we demonstrate that, like parental COX-2 containing MDA-MB-231 cells, COX-2-silenced cells maintain nuclear localization of β-catenin.

Mass Spectrometric Imaging (MSI) has become widely used in the analysis of a variety of biological surfaces. Biological samples are spatially, morphologically, and metabolically complex. Multimodal molecular imaging is an emerging approach that is capable of dealing with this complexity. In a multimodal approach, different imaging modalities can provide precise information about the local molecular composition of the surfaces. Images obtained by MSI can be co-registered with images obtained by other molecular imaging techniques such as microscopic images of fluorescent protein expression or histologically stained sections. In order to properly co-register images from different modalities, each tissue section must contain points of reference, which are visible in all data sets. Here, we report a newly developed co-registration technique using fiducial markers such as cresyl violet, Ponceau S, and bromophenol blue that possess a combination of optical and molecular properties that result in a clear mass spectrometric signature. We describe these fiducial markers and demonstrate an application that allows accurate co-registration and 3-dimensional reconstruction of serial histological and fluorescent microscopic images with MSI images of thin tissue sections from a breast tumor model.

Applications of molecular imaging in cancer and other diseases frequently require combining in vivo imaging modalities, such as magnetic resonance and optical imaging, with ex vivo optical, fluorescence, histology, and immunohistochemical (IHC) imaging, to investigate and relate molecular and biological processes to imaging parameters within the same region of interest. We have developed a multimodal image reconstruction and fusion framework that accurately combines in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI), ex vivo brightfield and fluorescence microscopic imaging, and ex vivo histology imaging. Ex vivo brightfield microscopic imaging was used as an intermediate modality to facilitate the ultimate link between ex vivo histology and in vivo MRI/MRSI. Tissue sectioning necessary for optical and histology imaging required generation of a three-dimensional (3D) reconstruction module for 2D ex vivo optical and histology imaging data. We developed an external fiducial marker based 3D reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. Registration of 3D tumor shape was pursued to combine in vivo MRI/MRSI and ex vivo optical brightfield and fluorescence imaging data. This registration strategy was applied to in vivo MRI/MRSI, ex vivo optical brightfield/fluorescence, as well as histology imaging data sets obtained from human breast tumor models. 3D human breast tumor data sets were successfully reconstructed and fused with this platform.