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Applications of molecular imaging in cancer and other diseases frequently require the combination of in vivo imaging modalities, such as MR and optical imaging, with ex vivo optical, fluorescence, histology and immunohistochemical 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 MRI and 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 the generation of a three-dimensional reconstruction module for two-dimensional ex vivo optical and histology imaging data. We developed an external fiducial marker-based three-dimensional reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. The registration of the three-dimensional 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 and histology imaging datasets obtained from human breast tumor models. Three-dimensional human breast tumor datasets were successfully reconstructed and fused with this platform. Copyright © 2012 John Wiley & Sons, Ltd.

Altered choline phospholipid metabolism is a metabolic hallmark of cancer. Malignant transformation of breast can-cer cells results in a switch from high glycerophosphocholine (GPC) and low phosphocholine (PC) to low GPC and high PC.Glycerophosphocholine phosphodiesterase (GPC-PDE; E.C. 3.1.4.2) catalyzes the degradation of GPC to choline (Cho) andglycerol-3-phosphate. The GPC-PDE gene(s) responsible for the relatively low GPC concentration in breast cancer cells havenot yet been characterized. Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) displays GPC-PDE ac-tivity, and is rapidly inhibited by sodium chloride and urea (NaCl/urea). We chemically inhibited GPC-PDE with NaCl/ureain nonmalignant MCF-12A breast epithelial cells, as well as in MCF-7 and MDA-MB-231 breast cancer cells. 1 H magneticresonance spectroscopy (MRS) of cell extracts demonstrated that exposure of MCF-12A, MCF-7 and MDA-MB-231 cells toNaCl/urea ( n = 5) significantly increased GPC and decreased PC, resulting in a low [PC]

PurposeTo assess the ability of a polarization transfer (PT) magnetic resonance spectroscopy (MRS) technique to improve the detection of the individual phospholipid metabolites phosphocholine (PC), phosphoethanolamine (PE), glycerophosphocholine (GPC), and glycerophosphoethanolamine (GPE) in vivo in breast tumor xenografts.Materials and methodsThe adiabatic version of refocused insensitive nuclei enhanced by polarization transfer (BINEPT) MRS was tested at 9.4 Tesla in phantoms and animal models. BINEPT and pulse-acquire (PA) 31P MRS was acquired consecutively from the same orthotopic MCF-7 (n = 10) and MDA-MB-231 (n = 10) breast tumor xenografts. After in vivo MRS measurements, animals were euthanized, tumors were extracted and high resolution (HR)-MRS was performed. Signal to noise ratios (SNRs) and metabolite ratios were compared for BINEPT and PA MRS, and were also measured and compared with that from HR-MRS.ResultsBINEPT exclusively detected metabolites with 1H-31P coupling such as PC, PE, GPC, and GPE, thereby creating a significantly improved, flat baseline because overlapping resonances from immobile and partly mobile phospholipids were removed without loss of sensitivity. GPE and GPC were more accurately detected by BINEPT in vivo, which enabled a reliable quantification of metabolite ratios such as PE/GPE and PC/GPC, which are important markers of tumor aggressiveness and treatment response.ConclusionBINEPT is advantageous over PA for detecting and quantifying the individual phospholipid metabolites PC, PE, GPC, and GPE in vivo at high magnetic field strength. As BINEPT can be used clinically, alterations in these phospholipid metabolites can be assessed in vivo for cancer diagnosis and treatment monitoring.

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.

Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of these NIR probes were investigated by dynamic optical imaging and immunofluorescence staining in human breast tumor xenografts. IR-1 and IR-2 demonstrated faster lysosome labeling rates in highly aggressive MDA-MB-231 and MDA-MB-435 cells compared with less aggressive MCF-7 and nontumorigenic MCF-12A cells. IR-1 and IR-2, but not IR-15, accumulated in human MDA-MB-231, MDA-MB-435, and MCF-7 breast tumor xenografts in vivo. IR-2 demonstrated the highest maximum fluorescence and tumor/normal tissue ratios in all tumor models. Specific lysosome labeling from IR-2 in vivo was validated by colocalization of the NIR fluorescence with CD63 immunofluorescence in tumor sections. IR-1 and IR-2 demonstrated high lysosome-labeling ability and breast tumor-targeting specificity in vitro and in vivo. They are promising for diagnosing malignant lesions and may provide a means for evaluating and monitoring future lysosome-targeted anticancer therapies.

[structure: see text] Two novel near-infrared (NIR) fluorescent probes have been synthesized by linking a carbocyanine fluorophore and glucosamine through different linkers. These probes demonstrated a high quantum yield, low cytotoxicity, reversible pH-dependent fluorescence in the physiological pH range, and a decreased aggregation tendency in aqueous solutions. In vitro NIR optical imaging studies revealed cellular uptake and strong intracellular NIR fluorescence of these two probes in four breast epithelial cell lines.

We have previously demonstrated that poorly differentiated MDA-MB-231 cells displayed delayed tumor onset, reduced frequency of occurrence and loss of their capacity for extrapulmonary colonization when transfected stably with a potent and specific cyclooxygenase (COX) −2 shRNA sequence. We attributed the reduction in tumor growth and extrapulmonary colonization to reduced invasive capabilities, reduced angiogenesis, altered metabolism and differences in the stabilization of hypoxia-inducible factor-1α. We also observed loss of expression of oncogenes, receptors and degradative enzymes associated with tumor growth and metastasis including matrix metalloproteinase-1, CXC Receptor-4, roundabout-1, hyaluronan synthase-2, and monocarboxylate transporter 2. In addition to these previously reported transcriptome changes, we observed that the most highly downregulated transcription factor was Lymphoid-Enhancer Factor-1 (LEF-1) a result validated by qPCR showing an 8-fold loss of LEF-1 in COX-2-silenced cells. Here, we present evidence demonstrating that LEF-1 accumulation in the nucleus of COX-2-silenced cells is reduced compared to COX-2 containing parental cells. Induction of COX-2 by phorbol esters resulted in nuclear accumulation of LEF-1 in COX-2 containing, but not in COX-2-silenced, cells. Addition of physiological concentrations of PGE2 to COX-2-silenced cells partially restored LEF-1 expression in the nucleus of these cells. LEF-1 is a member of the T-cell specific factor (TCF) family of transcription factors that direct transcription of downstream target genes upon nuclear translocation of β-catenin from the cytoplasm. β-catenin translocation to the nucleus occurs in response to canonical signaling of the ligand Wnt to its receptor Frizzled. An earlier study showed that the COX-2 product prostaglandin E2 promoted the translocation of β-catenin to the nucleus of colon cancer cells via G-protein coupled receptor signaling. As expected, immunofluorescence of COX-2 containing MDA-MB-231 breast cancer cells showed nuclear expression of β-catenin. However, the nuclear localization of β-catenin persisted even in the absence of PGE2, as observed in COX-2-silenced cells, suggesting that PGE2 expression is not necessary for the nuclear localization of β-catenin in MDA-MB-231 cells. Taken together, our results demonstrate that while PGE2 is not necessary for the nuclear localization of β-catenin in MDA-MB-231 cells, it can promote the nuclear accumulation of the TCF family member LEF-1, thereby further promoting the non-canonical activation of the Wnt pathway in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4276. doi:1538-7445.AM2012-4276