1H/31P polarization transfer at 9.4 Tesla for improved specificity of detecting phosphomonoesters and phosphodiesters in breast tumor models.

PURPOSE: To 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 METHODS: The 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. RESULTS: BINEPT 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. CONCLUSION: BINEPT 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.

Combined MR, fluorescence and histology imaging strategy in a human breast tumor xenograft model.

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.

Mass spectrometry images acylcarnitines, phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models.

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.

Mass spectrometric imaging of red fluorescent protein in breast tumor xenografts.

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.

Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models.

Tumor hypoxia triggers signaling cascades that significantly affect biologic outcomes such as resistance to radiotherapy and chemotherapy in breast cancer. Hypoxic regions in solid tumor are spatially heterogeneous. Therefore, delineating the origin and extent of hypoxia in tumors is critical. In this study, we have investigated the effect of hypoxia on different metabolic pathways, such as lipid and choline metabolism, in a human breast cancer model. Human MDA-MB-231 breast cancer cells and tumors, which were genetically engineered to express red fluorescent tdTomato protein under hypoxic conditions, were used to investigate hypoxia. Our data were obtained with a novel three-dimensional multimodal molecular imaging platform that combines magnetic resonance (MR) imaging, MR spectroscopic imaging (MRSI), and optical imaging of hypoxia and necrosis. A higher concentration of noninvasively detected total choline-containing metabolites (tCho) and lipid CH3 localized in the tdTomato-fluorescing hypoxic regions indicated that hypoxia can upregulate tCho and lipid CH3 levels in this breast tumor model. The increase in tCho under hypoxia was primarily due to elevated phosphocholine levels as shown by in vitro MR spectroscopy. Elevated lipid CH3 levels detected under hypoxia were caused by an increase in mobile MR-detectable lipid droplets, as demonstrated by Nile Red staining. Our findings demonstrate that noninvasive MRSI can help delineate hypoxic regions in solid tumors by means of detecting the metabolic outcome of tumor hypoxia, which is characterized by elevated tCho and lipid CH3.

Fiducial markers for combined 3-dimensional mass spectrometric and optical tissue imaging.

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 coregistered with images obtained by other molecular imaging techniques such as microscopic images of fluorescent protein expression or histologically stained sections. In order to properly coregister 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 coregistration 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 coregistration and 3-dimensional reconstruction of serial histological and fluorescent microscopic images with MSI images of thin tissue sections from a breast tumor model.

Glucosamine-bound near-infrared fluorescent probes with lysosomal specificity for breast tumor imaging.

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.

Synthesis and characterization of glucosamine-bound near-infrared probes for optical imaging.

[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.