Tiffany R. Greenwood

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

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.

Silencing of the glycerophosphocholine phosphodiesterase GDPD5 alters the phospholipid metabolite profile in a breast cancer model in vivo as monitored by 31P MRS

Abnormal choline phospholipid metabolism is an emerging hallmark of cancer, which is implicated in carcinogenesis and tumor progression. The malignant metabolic phenotype is characterized by high levels of phosphocholine (PC) and relatively low levels of glycerophosphocholine (GPC) in aggressive breast cancer cells. Phosphorus (31P) MRS is able to non‐invasively detect these water‐soluble metabolites of choline as well as ethanolamine phospholipid metabolism. Here we have investigated the effects of stably silencing glycerophosphoester diesterase domain containing 5 (GDPD5), which is an enzyme with glycerophosphocholine phosphodiesterase activity, in MDA‐MB‐231 breast cancer cells and orthotopic tumor xenografts. Tumors in which GDPD5 was stably silenced with GDPD5‐specific shRNA contained increased levels of GPC and phosphoethanolamine (PE) compared with control tumors. Copyright

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. Copyright

Combined magnetic resonance, 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. Copyright

Prostaglandin E2 promotes the nuclear accumulation of lymphoid enhancer factor-1 in poorly differentiated breast cancer cells ☆

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 PGE(2) 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.

GDPD5 inhibition alters the choline phospholipid metabolite profile of breast cancer cells toward a less malignant metabolic profile

Abstract. 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]

Silencing of the glycerophosphocholine phosphodiesterase GDPD5 alters the phospholipid metabolite profile in a breast cancer model in vivo as monitored by 31P Magnetic Resonance Spectroscopy

Abnormal choline phospholipid metabolism is an emerging hallmark of cancer, which is implicated in carcinogenesis and tumor progression. The malignant metabolic phenotype is characterized by high levels of phosphocholine (PC) and relatively low levels of glycerophosphocholine (GPC) in aggressive breast cancer cells. Phosphorus (31P) MRS is able to non‐invasively detect these water‐soluble metabolites of choline as well as ethanolamine phospholipid metabolism. Here we have investigated the effects of stably silencing glycerophosphoester diesterase domain containing 5 (GDPD5), which is an enzyme with glycerophosphocholine phosphodiesterase activity, in MDA‐MB‐231 breast cancer cells and orthotopic tumor xenografts. Tumors in which GDPD5 was stably silenced with GDPD5‐specific shRNA contained increased levels of GPC and phosphoethanolamine (PE) compared with control tumors. Copyright

Silencing of the glycerophosphocholine phosphodiesterase GDPD5 alters the phospholipid metabolite profile in a breast cancer modelin vivoas monitored by31P MRS: IN VIVO31P MRS ON THE EFFECTS OF SILENCING GDPD5 IN BREAST CANCER

Abnormal choline phospholipid metabolism is an emerging hallmark of cancer, which is implicated in carcinogenesis and tumor progression. The malignant metabolic phenotype is characterized by high levels of phosphocholine (PC) and relatively low levels of glycerophosphocholine (GPC) in aggressive breast cancer cells. Phosphorus (31P) MRS is able to non‐invasively detect these water‐soluble metabolites of choline as well as ethanolamine phospholipid metabolism. Here we have investigated the effects of stably silencing glycerophosphoester diesterase domain containing 5 (GDPD5), which is an enzyme with glycerophosphocholine phosphodiesterase activity, in MDA‐MB‐231 breast cancer cells and orthotopic tumor xenografts. Tumors in which GDPD5 was stably silenced with GDPD5‐specific shRNA contained increased levels of GPC and phosphoethanolamine (PE) compared with control tumors. Copyright