Characterization of Humanized Mouse Model of Organophosphate Poisoning and Detection of Countermeasures via MALDI-MSI.

Organophosphoate (OP) chemicals are known to inhibit the enzyme acetylcholinesterase (AChE). Studying OP poisoning is difficult because common small animal research models have serum carboxylesterase, which contributes to animals' resistance to OP poisoning. Historically, guinea pigs have been used for this research; however, a novel genetically modified mouse strain (KIKO) was developed with nonfunctional serum carboxylase (Es1 KO) and an altered acetylcholinesterase (AChE) gene, which expresses the amino acid sequence of the human form of the same protein (AChE KI). KIKO mice were injected with 1xLD50 of an OP nerve agent or vehicle control with or without atropine. After one to three minutes, animals were injected with 35 mg/kg of the currently fielded Reactivator countermeasure for OP poisoning. Postmortem brains were imaged on a Bruker RapifleX ToF/ToF instrument. Data confirmed the presence of increased acetylcholine in OP-exposed animals, regardless of treatment or atropine status. More interestingly, we detected a small amount of Reactivator within the brain of both exposed and unexposed animals; it is currently debated if reactivators can cross the blood-brain barrier. Further, we were able to simultaneously image acetylcholine, the primary affected neurotransmitter, as well as determine the location of both Reactivator and acetylcholine in the brain. This study, which utilized sensitive MALDI-MSI methods, characterized KIKO mice as a functional model for OP countermeasure development.

Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC. METHODS: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples. RESULTS: Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3ß and MAPK signaling. CONCLUSIONS: Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.

Lipidomic Phenotyping Of Human Small Intestinal Organoids Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

A multimodal pipeline using NMR spectroscopy and MALDI-TOF mass spectrometry imaging from the same tissue sample.

NMR spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple-negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several phospholipid metabolite species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy-MALDI imaging to advance metabolomic and lipidomic studies.

'On the Spot' Digital Pathology of Breast Cancer Based on Single-Cell Mass Spectrometry Imaging.

The molecular pathology of breast cancer is challenging due to the complex heterogeneity of cellular subtypes. The ability to directly identify and visualize cell subtype distribution at the single-cell level within a tissue section enables precise and rapid diagnosis and prognosis. Here, we applied mass spectrometry imaging (MSI) to acquire and visualize the molecular profiles at the single-cell and subcellular levels of 14 different breast cancer cell lines. We built a molecular library of genetically well-characterized cell lines. Multistep processing, including deep learning, resulted in a breast cancer subtype, the cancer's hormone status, and a genotypic recognition model based on metabolic phenotypes with cross-validation rates of up to 97%. Moreover, we applied our single-cell-based recognition models to complex tissue samples, identifying cell subtypes in tissue context within seconds during measurement. These data demonstrate "on the spot" digital pathology at the single-cell level using MSI, and they provide a framework for fast and accurate high spatial resolution diagnostics and prognostics.

CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs. METHODS AND RESULTS: We studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine. CONCLUSIONS: Taken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

Nanowire Assisted Mechanotyping of Cellular Metastatic Potential.

Nanotechnology has provided tools for next generation biomedical devices which rely on nanostructure interfaces with living cells. In vitro biomimetic structures have enabled observation of cell response to various mechanical and chemical cues, and there is a growing interest in isolating and harnessing the specific cues that three-dimensional microenvironments can provide without the requirement for such culture and the experimental drawbacks associated with it. Here we report a randomly oriented gold coated Si nanowire substrate with patterned hydrophobic-hydrophilic areas for differentiation of isogenic breast cancer cells of varying metastatic potential. When considering synthetic surfaces for the study of cell-nanotopography interfaces, randomly oriented nanowires more closely resemble the isotropic architecture of natural extracellular matrix as compared to currently more widely used vertical nanowire arrays. In the study reported here, we show that primary cancer cells preferably attach to the hydrophilic region of randomly oriented nanowire substrate while secondary cancer cells do not adhere. Using machine learning analysis of fluorescence images, cells were found to spread and elongate on the nanowire substrates as compared to a flat substrate, where they mostly remain round, when neither surface was coated with extracellular matrix (ECM) proteins. Such platforms can not only be used for developing bioassays but also as stepping stones for tissue printing technologies where cells can be selectively patterned at desired locations.

Cancer cells display increased migration and deformability in pace with metastatic progression.

In this study, we explored the relation between metastatic states vs the capacity of confined migration, amoeboid transition, and cellular stiffness. We compared across an isogenic panel of human breast cancer cells derived from MDA-MB-231 cells. It was observed that cells after lung metastasis have the fastest migration and lowest stiffness, with a significantly higher capacity to transition into an amoeboid mode. Our findings illustrate that metastasis is a selective process favoring motile and softer cells. Moreover, the observation that circulating tumor cells resemble the parental cell line, but not lung-metastatic cells, suggests that cells with higher deformability and motility are likely selected during extravasation and colonization.

Cellular resolution in clinical MALDI mass spectrometry imaging: the latest advancements and current challenges.

Mass spectrometry (MS) is the workhorse of metabolomics, proteomics and lipidomics. Mass spectrometry imaging (MSI), its extension to spatially resolved analysis of tissues, is a powerful tool for visualizing molecular information within the histological context of tissue. This review summarizes recent developments in MSI and highlights current challenges that remain to achieve molecular imaging at the cellular level of clinical specimens. We focus on matrix-assisted laser desorption/ionization (MALDI)-MSI. We discuss the current status of each of the analysis steps and remaining challenges to reach the desired level of cellular imaging. Currently, analyte delocalization and degradation, matrix crystal size, laser focus restrictions and detector sensitivity are factors that are limiting spatial resolution. New sample preparation devices and laser optic systems are being developed to push the boundaries of these limitations. Furthermore, we review the processing of cellular MSI data and images, and the systematic integration of these data in the light of available algorithms and databases. We discuss roadblocks in the data analysis pipeline and show how technology from other fields can be used to overcome these. Finally, we conclude with curative and community efforts that are needed to enable contextualization of the information obtained.

Focus on the glycerophosphocholine pathway in choline phospholipid metabolism of cancer.

Activated choline metabolism is a hallmark of carcinogenesis and tumor progression, which leads to elevated levels of phosphocholine and glycerophosphocholine in all types of cancer tested so far. Magnetic resonance spectroscopy applications have played a key role in detecting these elevated choline phospholipid metabolites. To date, the majority of cancer-related studies have focused on phosphocholine and the Kennedy pathway, which constitutes the biosynthesis pathway for membrane phosphatidylcholine. Fewer and more recent studies have reported on the importance of glycerophosphocholine in cancer. In this review article, we summarize the recent literature on glycerophosphocholine metabolism with respect to its cancer biology and its detection by magnetic resonance spectroscopy applications.

Molecular causes of elevated phosphoethanolamine in breast and pancreatic cancer cells.

Elevated phosphoethanolamine (PE) is frequently observed in MRS studies of human cancers and xenografts. The role of PE in cell survival and the molecular causes underlying this increase are, however, relatively underexplored. In this study, we investigated the roles of ethanolamine kinases (Etnk-1 and 2) and choline kinases (Chk-α and ß) in contributing to increased PE in human breast and pancreatic cancer cells. We investigated the effect of silencing Etnk-1 and Etnk-2 on cell viability as a potential therapeutic strategy. Both breast and pancreatic cancer cells showed higher PE compared with their nonmalignant counterparts. We identified Etnk-1 as a major cause of the elevated PE levels in these cancer cells, with little or no contribution from Chk-α, Chk-ß, or Etnk-2. The increase of PE observed in pancreatic cancer cells in culture was replicated in the corresponding tumor xenografts. Downregulation of Etnk-1 with siRNA resulted in cell cytotoxicity that correlated with PE levels in breast and pancreatic cancer cells. Etnk-1 may provide a potential therapeutic target in breast and pancreatic cancers.

Alterations in cellular metabolome after pharmacological inhibition of Notch in glioblastoma cells.

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

Mass Spectrometry Imaging of the Hypoxia Marker Pimonidazole in a Breast Tumor Model.

Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging (MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimonidazole as an exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimonidazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides, and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly colocalized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.

Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation, migration, and invasion.

Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF-7 and MDA-MB-231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC-specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF-7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA-MB-231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd.

CEST-MRI detects metabolite levels altered by breast cancer cell aggressiveness and chemotherapy response.

Chemical exchange saturation transfer (CEST) is an MRI contrast mechanism that detects the exchange of protons from distinct hydroxyl, amine, and amide groups to tissue water through the transfer of signal loss, with repeated exchange enhancing their effective signal. We applied CEST to detect systematically 15 common cellular metabolites in a panel of differentially aggressive human breast cancer cell lines. The highest CEST contrast was generated by creatine, myo-inositol, glutamate, and glycerophosphocholine, whose cellular concentrations decreased with increasing breast cancer aggressiveness. These decreased metabolite concentrations resulted in turn in a decreased CEST profile with increasing breast cancer aggressiveness in water-soluble extracts of breast cell lines. Treatment of both breast cancer cell lines with the chemotherapy drug doxorubicin resulted in increased metabolic CEST profiles, which correlated with significant increases in creatine, phosphocreatine, and glycerophosphocholine. CEST can detect breast cancer aggressiveness and response to chemotherapy in water-soluble extracts of breast cell lines. The presented results help shed light on possible contributions from CEST-active metabolites to the CEST contrast produced by breast cancers. The metabolic CEST profile may improve detection sensitivity over conventional MRS, and may have the potential to assess breast cancer aggressiveness and response to chemotherapy non-invasively using MRI if specialized metabolic CEST profile detection can be realized in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

MALDI-Mass Spectrometric Imaging Revealing Hypoxia-Driven Lipids and Proteins in a Breast Tumor Model.

Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of lipids and proteins by using principal component analysis-linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study, we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis, and telomere stress induced senescence. In parallel, we also identified colocalization of hypoxic regions and various lipid species such as PC(16:0/18:0), PC(16:0/18:1), PC(16:0/18:2), PC(16:1/18:4), PC(18:0/18:1), and PC(18:1/18:1), among others. Our findings shed light on the biomolecular composition of hypoxic tumor regions, which may be responsible for a given tumor's resistance to radiation or chemotherapy.

Choline kinase-α protein and phosphatidylcholine but not phosphocholine are required for breast cancer cell survival.

High levels of total choline and phosphocholine (PC) are consistently observed in aggressive cancers. Choline kinase (Chk) catalyzes choline phosphorylation to produce PC in phosphatidylcholine (PtdCho) biosynthesis. PtdCho is the most abundant phospholipid in eukaryotic cell membranes and plays a dual role as the structural component of membranes and as a substrate to produce lipid second messengers such as phosphatidic acid and diacylglycerol. Chk-α, but not Chk-ß, is overexpressed in various cancers, and is closely associated with tumor progression and invasiveness. We have previously shown that downregulation of mRNA using small interfering RNA (siRNA) against Chk-α (siRNA-Chk) or Chk short hairpin RNA, and the resultant decrease of Chk-α protein levels, significantly reduced proliferation in breast cancer cells and tumors. A novel potent and selective small-molecule Chk-α inhibitor, V-11-0711, that inhibits the catalytic activity of Chk has recently been developed. Here, we used triple negative MDA-MB-231 and SUM149 breast cancer cells to further investigate the role of Chk-α in cancer, by examining Chk-α protein levels, cell viability/proliferation, choline phospholipid and lipid metabolism, lipid droplet formation, and apoptosis, following treatment with V-11-0711. Under the conditions used in this study, treatment with V-11-0711 significantly decreased PC levels but did not reduce cell viability as long as Chk-α protein and PtdCho levels were not reduced, suggesting that Chk-α protein and PtdCho, but not PC, may be crucial for breast cancer cell survival. These data also support the approach of antitumor strategies that destabilize Chk-α protein or downregulate PtdCho in breast cancer treatment.

Glutamine transporter SLC38A3 promotes breast cancer metastasis via Gsk3ß/ß-catenin/EMT pathway.

Breast cancer is the leading cancer-related cause of death in women. Here we show that solute carrier family 38-member 3 (SLC38A3) is overexpressed in breast cancer, particularly in triple-negative breast cancer (TNBC) cells and tissues. Our study reveals that SLC38A3 regulates cellular glutamine, glutamate, asparagine, aspartate, alanine, and glutathione (GSH) levels in breast cancer cells. Our data demonstrate that SLC38A3 enhances cell viability, cell migration and invasion in vitro, and promotes tumor growth and metastasis in vivo, while reducing apoptosis and oxidative stress. Mechanistically, we show that SLC38A3 suppresses the activity of glycogen synthase kinase 3-ß (Gsk3ß), a negative regulator of ß-catenin, and increases protein levels of ß-catenin, leading to the upregulation of epithelial-to-mesenchymal-transition (EMT)-inducing transcription factors and EMT markers in breast cancer. In summary, we show that SLC38A3 is overexpressed in breast cancer and promotes breast cancer metastasis via the GSK3ß/ß-catenin/EMT pathway, presenting a novel therapeutic target to explore for breast cancer.

Mass Spectrometry Imaging of Organoids to Improve Preclinical Research.

Preclinical models are essential research tools before novel therapeutic or diagnostic methods can be applied to humans. These range from in vitro cell monocultures to vastly more complex animal models, but clinical translation to humans often fails to deliver significant results. Three-dimensional (3D) organoid systems are being increasingly studied to establish physiologically relevant in vitro platforms in a trade-off between the complexity of the research question and the complexity of practical experimental setups. The sensitivity and precision of analytical tools are yet another limiting factors in what can be investigated, and mass spectrometry (MS) is one of the most powerful analytical techniques available to the scientific community. Its innovative use to spatially resolve biological samples has opened many research avenues in the field of MS imaging (MSI). Here, this work aims to explore the current scientific landscape in the application of MSI on organoids, with an emphasis on their combined potential to facilitate and improve preclinical studies.