Polyunsaturated Fatty Acid-Enriched Lipid Fingerprint of Glioblastoma Proliferative Regions Is Differentially Regulated According to Glioblastoma Molecular Subtype.

Glioblastoma (GBM) represents one of the deadliest tumors owing to a lack of effective treatments. The adverse outcomes are worsened by high rates of treatment discontinuation, caused by the severe side effects of temozolomide (TMZ), the reference treatment. Therefore, understanding TMZ's effects on GBM and healthy brain tissue could reveal new approaches to address chemotherapy side effects. In this context, we have previously demonstrated the membrane lipidome is highly cell type-specific and very sensitive to pathophysiological states. However, little remains known as to how membrane lipids participate in GBM onset and progression. Hence, we employed an ex vivo model to assess the impact of TMZ treatment on healthy and GBM lipidome, which was established through imaging mass spectrometry techniques. This approach revealed that bioactive lipid metabolic hubs (phosphatidylinositol and phosphatidylethanolamine plasmalogen species) were altered in healthy brain tissue treated with TMZ. To better understand these changes, we interrogated RNA expression and DNA methylation datasets of the Cancer Genome Atlas database. The results enabled GBM subtypes and patient survival to be linked with the expression of enzymes accounting for the observed lipidome, thus proving that exploring the lipid changes could reveal promising therapeutic approaches for GBM, and ways to ameliorate TMZ side effects.

Confirmation of sub-cellular resolution using oversampling imaging mass spectrometry.

The use of oversampling in MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) to improve lateral resolution is a common practice. However, its application is still controversial and recent studies reported a spot size-dependent change in the relative intensity of the spectra. Previously, using oversampling, we described the lipidome of the human colon epithelium, a 20-30 µm wide cell monolayer; even assessing the changes occurring within this monolayer associated with complex biological processes. Interestingly, the K-means analysis of those experiments unveiled the presence of a third epithelial cluster that anatomically matched the nuclei position. Taking into account the nucleus size (9-12 µm of diameter) and its distinctive lipidome, we decided to test whether this cluster was really of nuclear origin. Hence, the spectra obtained directly from tissue sections were compared with those recorded from the nuclei isolated from colon biopsies. The highest correlation coefficient was obtained when comparing the spectrum of the isolated nuclei with that of the tissue nuclear cluster, demonstrating the successful identification of the nuclear lipidome in the MALDI-IMS experiments run using oversampling and a lateral resolution of 10 µm/pixel. Importantly, it was established that phosphatidylinositol 38:4 nuclear levels remained stable along the colon crypt. That is, it mimicked neither the regular decrease observed in the epithelium nor the regular increase observed in the stroma, eliminating the chance of inter-pixel contamination. Altogether, besides confirming the usefulness of the oversampling technique, these results strongly reinforce the pivotal role IMS may have in promising fields such as single-cell analysis. Graphical abstract.

Comparison of commercial 5-aminolevulinic acid (Gliolan®) and the pharmacy-compounded solution fluorescence in glioblastoma.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) has become an important assistant in glioblastoma (GB) surgery. Unfortunately, its price affects its widespread use. OBJECTIVE: The aim of this study was to compare commercial 5-ALA with the pharmacy-compounded solution. METHODS: Using first an in vitro experimental approach, different concentrations of the pharmacy-compounded solution and commercial 5-ALA were tested in U87MG, LN229, U373, and T98G commercial glioblastoma cell lines. Fluorescence intensity was compared for each concentration by flow cytometry. Mean fluorescence of culture supernatant and lysate samples were analyzed. In a second phase, both preparations were used for surgical glioblastoma resection and tumor samples were analyzed by confocal microscopy. Mean fluorescence intensity was analyzed for each preparation and compared. RESULTS: There was a high variability of fluorescence intensity between cell lines, but each cell line showed similar fluorescence for both preparations (compounded preparation and commercial 5-ALA). In the same way, both preparations had similar fluorescence intensity in glioblastoma samples. CONCLUSION: Both, compounded and commercial 5-ALA preparations produce equivalent fluorescent responses in human glioblastoma cells. Fluorescence intensity is cell line specific, but fluorescent properties of both preparations are undistinguishable.

Tissue-selective alteration of ethanolamine plasmalogen metabolism in dedifferentiated colon mucosa.

Human colon lipid analysis by imaging mass spectrometry (IMS) demonstrates that the lipid fingerprint is highly sensitive to a cell's pathophysiological state. Along the colon crypt axis, and concomitant to the differentiation process, certain lipid species tightly linked to signaling (phosphatidylinositols and arachidonic acid (AA)-containing diacylglycerophospholipids), change following a rather simple mathematical expression. We extend here our observations to ethanolamine plasmalogens (PlsEtn), a unique type of glycerophospholipid presenting a vinyl ether linkage at sn-1 position. PlsEtn distribution was studied in healthy, adenomatous, and carcinomatous colon mucosa sections by IMS. In epithelium, 75% of PlsEtn changed in a highly regular manner along the crypt axis, in clear contrast with diacyl species (67% of which remained constant). Consistently, AA-containing PlsEtn species were more abundant at the base, where stem cells reside, and decreased while ascending the crypt. In turn, mono-/diunsaturated species experienced the opposite change. These gradients were accompanied by a gradual expression of ether lipid synthesis enzymes. In lamina propria, 90% of stromal PlsEtn remained unchanged despite the high content of AA and the gradient in AA-containing diacylglycerophospholipids. Finally, both lipid and protein gradients were severely affected in polyps and carcinoma. These results link PlsEtn species regulation to cell differentiation for the first time and confirm that diacyl and ether species are differently regulated. Furthermore, they reaffirm the observations on cell lipid fingerprint image sensitivity to predict cell pathophysiological status, reinforcing the translational impact both lipidome and IMS might have in clinical research.

Lipid fingerprint image accurately conveys human colon cell pathophysiologic state: A solid candidate as biomarker.

Membrane lipids are gaining increasing attention in the clinical biomarker field, as they are associated with different pathologic processes such as cancer or neurodegenerative diseases. Analyzing human colonoscopic sections by matrix assisted laser/desorption ionization (MALDI) mass spectrometry imaging techniques, we identified a defined number of lipid species changing concomitant to the colonocyte differentiation and according to a quite simple mathematical expression. These species felt into two lipid families tightly associated in signaling: phosphatidylinositols and arachidonic acid-containing lipids. On the other hand, an opposed pattern was observed in lamina propria for AA-containing lipids, coinciding with the physiological distribution of the immunological response cells in this tissue. Importantly, the lipid gradient was accompanied by a gradient in expression of enzymes involved in lipid mobilization. Finally, both lipid and protein gradients were lost in adenomatous polyps. The latter allowed us to assess how different a single lipid species is handled in a pathological context depending on the cell type. The strict patterns of distribution in lipid species and lipid enzymes described here unveil the existence of fine regulatory mechanisms orchestrating the lipidome according to the physiological state of the cell. In addition, these results provide solid evidence that the cell lipid fingerprint image can be used to predict precisely the physiological and pathological status of a cell, reinforcing its translational impact in clinical research.

Lipid signature associated with chronic colon inflammation reveals a dysregulation in colonocyte differentiation process.

Inflammatory Bowel Disease (IBD) comprises a heterogeneous group of chronic inflammatory conditions of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease. Although the etiology is not well understood, IBD is characterized by a loss of the normal epithelium homeostasis that disrupts the intestinal barrier of these patients. Previous work by our group demonstrated that epithelial homeostasis along the colonic crypts involves a tight regulation of lipid profiles. To evaluate whether lipidomic profiles conveyed the functional alterations observed in the colonic epithelium of IBD, we performed matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) analyses of endoscopic biopsies from inflamed and non-inflamed segments obtained from UC patients. Our results indicated that lipid profiling of epithelial cells discriminated between healthy and UC patients. We also demonstrated that epithelial cells of the inflamed mucosa were characterized by a decrease in mono- and di-unsaturated fatty acid-containing phospholipids and higher levels of arachidonic acid-containing species, suggesting an alteration of the lipid gradients occurring concomitantly to the epithelial differentiation. This result was reinforced by the immunofluorescence analysis of EPHB2 and HPGD, markers of epithelial cell differentiation, sustaining that altered lipid profiles were at least partially due to a faulty differentiation process. Overall, our results showed that lipid profiling by MALDI-MSI faithfully conveys molecular and functional alterations associated with the inflamed epithelium, providing the foundation for a novel molecular characterization of UC patients.

A Drastic Shift in Lipid Adducts in Colon Cancer Detected by MALDI-IMS Exposes Alterations in Specific K+ Channels.

Even though colorectal cancer (CRC) is one of the most preventable cancers, it is one of the deadliest, and recent data show that the incidence in people <50 years has unexpectedly increased. While new techniques for CRC molecular classification are emerging, no molecular feature is as yet firmly associated with prognosis. Imaging mass spectrometry (IMS) lipidomic analyses have demonstrated the specificity of the lipid fingerprint in differentiating pathological from healthy tissues. During IMS lipidomic analysis, the formation of ionic adducts is common. Of particular interest is the [Na+]/[K+] adduct ratio, which already functions as a biomarker for homeostatic alterations. Herein, we show a drastic shift of the [Na+]/[K+] adduct ratio in adenomatous colon mucosa compared to healthy mucosa, suggesting a robust increase in K+ levels. Interrogating public databases, a strong association was found between poor diagnosis and voltage-gated potassium channel subunit beta-2 (KCNAB2) overexpression. We found this overexpression in three CRC molecular subtypes defined by the CRC Subtyping Consortium, making KCNAB2 an interesting pharmacological target. Consistently, its pharmacological inhibition resulted in a dramatic halt in commercial CRC cell proliferation. Identification of potential pharmacologic targets using lipid adduct information emphasizes the great potential of IMS lipidomic techniques in the clinical field.

Common and Differential Traits of the Membrane Lipidome of Colon Cancer Cell Lines and their Secreted Vesicles: Impact on Studies Using Cell Lines.

Colorectal cancer (CRC) is the fourth leading cause of cancer death in the world. Despite the screening programs, its incidence in the population below the 50s is increasing. Therefore, new stratification protocols based on multiparametric approaches are highly needed. In this scenario, the lipidome is emerging as a powerful tool to classify tumors, including CRC, wherein it has proven to be highly sensitive to cell malignization. Hence, the possibility to describe the lipidome at the level of lipid species has renewed the interest to investigate the role of specific lipid species in pathologic mechanisms, being commercial cell lines, a model still heavily used for this purpose. Herein, we characterize the membrane lipidome of five commercial colon cell lines and their extracellular vesicles (EVs). The results demonstrate that both cell and EVs lipidome was able to segregate cells according to their malignancy. Furthermore, all CRC lines shared a specific and strikingly homogenous impact on ether lipid species. Finally, this study also cautions about the need of being aware of the singularities of each cell line at the level of lipid species. Altogether, this study firmly lays the groundwork of using the lipidome as a solid source of tumor biomarkers.

Ins and Outs of Interpreting Lipidomic Results.

Membrane lipids are essential for life; however, research on how cells regulate cell lipid composition has been falling behind for quite some time. One reason was the difficulty in establishing analytical methods able to cope with the cell lipid repertoire. Development of a diversity of mass spectrometry-based technologies, including imaging mass spectrometry, has helped to demonstrate beyond doubt that the cell lipidome is not only greatly cell type dependent but also highly sensitive to any pathophysiological alteration such as differentiation or tumorigenesis. Interestingly, the current popularization of metabolomic studies among numerous disciplines has led many researchers to rediscover lipids. Hence, it is important to underscore the peculiarities of these metabolites and their metabolism, which are both radically different from protein and nucleic acid metabolism. Once differences in lipid composition have been established, researchers face a rather complex scenario, to investigate the signaling pathways and molecular mechanisms accounting for their results. Thus, a detail often overlooked, but of crucial relevance, is the complex networks of enzymes involved in controlling the level of each one of the lipid species present in the cell. In most cases, these enzymes are redundant and promiscuous, complicating any study on lipid metabolism, since the modification of one particular lipid enzyme impacts simultaneously on many species. Altogether, this review aims to describe the difficulties in delving into the regulatory mechanisms tailoring the lipidome at the activity, genetic, and epigenetic level, while conveying the numerous, stimulating, and sometimes unexpected research opportunities afforded by this type of studies.