Mass Spectrometry Imaging Research Articles

Spatial omics reveals molecular changes in focal cortical dysplasia type II

Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.

Mass Spectrometry Imaging (MALDI-MSI) as a molecular histology application for characterizing pulmonary lesions caused by PRRSV-1 strains of different virulence

Mass Spectrometry Imaging (MALDI-MSI) as a molecular histology application for characterizing pulmonary lesions caused by PRRSV-1 strains of different virulence

429 Spatial Investigation of the Extracellular Matrix Metastatic Niche in Invasive Breast Cancer by Mass Spectrometry Imaging

OBJECTIVES/GOALS: Metastasis to regional areas decreases invasive breast cancer (IBC) survival rate by 13%. Despite the clinical importance of lymph node involvement, the role of extracellular matrix (ECM) remodeling in metastases is unknown. We hypothesize that the spatial dysregulation of the collagen proteome facilitates pro-tumorigenic immune infiltration. METHODS/STUDY POPULATION: Lymph node metastases were compared to patient-matched primary tumor and normal lymph nodes using tissue microarrays (TMA) from 31 generational South Carolina women with IBC (black women, BW n=10, white women, WW n=21) and lumpectomies from 5 triple-negative breast cancer (TNBC) patients (BW n=3; WW n=2) by ECM-targeted mass spectrometry imaging. RESULTS/ANTICIPATED RESULTS: Between metastatic and normal lymph nodes, 10% of peptides, primarily from fibrillar collagens, were significantly different by area under the receiver operating curve (AUROC>70%; p-value< 0.01) within the TMAs. In a subsequent preliminary study of the TNBC metastatic niche, a segmentation analysis of 152 putatively identified peptides and 117,909 pixels revealed 10 uniquely localized proteomic groups. 12 peptides were found to have significantly decreased relative peak intensities in lymph node metastases compared to the primary tumor and normal lymph nodes by a one-way ANOVA test (p< 0.05). 7 peptides could discriminate between metastatic and normal lymph nodes, while 22 peptides could discriminate between metastatic lymph nodes and the primary tumor (AUROC>0.70; p-value < 0.05). DISCUSSION/SIGNIFICANCE: Our preliminary interrogation highlights emerging differences between lymph node metastases, the primary tumor, and normal lymph nodes. Future work is needed to connect these discrete ECM proteomes to immune infiltration alterations, which could contribute to disparate patient outcomes.

Unveiling the regulatory mechanisms of nodules development and quality formation in Panax notoginseng using multi-omics and MALDI-MSI

IntroductionRenowned for its role in traditional Chinese medicine, Panax notoginseng exhibits healing properties including bidirectional regulatory effects on hematological system diseases. However, the presence of nodular structures near the top of the main root, known as nail heads, may impact the quality of the plant's valuable roots. ObjectivesIn this paper, we aim to systematically analyze nail heads to identify their potential correlation with P. notoginseng quality. Additionally, we will investigate the molecular mechanisms behind nail head development. MethodsMorphological characteristics and anatomical features were analyzed to determine the biological properties of nail heads. Active component analysis and MALDI mass spectrometry imaging (MALDI-MSI) were performed to determine the correlation between nail heads and P. notoginseng quality. Phytohormone quantitation, MALDI-MSI, RNA-seq, and Arabidopsis transformation were conducted to elucidate the mechanisms of nail head formation. Finally, protein-nucleic acid and protein–protein interactions were investigated to construct a transcriptional regulatory network of nodule development and quality formation. ResultsOur analyses have revealed that nail heads originate from an undeveloped lateral root. The content of ginsenosides was found to be positively associated with the amount of nail heads. Ginsenoside Rb1 specifically accumulated in the cortex of nail heads, while IAA, tZR and JAs also showed highest accumulation in the nodule. RNA-seq analysis identified PnIAA14 and PnCYP735A1 as inhibitors of lateral root development. PnMYB31 and PnMYB78 were found to form binary complexes with PnbHLH31 to synergistically regulate the expression of PnIAA14, PnCYP735A1, PnSS, and PnFPS. ConclusionOur study details the major biological properties of nodular structures in P. notoginseng and outlines their impact on the quality of the herb. It was also determined that PnMYB31- and PnMYB78-PnbHLH31 regulate phytohormones and ginsenosides accumulation, further affecting plant development and quality. This research provides insights for quality evaluation and clinical applications of P. notoginseng.

Spatial mapping of gemcitabine and its pharmacologically active metabolites in tissues using MALDI mass spectrometry imaging

Spatial mapping of gemcitabine and its pharmacologically active metabolites in tissues using MALDI mass spectrometry imaging

Exploring the Small-Molecule Composition of Voacanga africana Seeds Using LADI-MS: An Interview with Rabi Musah

Plant seeds are a source of medicinal components used in human therapeutics. As a result, it is important that extraction methods are developed that can remove the bioactive compounds used for these therapeutics without damaging the plant tissue. In this “Focus on Environmental Analysis” column, LCGC sat down with Dr. Rabi Ann Musah from the University of Albany, who is currently researching methods to protect seed viability while isolating specific compounds. Musah's team used a technique called laser ablation direct analysis in real-time imaging–mass spectrometry (LADI–MS) to analyze the distribution of molecules, such as alkaloids, in Voacanga africana seeds.

Molecularly Imprinted Heterostructure-Assisted Laser Desorption Ionization Mass Spectrometry Analysis and Imaging of Quinolones.

Quinolone residues resulting from body metabolism and waste discharge pose a significant threat to the ecological environment and to human health. Therefore, it is essential to monitor quinolone residues in the environment. Herein, an efficient and sensitive matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) method was devised by using a novel molecularly imprinted heterojunction (MIP-TNs@GCNs) as the matrix. Molecularly imprinted titanium dioxide nanosheets (MIP-TNs) and graphene-like carbon nitrides (GCNs) were associated at the heterojunction interface, allowing for the specific, rapid, and high-throughput ionization of quinolones. The mechanism of MIP-TNs@GCNs was clarified using their adsorption properties and laser desorption/ionization capability. The prepared oxygen-vacancy-rich MIP-TNs@GCNs heterojunction exhibited higher light absorption and ionization efficiencies than TNs and GCNs. The good linearity (in the quinolone concentration range of 0.5-50 pg/μL, R2 > 0.99), low limit of detection (0.1 pg/μL), good reproducibility (n = 8, relative standard deviation [RSD] < 15%), and high salt and protein resistance for quinolones in groundwater samples were achieved using the established MIP-TNs@GCNs-MALDI/MS method. Moreover, the spatial distributions of endogenous compounds (e.g., amino acids, organic acids, and flavonoids) and xenobiotic quinolones from Rhizoma Phragmitis and Rhizoma Nelumbinis were visualized using the MIP-TNs@GCNs film as the MALDI/MS imaging matrix. Because of its superior advantages, the MIP-TNs@GCNs-MALDI/MS method is promising for the analysis and imaging of quinolones and small molecules.

Spatiotemporal Study of Galactolipid Biosynthesis in Duckweedwith Mass Spectrometry Imaging and in vivo Isotope Labeling.

Isotope labeling coupled with mass spectrometry imaging (MSI) presents a potent strategy for elucidating the dynamics of metabolism in cellular resolution, yet its application to plant systems is scarce. It has the potential to reveal the spatiotemporal dynamics in lipid biosynthesis during plant development. In this study, we explore its application to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization (MALDI) MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied after growing in 50% D2O media over fifteen-day time period. When they were partially labeled after five days, three distinct binomial isotopologue distributions were observed corresponding to the labeling of partial structural moieties: galactose only, galactose and a fatty acyl chain, and the entire molecule. The temporal change of the relative abundance of these distributions follows the expected linear pathway of galactolipid biosynthesis. Notably, their MS images revealed the localization of each isotopologue group to the old parent frond, the intermediate tissues, and the newly grown daughter fronds. Besides, two additional labeling experiments, 1) 13CO2 labeling and 2) backward labeling of completely 50% D2O labeled L. minor in H2O media, confirm the observations in forward labeling. Further, these experiments unveiled hidden isotopologue distributions indicative of membrane lipid restructuring. This study suggests the potential of isotope labeling with MSI to provide spatiotemporal details in lipid biosynthesis in plant development.

Development of a Mass Spectrometry Imaging Method to Evaluate the Penetration of Moisturizing Components Coated on Surgical Gloves into Artificial Membranes.

Skin dryness and irritant contact dermatitis induced by the prolonged use of surgical gloves are issues faced by physicians. To address these concerns, manufacturers have introduced surgical gloves that incorporate a moisturizing component on their inner surface, resulting in documented results showing a reduction in hand dermatitis. However, the spatial distribution of moisturizers applied to surgical gloves within the integument remains unclear. Using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI), we investigated the spatial distribution of moisturizers in surgical gloves within artificial membranes. Recently, dermal permeation assessments using three-dimensional models, silicone membranes, and Strat-M have gained attention as alternative approaches to animal testing. Therefore, in this study, we established an in vitro dermal permeation assessment of commercially available moisturizers in surgical gloves using artificial membranes. In this study, we offer a methodology to visualize the infiltration of moisturizers applied to surgical gloves into an artificial membrane using MALDI-MSI, while evaluating commercially available moisturizer-coated surgical gloves. Using our penetration evaluation method, we confirmed the infiltration of the moisturizers into the polyethersulfone 2 and polyolefin layers, which correspond to the epidermis and dermis of the skin, after the use of surgical gloves. The MSI-based method presented herein demonstrated the efficacy of evaluating the permeation of samples containing active ingredients.

Developing a novel strategy for fabricating matrix film to assess the distribution of potassium perfluorooctanic sulfonate by matrix-assisted laser desorption/ionization mass spectrometry imaging

Matrix deposition plays a critical role in image quality of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). To improve the ionization efficiency and overcome the limitation of traditional matrix deposition methods in the face of difficult-to-sublimate or difficult-to-dissolve matrix, covalent organic frameworks (COFs) named COF-DhaTab was successfully synthesized and firstly used as matrix film. It was fabricated by imprinting of sieved COF-DhaTab powder on the surface of a double-sided adhesive tape. Outstanding reproducibility and uniformity of COF-DhaTab film were demonstrated by relative standard deviation (RSD) within 8.37% and 7.71% from dot-to-dot and plate-to-plate, respectively. With the introduction of double-sided adhesive tape, water contact angle (WCA) of COF-DhaTab film increased from 55° to 141°, resulting in significant suppression of analyte diffusion. Moreover, the intensity of potassium perfluorooctanic sulfonate (PFOS, C8F17SO3−, m/z 498.93) was 9.3 × 105, more than six hundred times higher than that using DHB matrix. This enhancement was attributed to the rough surface and multiple branches of the synthesized COF-DhaTab. To verify the ability of COF-DhaTab film as substrate, the spatial distribution of PFOS in zebrafish, rat liver and kidney tissues was explored. Superior imaging capability was displayed with high-spatial resolution and reliable location distribution. These results not only demonstrate the outstanding ability of COF-DhaTab as matrix for MALDI-MS and MALDI-MSI, but also provide a facile approach for fabrication of novel matrix films for MALDI-MSI.

Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 μm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Development of Transmission Ambient Pressure Laser Desorption Ionization/Postphotoionization Mass Spectrometry Imaging.

Laser-based high-resolution mass spectrometry imaging at ambient conditions has promising applications in life science. However, the ion yield during laser desorption/ablation is poor. Here, transmission atmospheric pressure laser desorption ionization combined with a compact postphotoionization (t-AP-LDI/PI) assembly with a krypton discharge lamp was developed for the untargeted imaging of various biomolecules. The spatial distributions of numerous lipid classes, fatty acids, neurotransmitters, and amino acids in the subregions of mouse cerebellum tissue were obtained. Compared with single laser ablation, the sensitivities for most analytes were increased by 1 to 3 orders of magnitude by dopant-assisted postphotoionization. After careful optimization, a spatial resolution of 4 μm could be achieved for the metabolites in mouse hippocampus tissue. Finally, the melanoma tissue slices were analyzed using t-AP-LDI/PI MSI, which revealed the metabolic heterogeneity of the melanoma microenvironment and exhibited the phenomenon of abnormal proliferation and invasion trends in tumor cells.

Identification and quantification of microplastics in salts by complementary approaches using pyrolysis-gas chromatography/quadrupole-time of flight mass spectrometry (Py-GC/QTOFMS) and laser direct infrared (LDIR) chemical imaging analysis

Microplastics (MPs) and nanoplastics (NPs) have been suspected as contaminants in various foodstuffs, including salts, all over the world. Regarding the different sizes and polymer types, the mass concentrations of actual plastic particles in salt are not very clear. The purpose of this study is to develop a scalable method for qualitative and quantitative analysis of MPs and NPs by using Pyrolysis Gas Chromatography Quadrupole-Time of Flight mass spectrometry (Py-GC/QTOFMS) to detect their mass concentrations in salt samples. The targeted and suspected lists of polymers in salts were compiled based on the combined results of the high-resolution mass spectrometry (HRMS) full scanning with auxiliary MS dataset and the laser direct infrared (LDIR) chemical imaging analysis. The seven targeted MPs with polymer standards, i.e., polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), polypropylene (PP), polystyrene (PS), polyethylene (PE), polyethylene terephthalate (PET), and polycarbonate (PC), were first subjected to a full MS scanning mode of the Py-GC/QTOFMS analysis. Subsequently, the parental masses of their pyrolysis compounds were used as the seeds to generate the related daughter masses. This process established both retention time and mass-pairs matching for the target MS/MS mode for enabling the identification and quantification of the particles. The suspected MPs with a matching degree >0.65 in the LDIR list were explored either by the full scan MS. Only PVC was identified, and PET was suspected. The Py-GC/QTOFMS result is complementary and comparable to the LDIR detection with the matching degree >0.85. We identified that PVC and PET (suspected) can be measured in both commercial and bulk sea salts, and their concentrations in sea salts are much higher than in rock salts, implying heavy contamination of the seawater.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 μm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

Abstract 6188: Pathomics of prostate cancer incorporating histology and mass spectrometry imaging

Abstract Background: Recent evidence shows that interactions between tumor cells, immune cells, and extracellular matrix (ECM) proteins in the stroma play an important role in prostate cancer (PCa) progression. However, to date these factors are not accounted for in clinical evaluation of PCa patients. Our team has developed methods based on matrix-assisted laser desorption/ionization (MALDI) imaging to identify sets of ECM glycan and collagen molecules associated with PCa. In this study, we leverage computational imaging methods and pathomic textural features to assess the potential of MALDI imaging to guide the identification of regions in PCa hematoxylin and eosin stain (H&amp;E) images as tumor or non-tumor. Methods: We used a cohort of PCa patients (n=10), who have undergone radical prostatectomy, with grade groups GG3−GG5, and with available H&amp;E and N-glycan MALDI images. We used five MALDI images per patient representing the distribution of N-glycan species at 1419, 2539, 2686, 1663, and 1809 m/z. For each MALDI image, an N-glycan mask was generated and co-registered with the corresponding H&amp;E image via computational imaging methods (i.e., Otsu thresholding, largest connected components labeling, sequential affine and B-spline registration using mutual information). To quantify the overlap of each mask with tumor regions, we calculated Dice scores between the masks and the pathology annotation in H&amp;E. To assess differences in imaging patterns of regions indicated by the different masks, we extracted a set of pathomic textural features (n=70) from the H&amp;E image within each mask using PyRadiomics, and we performed Wilcoxon signed rank tests across all pathomic textural features for all paired combinations of the five masks. Results: The mean Dice scores between the pathology tumor annotation and the N-glycan masks from 1419 (0.56 [0.44‒0.65]) and 2539 m/z (0.64 [0.59‒0.82]) was significantly higher (p&amp;lt;0.008) than the Dice score of 1663 (0.24 [0.17‒0.36]), 1809 (0.31 [0.13‒0.45]), and 2686 m/z (0.32 [0.19‒0.37]). This suggests a large overlap between 1419 and 2539 m/z and tumor, and large overlap between 1663 and 1809 m/z and non-tumor regions. Consistent with these results, our pathomic textural analysis showed that 50 out of 70 features were significantly different (p&amp;lt;0.05) between the masks derived from 1419 and 1663 m/z, 52 between 1419 and 1809 m/z, 62 between 2539 and 1663 m/z, and 57 between 2539 and 1809 m/z (p&amp;lt;0.05). Moreover, there were only 23 significantly different features between 1419 and 2539 m/z (p&amp;lt;0.05), and 0 features between 1663 and 1809 m/z. Further evaluation is needed for 2686 m/z for which a mean of 54 [45‒64] significantly different features (p&amp;lt;0.05) from all other masks were found. Conclusions: We provide preliminary evidence that MALDI imaging can elucidate tissue properties and guide pathomic textural analysis in H&amp;E images, with potential applications in treatment guidance and prognostication of PCa. Citation Format: José Marcio Luna, Hani Nakhoul, Cody Weimholt, Eric H. Kim, Sheng-Kwei Song, Peggi M. Angel, Richard R. Drake, Joseph E. Ippolito. Pathomics of prostate cancer incorporating histology and mass spectrometry imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6188.

Abstract 6222: Exposing the heterogeneity of the lipidome in the TME of cutaneous melanoma following treatment with NUC-7738 in combination with anti-PD-1 therapy

Abstract Background. Malignant melanoma exhibits distinct lipid profiles compared to normal tissue and changes in the lipidome influence multiple aspects of tumor biology. Lipidomic alterations within the tumor microenvironment (TME) may influence the efficacy of anti-PD-1 therapy. In combination with anti-PD-1 agent pembrolizumab, NUC-7738 (3'-deoxyadenosine [3’-dA] which is phosphorylated and protected with a phosphoramidate moiety attached at the 5’-position) has shown promising activity in anti-PD-1 refractory melanoma (Phase 2; NCT03829254). NUC-7738 generates sustained intracellular levels of 3’-dATP, which profoundly alters RNA regulatory processes in tumor cells, resulting in impaired cellular responses and changes in expression of proteins related to lipid biosynthesis. To interrogate components of the TME before and after NUC-7738 + pembrolizumab treatment, we developed a novel workflow incorporating Mass Spectrometry Imaging (MSI) to co-register lipidomic changes with molecular markers. Machine learning accelerated the interpretation of complex data obtained from paired patient biopsies. Methods. Snap frozen paired biopsies from patients with advanced cutaneous melanoma treated with NUC-7738 ±pembrolizumab were analyzed by desorption electrospray ionization (DESI)-MSI. Specific metabolic (e.g., ASAH1) and immune (e.g., CD8, CD20) markers were investigated by multiplex immunofluorescence (mIF) post DESI-MSI. Target selection was guided by bulk RNA-seq data. Raw MSI data was visualized using Waters HDI® software. Data extracted from selected regions of interest was exported into MetaboAnalyst 5.0 for statistical analysis. LIPID MAPS® Structure Database was used to assign lipid species. mIF/mIHC images were analyzed by HALO® AI. Results. Single 10 µm tissue sections allowed co-registered DESI-MSI and mIF, suitable for further informatic and AI-enabled image analysis. This facilitated direct comparison of lipids, cells, and protein expression in the TME, before and after treatment with NUC-7738, a compound known to cause profound changes in poly(A) tail regulation. MSI data analyses of the TME uncovered intra-tumoral heterogeneity of the lipidome and demonstrated changes following treatment. Conclusion. This platform provides a powerful investigative tool to simultaneously explore the myriad of factors in the TME that contribute to tumorigenesis and therapeutic response. Further analyses of the role of the lipidome in the TME and intra-tumoral heterogeneity will be explored in paired biopsies from patients with PD-1 inhibitor refractory melanoma. Citation Format: Alison L. Dickson, Greice M. Zickuhr, In Hwa Um, Ying Zhang, Ruth Plummer, Sarah P. Blagden, Stefan N. Symeonides, Natalie Cook, T.R. Jeffry Evans, Mustafa Elshani, David J. Harrison. Exposing the heterogeneity of the lipidome in the TME of cutaneous melanoma following treatment with NUC-7738 in combination with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6222.

Abstract 4779: The effects of glutamine deprivation on ovarian cancer initiation and progression

Abstract High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. Most patients with HGSC are diagnosed at advanced stage diseases. Despite the high response rate to platinum/taxane-based chemotherapy, most of these patients will develop recurrent chemoresistant disease. Recent studies showed the cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) play an important role in modulating the malignant phenotypes of HGSC. CAFs are primarily responsible for producing factors such as cytokines and growth factors to modify the TME niche to promote the malignant phenotypes of cancer cells. Recently, we have identified a previously unrecognized mechanism in which metabolism of reactive CAFs is reprogrammed through an upregulated glutamine anabolic pathway in HGSC. This dysfunctional CAF metabolism confers atypical metabolic flexibility and adaptive mechanisms in CAFs, allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in the TME. Our preliminary data show that tumors developed from syngeneic fallopian epithelial cell-derived cancer cells injected IP into C57BL/6 mice, which started to be fed with normal diet two weeks before tumor cells injection had significantly higher tumor burden than those fed with the glutamine-deprived diet. Moreover, tumor-bearing mice treated with glutaminase inhibitor for 3 weeks demonstrated significantly lower tumor burden than those treated with the control vehicle. These findings suggest that the interplay between CAFs and HGSC cells enriches glutamine in the ovarian TME; and glutamine depletion can not only suppress ovarian tumor growth but also prevent the development of advanced progressive diseases. In addition to the direct promoting effect of glutamine on the glutamine addicted HGSC cells, increased CAF-derived glutamine can also affect the activity of other stromal cell types such as immune cells in the TME, which subsequently modulate HGSC cell growth. Imaging mass cytometry (IMC) and Mass Spectrometry Imaging (MSI) were performed on serial tissue sections of tissue microarrays generated from the abovementioned murine samples. Mice fed with a glutamine-deprived diet or treated with the glutaminase inhibitor not only have a significantly lower tumor burden but also induced dramatic changes in the cellular and molecular composition within the TME. In particular, mice fed with a glutamine-deprived diet had greater B-cell-related immune response, reduced stemness, and EMT of ovarian cancer cells, and more activated CAFs, which may result in increased stiffness of the ECM and subsequently enhance the malignant phenotype of HGSC cells. Further studies to delineate the molecular mechanisms by which spatial cellular and metabolic profiles modulate HGSC pathogenesis and progression are warranted. Citation Format: Sammy Ferri-Borgogno, Kathryn Lu, Basant T. Gamal, Chi Lam Au Yeung, Erin Seeley, Javier A. Gomez, Akshay Basi, Jared K. Burks, Olamide Animasahun, Deepak Nagrath, Samuel C. Mok. The effects of glutamine deprivation on ovarian cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4779.

Abstract 133: GMDS is a key driver of MYCN-amplified neuroblastoma core fucosylation and tumorigenesis

Abstract Introduction: MYCN-amplification (MYCN-amp) is a genetic hallmark of ~40% of high-risk neuroblastomas (NBs). N-MYC is an oncogenic transcription factor and master regulator of metabolism. Glycosylation is a major post-translational modification that is critical for cancer progression, spread, and immune evasion. How genetic signatures such as MYCN-amp alter glycosylation profiles is unknown. Herein, we utilized matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to define the N-linked glycome of MYCN-amp NBs in situ identifying enrichment of core fucosylated glycans. GDP-mannose 4,6-dehydratase (GMDS) is the first enzyme responsible for de novo GDP-fucose synthesis. We hypothesized that GMDS is a key mediator of NB core fucosylation and tumorigenesis. Methods: MALDI-MSI was performed on five MYCN-amp primary NBs and six MYCN non-amplified primary NBs using a timsTOF fleX mass spectrometry imager. Significance analysis of metabolites (SAM) was performed with a q-value &amp;lt;0.05 used to define significance. Kaplan-Meier analysis was performed to determine whether GMDS expression correlated with overall survival using the R2 Platform. GMDS blockade was performed using genetic knockdown and chemical inhibition with 2-fluorofucose (2-FF). Core fucosylated glycan abundance was measured by western blotting, ELISA, and flow cytometry using Aleuria aurantia lectin. MYCN tet-inducible NB cells were used to investigate the interplay of N-MYC and GMDS expression. Chromatin immunoprecipitation (ChIP) and luciferase promoter assay were performed to investigate the direct interplay of N-MYC and the GMDS promoter. Subcutaneous tumor formation was our measure of in vivo tumorigenesis. Results: MALDI-MSI revealed increased expression of eleven N-linked glycans within MYCN-amp NBs, and six of these glycans feature core fucosylation. High GMDS expression is associated with poor overall patient survival (p=3.5 × 10−22) and correlates with MYCN-amp in human NB tumors (p=7.4 × 10−27). Genetic and chemical inhibition of GMDS blocks core fucosylated glycan abundance and secretion, while impeding NB cell growth and adhesion in vitro. Induction and blockade of N-MYC directly correlates with GMDS expression. ChIP and promoter luciferase reporter assay demonstrates direct binding and activation of the GMDS promoter by N-MYC. Importantly, GMDS knockdown blocks NB tumor formation (p&amp;lt;0.01), and 2-FF impedes established tumor progression (p&amp;lt;0.05). Conclusions: This multi-institutional study utilized MALDI-MSI to reveal that core fucosylated glycans are enriched in MYCN-amp NBs. GMDS is a key regulator of NB core fucosylation that is driven by N-MYC. GMDS blockade impairs NB growth in vitro and in vivo. These critical findings identify de novo GDP-fucose production as a metabolic vulnerability that may be exploited in designing novel treatment paradigms for high-risk NBs. Citation Format: Beibei Zhu, Michelle Pitts, Michael Buoncristiani, Lindsay Bryant, Oscar Lopez-Nunez, Juan Gurria, Cameron Shedlock, Roberto Ribas, Shannon Keohane, Jinpeng Liu, Chi Wang, Matt Gentry, Nathan Shelman, Derek Allison, B. Mark Evers, Ramon Sun, Eric J. Rellinger. GMDS is a key driver of MYCN-amplified neuroblastoma core fucosylation and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 133.

Abstract 7088: MALDI-IHC high-plex protein biomarker imaging for fast, non-iterative identification of lung carcinoma subtypes

Abstract Lung cancer is the second most common cancer worldwide, with over 2.2 million new cases in 2020 and is the leading cause of cancer-related deaths worldwide. Most lung cancer (80-85%) is non-small cell (NSCLC), comprising mostly adenocarcinoma and squamous cell carcinoma, while roughly 10-15% of lung cancer is small cell lung cancer (SCLC), also termed neuroendocrine lung cancer. Subtype identification is critical for determining treatment, and with limited samples, it is critical to get the most data possible from each tissue section. NSCLC subtypes can be treated with chemotherapy or targeted drugs, whereas SCLC tends to respond best to a combination of chemotherapy and radiation. While there have been many improvements in the detection and treatment of lung carcinoma, the immuno-histochemical (IHC) confirmation of subtype remains a prolonged and often iterative process. To overcome this limitation, we have developed a new IHC approach which uses antibodies labeled with novel photocleavable mass-tags combined with matrix assisted laser desorption ionization (MALDI) mass spectrometry imaging. The approach, termed MALDI-IHC, provides fast (1 hr/cm2 imaging time) and highly multiplexed IHC (100+-plex) without the need for iterative cycling procedures. Here, we present a 20+-plex panel for the determination of subtype in lung cancer. An antibody panel comprised of established clinical biomarkers was developed to differentiate between the lung carcinoma subtypes and provide potentially prognostic information. This included markers for adenocarcinoma (TTF-1, Napsin A), squamous cell carcinoma (p40, CK5), and small cell lung cancer (CD56, Chromogranin A and Synaptophysin), as well as general carcinoma markers for t cells, b cells, macrophages, and fibroblasts These markers were first validated individually by immunofluorescence and then compared to MALDI-IHC on annotated tissues. Here we present an overview of the antibody validation for the 20+-plex lung carcinoma panel. Biomarker presence and peak signal intensity for the various subtypes was then determined using pathologist classified tissues for normal lung, adenocarcinoma, squamous cell carcinoma and small cell lung cancer. We report application of the MALDI-IHC approach to the simultaneous determination of lung carcinoma subtypes on a 99-core human lung tissue microarray (TMA) comprised of normal, normal adjacent, cancer adjacent, and cancerous tissues. Moreover, because MALDI-IHC is a non-destructive process, it can be followed by conventional H&amp;E staining on the same tissue section for accurate co-registration of traditional pathology annotations with the highly multiplexed MALDI-IHC results. In the future, even larger, 100+-plex antibody panels can be used for precision medicine on a wide range of cancers, in order to determine the molecular subtypes and guide treatment paths. Citation Format: Catherine A. Kita, Kristina Schwamborn, Gargey Yagnik, Kenneth J. Rothschild, Mark J. Lim. MALDI-IHC high-plex protein biomarker imaging for fast, non-iterative identification of lung carcinoma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7088.

Abstract 4436: Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics

Abstract Introduction: The high chemoresistance of pancreatic ductal adenocarcinoma (PDAC) is partly attributed to the phenotypic plasticity and high adaptability of PDAC cells to the challenges of the harsh microenviroment. Phenotypic presentation of the PDAC cell is also determined by the lipid composition of the plasma membrane and lipid species that organize the rigid or more flexible lipid bilayer and determine the cellular mobility and plasticity. Lipids are also important energy and signaling molecules that contribute to the aggressiveness of the cell. Here we focus on differences in lipid profile of PDAC cells in epithelial and mesenchymal cell state using single-cell lipidomics approach (SpaceM). Methods: Lipid profile of 2D cultures of eight conventional and ten primary patient derived cell lines of different source (patient derived xenograft, ascites, primary tumor, organoid derived) was investigated. Epithelial (E) or mesenchymal (M) cell state was evaluated by gene and protein expression of EMT markers. Matrix-Assisted Laser Desorption/Ionization (MALDI)-imaging MS was used for data acquisition on positive ion mode at 25 µm step size and m/z range of 600-1000. For annotating lipids in MALDI-imaging data, we used the METASPACE cloud software. For constructing single-cell lipidomic profiles we used the SpaceM method. In total, around 150.000 cells were analyzed and 1400 lipid ions detected. Results: Gene expression analysis suggested prominent metabolism of structural lipids in the conventional epithelial cells. In UMAP analysis of single cell lipidomics data, each cell line, conventional and primary, presented a unique lipid fingerprint with however prominent clustering into the two phenotypes, epithelial and mesenchymal. We detected enrichment of alkyl-acyl phosphatidylcholines, alkyl-acyl phospatidylethanolamines, ceramides and sphingolipids in the epithelial phenotype. Mesenchymal phenotype was enriched for lipids involved in lateral diffusion and low bilayer thickness supporting the increased fluidity of the mesenchymal cell membrane. Furthermore, mesenchymal phenotype was enriched for lipid droplets and energy storing triacylglycerols, also confirmed by histological lipid droplet staining. Conclusions: Using the single cell lipid information, a successful grouping to epithelial or mesenchymal subtype could be performed regardless of sample source, suggesting a conserved lipid profile typical for the two states. Further detailed investigation of single cell lipidomics data aims to unveil the prominent epithelial-mesenchymal state lipid markers and open a niche for functional investigations of cell-state specific markers and potential cell-state targeting. Citation Format: Marija Trajkovic-Arsic, Jeany Delafiori, Mohammed Shahraz, Corinna Münch, Smiths S. Lueong, Sven T. Liffers, Doris Hellerschmied, Theodore Alexandrov, Jens T. Siveke. Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4436.