Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows.

The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.

Accelerating Drug Development Using Spatial Multi-omics.

SUMMARY: Spatial biology approaches enabled by innovations in imaging biomarker platforms and artificial intelligence-enabled data integration and analysis provide an assessment of patient and disease heterogeneity at ever-increasing resolution. The utility of spatial biology data in accelerating drug programs, however, requires balancing exploratory discovery investigations against scalable and clinically applicable spatial biomarker analysis.

Vitamin B5 supports MYC oncogenic metabolism and tumor progression in breast cancer.

Tumors are intrinsically heterogeneous and it is well established that this directs their evolution, hinders their classification and frustrates therapy1-3. Consequently, spatially resolved omics-level analyses are gaining traction4-9. Despite considerable therapeutic interest, tumor metabolism has been lagging behind this development and there is a paucity of data regarding its spatial organization. To address this shortcoming, we set out to study the local metabolic effects of the oncogene c-MYC, a pleiotropic transcription factor that accumulates with tumor progression and influences metabolism10,11. Through correlative mass spectrometry imaging, we show that pantothenic acid (vitamin B5) associates with MYC-high areas within both human and murine mammary tumors, where its conversion to coenzyme A fuels Krebs cycle activity. Mechanistically, we show that this is accomplished by MYC-mediated upregulation of its multivitamin transporter SLC5A6. Notably, we show that SLC5A6 over-expression alone can induce increased cell growth and a shift toward biosynthesis, whereas conversely, dietary restriction of pantothenic acid leads to a reversal of many MYC-mediated metabolic changes and results in hampered tumor growth. Our work thus establishes the availability of vitamins and cofactors as a potential bottleneck in tumor progression, which can be exploited therapeutically. Overall, we show that a spatial understanding of local metabolism facilitates the identification of clinically relevant, tractable metabolic targets.

Imaging tumor lactate is feasible for identifying intermediate-risk prostate cancer patients with postsurgical biochemical recurrence.

While radical prostatectomy remains the mainstay of prostate cancer (PCa) treatment, 20 to 40% of patients develop postsurgical biochemical recurrence (BCR). A particularly challenging clinical cohort includes patients with intermediate-risk disease whose risk stratification would benefit from advanced approaches that complement standard-of-care diagnostic tools. Here, we show that imaging tumor lactate using hyperpolarized 13C MRI and spatial metabolomics identifies BCR-positive patients in two prospective intermediate-risk surgical cohorts. Supported by spatially resolved tissue analysis of established glycolytic biomarkers, this study provides the rationale for multicenter trials of tumor metabolic imaging as an auxiliary tool to support PCa treatment decision-making.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models.

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB- T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB- resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

Targeted Desorption Electrospray Ionization Mass Spectrometry Imaging for Drug Distribution, Toxicity, and Tissue Classification Studies.

With increased use of mass spectrometry imaging (MSI) in support of pharmaceutical research and development, there are opportunities to develop analytical pipelines that incorporate exploratory high-performance analysis with higher capacity and faster targeted MSI. Therefore, to enable faster MSI data acquisition we present analyte-targeted desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) utilizing a triple-quadrupole (TQ) mass analyzer. The evaluated platform configuration provided superior sensitivity compared to a conventional time-of-flight (TOF) mass analyzer and thus holds the potential to generate data applicable to pharmaceutical research and development. The platform was successfully operated with sampling rates up to 10 scans/s, comparing positively to the 1 scan/s commonly used on comparable DESI-TOF setups. The higher scan rate enabled investigation of the desorption/ionization processes of endogenous lipid species such as phosphatidylcholines and a co-administered cassette of four orally dosed drugs-erlotininb, moxifloxacin, olanzapine, and terfenadine. This was used to enable understanding of the impact of the desorption/ionization processes in order to optimize the operational parameters, resulting in improved compound coverage for olanzapine and the main olanzapine metabolite, hydroxy-olanzapine, in brain tissue sections compared to DESI-TOF analysis or matrix-assisted laser desorption/ionization (MALDI) platforms. The approach allowed reducing the amount of recorded information, thus reducing the size of datasets from up to 150 GB per experiment down to several hundred MB. The improved performance was demonstrated in case studies investigating the suitability of this approach for mapping drug distribution, spatially resolved profiling of drug-induced nephrotoxicity, and molecular-histological tissue classification of ovarian tumors specimens.

Brain metastatic outgrowth and osimertinib resistance are potentiated by RhoA in EGFR-mutant lung cancer.

The brain is a major sanctuary site for metastatic cancer cells that evade systemic therapies. Through pre-clinical pharmacological, biological, and molecular studies, we characterize the functional link between drug resistance and central nervous system (CNS) relapse in Epidermal Growth Factor Receptor- (EGFR-) mutant non-small cell lung cancer, which can progress in the brain when treated with the CNS-penetrant EGFR inhibitor osimertinib. Despite widespread osimertinib distribution in vivo, the brain microvascular tumor microenvironment (TME) is associated with the persistence of malignant cell sub-populations, which are poised to proliferate in the brain as osimertinib-resistant lesions over time. Cellular and molecular features of this poised state are regulated through a Ras homolog family member A (RhoA) and Serum Responsive Factor (SRF) gene expression program. RhoA potentiates the outgrowth of disseminated tumor cells on osimertinib treatment, preferentially in response to extracellular laminin and in the brain. Thus, we identify pre-existing and adaptive features of metastatic and drug-resistant cancer cells, which are enhanced by RhoA/SRF signaling and the brain TME during the evolution of osimertinib-resistant disease.

Evaluation of Formalin-Fixed and FFPE Tissues for Spatially Resolved Metabolomics and Drug Distribution Studies.

Fixation of samples is broadly used prior to the histological evaluation of tissue samples. Though recent reports demonstrated the ability to use fixed tissues for mass spectrometry imaging (MSI) based proteomics, glycomics and tumor classification studies, to date comprehensive evaluation of fixation-related effects for spatially resolved metabolomics and drug disposition studies is still missing. In this study we used matrix assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) MSI to investigate the effect of formalin-fixation and formalin-fixation combined with paraffin embedding on the detectable metabolome including xenobiotics. Formalin fixation was found to cause significant washout of polar molecular species, including inorganic salts, amino acids, organic acids and carnitine species, oxidation of endogenous lipids and formation of reaction products between lipids and fixative ingredients. The slow fixation kinetics under ambient conditions resulted in increased lipid hydrolysis in the tissue core, correlating with the time-dependent progression of the fixation. Paraffin embedding resulted in subsequent partial removal of structural lipids resulting in the distortion of the elucidated biodistributions.

Exploring New Methods to Study and Moderate Proton Beam Damage for Multimodal Imaging on a Single Tissue Section.

Characterizing proton beam damage in biological materials is of interest to enable the integration of proton microprobe elemental mapping techniques with other imaging modalities. It is also of relevance to obtain a deeper understanding of mechanical damage to lipids in tissues during proton beam cancer therapy. We have developed a novel strategy to characterize proton beam damage to lipids in biological tissues based on mass spectrometry imaging. This methodology is applied to characterize changes to lipids in tissues ex vivo, irradiated under different conditions designed to mitigate beam damage. This work shows that performing proton beam irradiation at ambient pressure, as well as including the application of an organic matrix prior to irradiation, can reduce damage to lipids in tissues. We also discovered that, irrespective of proton beam irradiation, placing a sample in a vacuum prior to desorption electrospray ionization imaging can enhance lipid signals, a conclusion that may be of future benefit to the mass spectrometry imaging community.

Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury.

BACKGROUND: Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored. METHODS: C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining. RESULTS: Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate. CONCLUSIONS: Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.

Correction: Brüning-Richardson et al. GSK-3 Inhibition Is Cytotoxic in Glioma Stem Cells through Centrosome Destabilization and Enhances the Effect of Radiotherapy in Orthotopic Models. Cancers 2021, 13, 5939.

The authors wish to make the following corrections to this paper [1][...].

Atmospheric-Pressure Infrared Laser-Ablation Plasma-Postionization Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded (FFPE) and Fresh-Frozen Tissue Sections with No Sample Preparation.

Mass spectrometry imaging (MSI) encompasses a powerful suit of techniques which provide spatially resolved atomic and molecular information from almost any sample type. MSI is now widely used in preclinical research to provide insight into metabolic phenotypes of disease. Typically, fresh-frozen tissue preparations are considered optimal for biological MSI and other traditional preservation methods such as formalin fixation, alone or with paraffin embedding (FFPE), are considered less optimal or even incompatible. Due to the prevalence of FFPE tissue storage, particularly for rare and therefore high-value tissue samples, there is substantial motivation for optimizing MSI methods for analysis of FFPE tissue. Here, we present a novel modality, atmospheric-pressure infrared laser-ablation plasma postionization (AP-IR-LA-PPI), with the first proof-of-concept examples of MSI for FFPE and fresh-frozen tissues, with no post-sectioning sample preparation. We present ion images from FFPE and fresh tissues in positive and negative ion modes. Molecular annotations (via the Metaspace annotation engine) and on-tissue MS/MS provide additional confidence that the detected ions arise from a broad range of metabolite and lipid classes from both FFPE and fresh-frozen tissues.

Mass Spectrometry Detection and Imaging of a Non-Covalent Protein-Drug Complex in Tissue from Orally Dosed Rats.

Here, we demonstrate detection by mass spectrometry of an intact protein-drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein-drug complex comprised fatty acid binding protein 1, FABP1, non-covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non-covalent protein-drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target-drug characterization directly from the physiological environment.

Quantitative Characterization of Three Carbonic Anhydrase Inhibitors by LESA Mass Spectrometry.

Liquid extraction surface analysis (LESA) coupled to native mass spectrometry (MS) presents unique analytical opportunities due to its sensitivity, speed, and automation. Here, we examine whether this tool can be used to quantitatively probe protein-ligand interactions through calculation of equilibrium dissociation constants (Kd values). We performed native LESA MS analyses for a well-characterized system comprising bovine carbonic anhydrase II and the ligands chlorothiazide, dansylamide, and sulfanilamide, and compared the results with those obtained from direct infusion mass spectrometry and surface plasmon resonance measurements. Two LESA approaches were considered: In one approach, the protein and ligand were premixed in solution before being deposited and dried onto a solid substrate for LESA sampling, and in the second, the protein alone was dried onto the substrate and the ligand was included in the LESA sampling solvent. Good agreement was found between the Kd values derived from direct infusion MS and LESA MS when the protein and ligand were premixed; however, Kd values determined from LESA MS measurements where the ligand was in the sampling solvent were inconsistent. Our results suggest that LESA MS is a suitable tool for quantitative analysis of protein-ligand interactions when the dried sample comprises both protein and ligand.

Mapping the Influence of the Gut Microbiota on Small Molecules across the Microbiome Gut Brain Axis.

Microbes exert influence across the microbiome-gut-brain axis through neurotransmitter production, induction of host immunomodulators, or the release or induction of other microbial or host molecules. Here, we used mass spectrometry imaging (MSI), a label-free imaging tool, to map molecular changes in the gut and brain in germ-free, antibiotic-treated and control mice. We determined spatial distribution and relative quantification of neurotransmitters and their precursors in response to the microbiome. Using untargeted MSI, we detected a significant change in the levels of four identified small molecules in the brains of germ-free animals compared to controls. However, antibiotic treatment induced no significant changes in these same metabolites in the brain after 1 week of treatment. This work exemplifies the utility of MSI as a tool for the study of known and discovery of novel, mediators of microbiome-gut-brain axis communication.

Correlating Mass Spectrometry Imaging and Liquid Chromatography-Tandem Mass Spectrometry for Tissue-Based Pharmacokinetic Studies.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a standard tool used for absolute quantification of drugs in pharmacokinetic (PK) studies. However, all spatial information is lost during the extraction and elucidation of a drugs biodistribution within the tissue is impossible. In the study presented here we used a sample embedding protocol optimized for mass spectrometry imaging (MSI) to prepare up to 15 rat intestine specimens at once. Desorption electrospray ionization (DESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) were employed to determine the distributions and relative abundances of four benchmarking compounds in the intestinal segments. High resolution MALDI-MSI experiments performed at 10 µm spatial resolution allowed to determine the drug distribution in the different intestinal histological compartments to determine the absorbed and tissue bound fractions of the drugs. The low tissue bound drug fractions, which were determined to account for 56-66% of the total drug, highlight the importance to understand the spatial distribution of drugs within the histological compartments of a given tissue to rationalize concentration differences found in PK studies. The mean drug abundances of four benchmark compounds determined by MSI were correlated with the absolute drug concentrations. Linear regression resulted in coefficients of determination (R2) ranging from 0.532 to 0.926 for MALDI-MSI and R2 values ranging from 0.585 to 0.945 for DESI-MSI, validating a quantitative relation of the imaging data. The good correlation of the absolute tissue concentrations of the benchmark compounds and the MSI data provides a bases for relative quantification of compounds within and between tissues, without normalization to an isotopically labelled standard, provided that the compared tissues have inherently similar ion suppression effects.