Metabolic imaging across scales reveals distinct prostate cancer phenotypes.

Hyperpolarised magnetic resonance imaging (HP-13C-MRI) has shown promise as a clinical tool for detecting and characterising prostate cancer. Here we use a range of spatially resolved histological techniques to identify the biological mechanisms underpinning differential [1-13C]lactate labelling between benign and malignant prostate, as well as in tumours containing cribriform and non-cribriform Gleason pattern 4 disease. Here we show that elevated hyperpolarised [1-13C]lactate signal in prostate cancer compared to the benign prostate is primarily driven by increased tumour epithelial cell density and vascularity, rather than differences in epithelial lactate concentration between tumour and normal. We also demonstrate that some tumours of the cribriform subtype may lack [1-13C]lactate labelling, which is explained by lower epithelial lactate dehydrogenase expression, higher mitochondrial pyruvate carrier density, and increased lipid abundance compared to lactate-rich non-cribriform lesions. These findings highlight the potential of combining spatial metabolic imaging tools across scales to identify clinically significant metabolic phenotypes in prostate cancer.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

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.

Multimodal Mass Spectrometry Imaging of an Aggregated 3D Cell Culture Model.

Multimodal mass spectrometry imaging (MSI) is a leading approach for investigating the molecular processes within biological samples. The parallel detection of compounds including metabolites, lipids, proteins, and metal isotopes allows for a more holistic understanding of tissue microenvironments. Universal sample preparation is a primary enabler for samples of the same set to be run across multiple modalities. Using the same method and materials for a cohort of samples reduces any potential variability during sample preparation and allows for comparable analysis across analytical imaging techniques. Here, the MSI workflow is describing a sample preparation protocol for the analysis of three-dimensional (3D) cell culture models. The analysis of biologically relevant cultures by multimodal MSI offers a method in which models of cancer and disease can be studied for the use in early-stage drug development.

Comparison of Osteosarcoma Aggregated Tumour Models with Human Tissue by Multimodal Mass Spectrometry Imaging.

Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade.

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 µm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.

Role of MALDI-MSI in combination with 3D tissue models for early stage efficacy and safety testing of drugs and toxicants.

Introduction: Three-dimensional (3D) cell cultures have become increasingly important materials to investigate biological processes and drug efficacy and toxicity. The ability of 3D cultures to mimic the physiology of primary tissues and organs in the human body enables further insight into cellular behavior and is hence highly desirable in early-stage drug development. Analyzing the spatial distribution of drug compounds and endogenous molecules provides an insight into the efficacy of a drug whilst simultaneously giving information on biological responses. Areas Covered: In this review we will examine the main 3D cell culture systems employed and applications, which describe their integration with mass spectrometry imaging (MSI). Expert Opinion: MSI is a powerful technique that can map a vast range of molecules simultaneously in tissues without the addition of labels that can provide insights into the efficacy and safety of a new drug. The combination of MSI and 3D cell cultures has emerged as a promising tool in early-stage drug analysis. However, the most common administration route for pharmaceutical drugs is via oral delivery. The use of MSI in combination with models of the GI tract is an area that has been little explored to date, the reasons for this are discussed.

Mass Spectrometry Imaging of 3D Tissue Models.

A 3D cell culture is an artificially created environment in which cells are permitted to grow/interact with their surroundings in all three dimensions. Derived from 3D cell culture, organoids are generally small-scale constructs of cells that are fabricated in the laboratory to serve as 3D representations of in vivo tissues and organs. Due to regulatory, economic and societal issues concerning the use of animals in scientific research, it seems clear that the use of 3D cell culture and organoids in for example early stage studies of drug efficacy and toxicity will increase. The combination of such 3D tissue models with mass spectrometry imaging provides a label-free methodology for the study of drug absorption/penetration, drug efficacy/toxicity, and drug biotransformation. In this article, some of the successes achieved to date and challenges to be overcome before this methodology is more widely adopted are discussed.

A comparative study on the raft chemical properties of various alginate antacid raft-forming products.

OBJECTIVE: Research to measure the chemical characterization of alginate rafts for good raft performance and ascertain how formulation can affect chemical parameters. SIGNIFICANCE: A selection of alginate formulations was investigated all claiming to be proficient raft formers with significance between products established and ranked. METHODS: Procedures were selected which demonstrated the chemical characterization allowing rafts to effectively impede the reflux into the esophagus or in severe cases to be refluxed preferentially into the esophagus and exert a demulcent effect, with focus of current research on methods which complement previous studies centered on physical properties. The alginate content was analyzed by a newly developed HPLC method. Methods were used to determine the neutralization profile and the acid neutralization within the raft determined along with how raft structure affects neutralization. RESULTS: Alginate content of Gaviscon Double Action (GDA) within the raft was significantly superior (p < .0001) to all competitor products. The two products with the highest raft acid neutralization capacity were GDA and Rennie Duo, the latter product not being a raft former. Raft structure was key and GDA had the right level of porosity to allow for longer duration of neutralization. CONCLUSION: Alginate formulations require three chemical reactions to take place simultaneously: transformation to alginic acid, sodium carbonate reacting to form carbon dioxide, calcium releasing free calcium ions to bind with alginic acid providing strength to raft formation. GDA was significantly superior (p <.0001) to all other comparators.