Tissue analysis workflow by mass spectrometry imaging
Mass spectrometry imaging (MSI) is a unique analytical technique enabling a qualitative and quantitative label-free MS analysis of multiple biomolecules in the context of tissue morphology. The preparation procedure of tissue specimens requires sectioning by microtome (FFPE) or cryostat (fresh frozen) followed by mounting the tissue section onto a conductive MSI compatible carrier. We are preferably using transparent indium-tin oxide (ITO) coated glass slides which allow us to perform morphological assessment of the analyzed tissue after MSI, e.g. by optical microscopy.
During an MSI experiment, the tissue section is raster scanned in two dimensions (x & y axes) with a laser to create a mass spectrum at each measurement position. The MS dataset with the associated information about the localization of each mass spectrum on the investigated sample surface is computationally integrated post-acquisition to generate two-dimensional ion intensity maps.
The classical morphological assessment of tissues and cells is an important step towards a precise categorization of molecular changes at the level of individual tissue and cell types. Routine hematoxylin and eosin staining or other special stains play a critical role in our MSI analyses workflow. For instance, pancreatic alpha and beta cells are histologically indistinguishable, but they fulfill different functions with regard to glucose metabolism. The immunohistochemical staining of alpha and beta cells with glucagon and insulin allows comprehensive and spatially resolved in situ analyses of metabolic profiles on basis of cellular features.
Another clinically relevant example for the use of immunohistochemical stainings for tissue annotation are pan-cytokeratine as an epithelial marker to first annotate potential tumor cells and HER2/neu stainings to then identify HER2/neu postive and negative tumor regions (images on the left) – an information important for optimal therapy choice.
Our approach allows pixel-accurate, objective, semantic-based and functional tissue analyses. The use of morphological image information together with mass spectrometry can help to illuminate underlying phenotypic mechanisms to expand our knowledge of cell function or new therapeutic strategies.
Tissue microarrays permit a simultaneous and comparative analysis of tissue samples from hundreds to thousands of patients. We can use clinical data and state-of-the-art machine learning algorithms to identify biomarkers that help us discriminate between patients with different pathophysiological conditions. Using the molecular signatures, we can predict the impact of each molecule on prognosis or survival and can even suggest the treatment with optimal chances of success.
The use of high-resolution MSI enables the in situ detection and spatial imaging of hundreds to thousands of metabolites with minimum demands on FFPE tissue sample volumes. The new access to metabolic information, an application first published by the group of Prof. Axel Walch (Buck et al., J. Pathol. 2015; Ly et al., Nat. Protoc. 2016) offers enormous unexploited potential for tissue-based research and molecular diagnostics.
Showcase of a MALDI imaged adrenal gland
The in situ imaging of biomolecules and hormones by mass spectrometry imaging allows a more detailed and novel view of adrenal cortical zonation and its function in comparison with classical histological definition. The adrenal glands are two small organs, one located on the top of each kidney. They are part of the endocrine system, a collection of glands that secrete hormones directly into the circulatory system. The adrenal gland is composed of the outer cortex and the inner medulla as main parts. The adrenal cortex is the largest part of an adrenal gland. The cortex itself is divided into three separate zones: zona glomerulosa, zona fasciculata and zona reticularis. Each zone is responsible for producing specific hormones. The other part is the adrenal medulla which is located inside the adrenal cortex in the center of an adrenal gland. It produces “stress hormones,” including adrenaline and noradrenaline. The adrenal cortex and adrenal medulla are enveloped in an adipose capsule that forms a protective layer around an adrenal gland.
Using MSI, the in situ distribution of endogenous metabolites such as catecholamines, sterol and steroid metabolites, nucleotide derivatives, intermediates of glycolysis and tricarboxylic acid cycle, lipids and fatty acids can be visualized and subjected to pathway analyses.
The hormones produced by the adrenal glands help to regulate metabolism, immune system, blood pressure, response to stress and other essential functions. Disorders of the adrenal gland can involve the secretion of too little or too much hormone. A number of endocrine diseases involve dysfunctions of the adrenal gland:
- Cushing syndrome (oversecretion of glucocorticoids)
- Hyperaldosteronism (oversecretion of aldosterone)
- Pheochromocytoma (oversecretion of epinephrine and norepinephrine)
- Virilization (oversecretion of androgens)