Tumor classification with MALDI-MSI data of tissue microarrays: A case study.

With mass spectrometry imaging (MSI) on tissue microarrays (TMAs) a large number of biomolecules can be studied for many patients at the same time, making it an attractive tool for biomarker discovery. Here we investigate whether lymph node metastasis can be predicted from MALDI-MSI data. Measurements are performed on TMAs and then filtered based on spectral intensity and the percentage of tumor cells, after which the resulting data for 122 patients is further preprocessed. We assume differences between patients with and without metastasis are expressed in a limited number of features. Two univariate feature selection methods are applied to reduce the dimensionality of the MALDI-MSI data. The selected features are then used in combination with three classifiers. The best classification scores are obtained with a decision tree classifier, which classifies about 72% of patients correctly. Almost all the predictive power comes from a single peak (m/z 718.4). The sensitivity of our classification approach, which can be generically used to search for biomarkers, is investigated using artificially modified data.

Mass Spectrometry Imaging of the Hypoxia Marker Pimonidazole in a Breast Tumor Model.

Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging (MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimonidazole as an exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimonidazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides, and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly colocalized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.

The use of mass spectrometry imaging to predict treatment response of patient-derived xenograft models of triple-negative breast cancer.

In recent years, mass spectrometry imaging (MSI) has been shown to be a promising technique in oncology. The effective application of MSI, however, is hampered by the complexity of the generated data. Bioinformatic approaches that reduce the complexity of these data are needed for the effective use in a (bio)medical setting. This holds especially for the analysis of tissue microarrays (TMA), which consist of hundreds of small tissue cores. Here we present an approach that combines MSI on tissue microarrays with principal component linear discriminant analysis (PCA-LDA) to predict treatment response. The feasibility of such an approach was evaluated on a set of patient-derived xenograft models of triple-negative breast cancer (TNBC). PCA-LDA was used to classify TNBC tumor tissues based on the proteomic information obtained with matrix-assisted laser desorption ionization (MALDI) MSI from the TMA surface. Classifiers based on two different tissue microarrays from the same tumor models showed overall classification accuracies between 59 and 77%, as determined by cross-validation. Reproducibility tests revealed that the two models were similar. A clear effect of intratumor heterogeneity of the classification scores was observed. These results demonstrate that the analysis of MALDI-MSI data by PCA-LDA is a valuable approach for the classification of treatment response and tumor heterogeneity in breast cancer.

A novel taxonomic marker that discriminates between morphologically complex actinomycetes.

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.

A MASSive laboratory tour. An interactive mass spectrometry outreach activity for children.

It is imperative to fascinate young children at an early stage in their education for the analytical sciences. The exposure of the public to mass spectrometry presently increases rapidly through the common media. Outreach activities can take advantage of this exposure and employ mass spectrometry as an exquisite example of an analytical science in which children can be fascinated. The presented teaching modules introduce children to mass spectrometry and give them the opportunity to experience a modern research laboratory. The modules are highly adaptable and can be applied to young children from the age of 6 to 14 y. In an interactive tour, the students explore three major scientific concepts related to mass spectrometry; the building blocks of matter, charged particle manipulation by electrostatic fields, and analyte identification by mass analysis. Also, the students carry out a mass spectrometry experiment and learn to interpret the resulting mass spectra. The multistage, inquiry-based tour contains flexible methods, which teach the students current-day research techniques and possible applications to real research topics. Besides the scientific concepts, laboratory safety and hygiene are stressed and the students are enthused for the analytical sciences by participating in "hands-on" work. The presented modules have repeatedly been successfully employed during laboratory open days. They are also found to be extremely suitable for (early) high school science classes during laboratory visit-focused field trips.