Targeted imaging of integrins in cancer tissues using photocleavable Ru(ii) polypyridine complexes as mass-tags

Targeted epitope-based mass spectrometry imaging (MSI) utilizes laser cleavable mass-tags bound to targeting moieties for detecting proteins in tissue sections. Our work constitutes the first proof-of-concept of a novel laser desorption ionization (LDI)-MSI strategy using photocleavable Ru(ii) polypyridine complexes as mass-tags for imaging of integrins avß3 in human cancer tissues

Multiple Statistical Analysis Techniques Corroborate Intratumor Heterogeneity in Imaging Mass Spectrometry Datasets of Myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information

miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility

New methods for multimodal MS imaging of histological tissue sections

The insights derived from spatial localization of molecules in tissue sections are of great value for understanding and treating cancer and other diseases. These insights can relate to molecules linked to a disease as well as to drug molecules distributed across organs of interest. Mass spectrometry (MS) imaging can not only provide the molecular and spatial information of single molecules, but can do so for a broad range of substances, from elements to proteins. In one single measurement, the impact of these molecules in multiple physiological and pathological pathways can be analyzed. The value of MS imaging for cancer research is demonstrated by the insights in tumor malignancy generated with MS imaging in this research. Two breast cancer xenograft models, the nonmetastatic MCF-7 and highly metastatic MDA-MB-231 tumors, have been studied with MS imaging in combination with magnetic resonance spectroscopic imaging (MRSI). This study focused on the distribution of phosphocholine (PC), total choline (tCho), free choline (Cho), sodium and potassium in the two different tumors. In vivo 3-dimensional MRSI of MDA-MB-231 tumors demonstrated that high tCho levels (Cho, glycerophosphocholine (GPC) and PC), displayed a heterogeneous spatial distribution in the tumor. MS imaging data provided information about each individual molecule, with increased PC and Cho in viable tumor regions compared to necrotic regions in the highly metastatic MDA-MB-231 tumor type. The same molecules were homogeneously distributed in the nonmetastatic MCF-7 tumor type. Also, significant differences in the spatial distribution of Na+ and K+ in the these two tumor types suggested differential Na+/K+ pump functions and K+ channel expressions. The combined results provide a link to the impact of multiple pathways in tumor malignancy in one single study. In drug discovery research, it is important to know not only in which organs drug and drug metabolites can be found after drug administration, but also how much of drug and metabolites are present in each organ. However, quantification by MS imaging is challenging, with several factors affecting the results of quantitation of drugs in tissue sections. Therefore an experimental setting was specially designed to address the impact of several factors on drug quantitation, such as ion suppression, sample preparation and organ dependence on the mass spectrometric response of the drug using organ homogenates and drugs. Because sample handling is of such importance to the success of MS imaging experiments, a new method was developed for fragile tissue sections and localized washing, which consists of a wetted paper with the solvent of choice applied directly onto the biological tissue surface. The washing solution is spatially contained by the paper structure, thus the formation of a liquid film on the tissue surface is avoided. An additional advantage is that the paper used in the washing step can also be analyzed by MS imaging, providing additional information of components removed from the tissue surface while retaining their spatial organization

Isocyanate-Free Polyurea Synthesis via Ru-Catalyzed Carbene Insertion into the N-H Bonds of Urea

Polyureas have widespread applications due to their unique material properties. Because of the toxicity of isocyanates, sustainable isocyanate-free routes to prepare polyureas are a field of active research. Current routes to isocyanate-free polyureas focus on constructing the urea moiety in the final polymerizing step. In this study we present a new isocyanate-free method to produce polyureas by Ru-catalyzed carbene insertion into the N-H bonds of urea itself in combination with a series of bis-diazo compounds as carbene precursors. The mechanism was investigated by kinetics and DFT studies, revealing the rate-determining step to be nucleophilic attack on a Ru-carbene moiety by urea. This study paves the way to use transition-metal-catalyzed reactions in alternative routes to polyureas

Fiducial Markers for Combined 3-Dimensional Mass Spectrometric and Optical Tissue Imaging

Mass spectrometric imaging (MSI) has become widely used in the analysis of a variety of biological surfaces. Biological samples are spatially, morphologically, and metabolically complex. Multimodal molecular imaging is an emerging approach that is capable of dealing with this complexity. In a multimodal approach, different imaging modalities can provide precise information about the local molecular composition of the surfaces. Images obtained by MSI can be coregistered with images obtained by other molecular imaging techniques such as microscopic images of fluorescent protein expression or histologically stained sections. In order to properly coregister images from different modalities, each tissue section must contain points of reference, which are visible in all data sets. Here, we report a newly developed coregistration technique using fiducial markers such as cresyl violet, Ponceau S, and bromophenol blue that possess a combination of optical and molecular properties that result in a clear mass spectrometric signature. We describe these fiducial markers and demonstrate an application that allows accurate coregistration and 3-dimensional reconstruction of serial histological and fluorescent microscopic images with MSI images of thin tissue sections from a breast tumor model