PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples

Noberini et al. Clinical Epigenetics (2017) 9:69 DOI 10.1186/s13148-017-0369-8 METHODOLOGY Open Access PAT-H-MS coupled with laser microdissection to study histone posttranslational modifications in selected cell populations from pathology samples Roberta Noberini1*, Rémi Longuespée2, Cristina Richichi3, Giancarlo Pruneri4,5, Mark Kriegsmann2, Giuliana Pelicci3,6 and Tiziana Bonaldi3* Abstract Background: Aberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could not only represent useful biomarkers but also suggest possible targetable epigenetic mechanisms. We have recently developed an approach, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS), that allows performing a comprehensive and quantitative analysis of histone PTMs from formalin-fixed paraffin-embedded pathology samples. Despite its great potential, the application of this technique is limited by tissue heterogeneity. Methods: In this study, we further implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD). Results: When applied to the analysis of a small set of breast cancer samples, LMD-PAT-H-MS allowed detecting more marked changes between luminal A-like and triple negative patients as compared with the classical approach. These changes included not only the already known H3 K27me3 and K9me3 marks, but also H3 K36me1, which was found increased in triple negative samples and validated on a larger cohort of patients, and could represent a potential novel marker distinguishing breast cancer subtypes. Conclusions: These results show the feasibility of applying techniques to reduce sample heterogeneity, including laser microdissection, to the PAT-H-MS protocol, providing new tools in clinical epigenetics and opening new avenues for the comprehensive analysis of histone post-translational modifications in selected cell populations. Keywords: Histone post-translational modifications, PAT-H-MS, Laser microdissection, Proteomics, Epigenetic marker, Formalin-fixed paraffin embedded, Mass spectrometry Background Histone post-translational modifications (hPTMs) generate a complex combinatorial code that is crucial to regulate gene expression and determine cell fate [1]. Increasing evidence has linked aberrations in hPTMs with various pathologies, including cancer, suggesting * Correspondence: ; 1 Center for Genomic Science of IIT@ SEMM, Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milan, Italy 3 Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy Full list of author information is available at the end of the article that they could represent useful biomarkers for patient stratification. Indeed, after the landmark discoveries of the loss of H4-lysine 16 acetylation (H4K16ac) and H4lysine 20 trimethylation (H4K20me3) in cancer [2], and of the prognostic value of hPTMs in various types of cancers [3, 4], many more histone marks have been recognized as possible biomarkers in different diseases, and particularly in cancer [5]. In addition, because changes in hPTM levels are usually the result of the aberrant expression or mislocalization of histone modifying enzymes [6], profiling histone modification in disease © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Noberini et al. Clinical Epigenetics (2017) 9:69 could not only help uncover possible epigenetic mechanisms underlying different pathologies but also provide novel epigenetic pathways targetable for therapy. Indeed, since epigenetic changes, unlike genetic ones, are intrinsically reversible and can be overturned, epigenetic therapies are a promising avenue in translational research. In recent years mass spectrometry (MS) has emerged as a powerful method to analyze hPTMs, thanks to its accuracy, its unbiased nature, and its ability to accurately quantify modifications and their combinations [7], which represent important advantages over traditional antibody basedmethods. However, the potential offered by the MS-based analysis of hPTMs in clinical cancer samples has been left largely unexploited. Indeed, most of the studies employing MS-based techniques, which can provide a comprehensive and quantitative view on hPTM patters, have focused on cell lines and animal tissue, while clinical samples are usually tested through antibody-based techniques. Recently, we have reported for the first time a method that allows the MS-based analysis of hPTMs from human pathology tissues, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS) [8], which combines protocols used for global proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues [9] with a proteomic workflow optimized for hPTM analysis [10]. By using this method, we revealed significant changes in histone H3 methylation patterns between luminal A-like and triple negative breast cancer subtypes. By combining the power of the MS-based analysis of hPTMs with the enormous amount of clinical information contained in FFPE archives, which represent the storage method of choice for clinical specimens, PAT-H-MS is a significant technological advancement in clinical epigenetics. However, a limitation of this approach, which is shared by any other application in which FFPE sections are used as starting material, is tissue heterogeneity. This is a particular issue for tumor specimens, which often contain nontumoral cells that can mask or reduce the contribution of the tumor cells. In this study, we implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD), and show the potential of LMD-PAT-H-MS by applying it to a small panel of breast cancer luminal A-like and triple negative samples. Methods Collection of specimens and preparation of FFPE tissues Spleen tissue was collected from a leukemic mouse with splenomegaly, washed in PBS and incubated for 16 h at room temperature in a 4% paraformaldehyde solution. The fixed spleen was then routinely dehydrated with increasing concentrations of ethanol (70, 80, 90, and P (...truncated)