Comprehensive analysis of histone related modifications of formaldehyde fixed paraffin embedded and fresh frozen pancreatic tumor xenografts using LC-MS/MS

Background

Post translational modifications (PTMs) of histones including acetylation, methylation or ubiquitination are known to be involved in the epigenetic regulation of gene expression and thus can play an important role in tumorigenesis. Some PTMs have been linked to pancreatic cancer and are frequently studied as a potential target for cancer therapy. Tissue samples can be stored as formaldehyde fixed paraffin embedded (FFPE) blocks or fresh frozen (FF). It is however important to consider that the treatment with formaldehyde may induce a variety of chemical modifications in the proteins. The aim of this study is to evaluate whether the FFPE tissue processing induced chemical modifications on the histone proteins originating from pancreatic tumor xenografts.

Methods

Tissues were obtained from human pancreatic tumor xenografts developed from inoculated human pancreatic cancer cell line, capan-1. This study comprised two sample cohorts, including nine FFPE or FF samples, respectively. Peptides obtained after protein extraction, reduction, alkylation, and digestion were separated by capillary HPLC with a 150 min 5-40% acetonitrile in 0.10 % FA gradient and identified by quadrupole-Orbitrap mass spectrometry.

Results

In total sixteen individual modification sites located on lysine residues have been characterized in FFPE samples compared to five matched modification sites identified in FF samples. We speculate, based on the obtained results that only the three equivalent modifications found uniformly in both groups, identified as H1.1K55me2, H1.2K34me and H3K80me together with H3K80me2 and H3K24Ac detected only in FF samples might be of biological origin. All the remaining identified modifications occurring exclusively in FFPE material were considered as PTM artifacts resulting from processing.

Conclusions

Our results indicate that FFPE tissue processing can induce chemical modifications on lysine residues of histones originating from pancreatic tumor xenografts. A current experiment about the analysis of histone related PTMs between patient and control pancreatic cancer FF tissues should contribute to the discovery of novel biomarkers.

Clinical trial identification

Legal entity responsible for the study

Lund University Medical Faculty

Funding

Lund University Medical Faculty

Disclosure

All authors have declared no conflicts of interest.