Proteomics Analysis of Formalin Fixed Paraffin Embedded Tissues in the Investigation of Prostate Cancer

Abstract

Prostate cancer (PCa) is one of the leading causes of death in men worldwide. The molecular features, associated 8 with the onset and progression of the disease, are under vigorous investigation. Formalin-fixed paraffin-9 embedded (FFPE) tissues are valuable resources for large-scale studies, however, their application in proteomics 10 is limited due to protein cross-linking. In this study, the adjustment of a protocol for the proteomic analysis of 11 FFPE tissues was performed which was followed by a pilot application on FFPE PCa clinical samples to investigate 12 whether the optimized protocol can provide biologically relevant data for the investigation of PCa. For the 13 optimization, FFPE mouse tissues were processed using eight seven protein extraction protocols including 14 combinations of homogenization methods (beads, sonication, boiling) and buffers (SDS based and Urea-15 Thiourea based). The proteome extraction efficaiency was then evaluated based on protein identifications and 16 reproducibility using SDS electrophoresis and high resolution LC-MS/MS analysis. Comparison between the FFPE 17 and matched fresh frozen (FF) tissues, using an optimized protocol involving protein extraction with an SDS-18 based buffer following beads homogenization and boiling, showed a substantial overlap in protein 19 identifications (1106 common between the 1214 identified in FF and 1249 identified in FFPE) with a strong 20 correlation in relative abundances (r s =0.7820.819, p <0.001). Next, FFPE tissues (3 sections, 15μm each per 21 sample) from 10 patients with PCa corresponding to tumor (GS=6 or GS≥8) and adjacent benign regions were 22 processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical 23 and bioinformatics analysis. Proteins significantly deregulated between PCa GS≥8 and PCa GS=6 represented 24 extracellular matrix organisation, gluconeogenesis and phosphorylation pathways. Proteins deregulated 25 between cancerous tissues and adjacent benign tissues counterparts, showed reflected increased translation, 26 peptide synthesis and protein metabolism in the former, which is consistent to with the literature. In conclusion, 27 the results support the relevance of the proteomic findings in the context of PCa and the reliability of the 28 optimized protocol for proteomics analysis of FFPE material.

Proteomic characterization of primary and metastatic prostate cancer reveals reduced proteinase activity in aggressive tumors

“…In a comparative study of PCa cell lines with PCa tumor tissues, 12 mutant peptides were identified to be differentially expressed in PCa tumor tissue 38 . Similarly, several proteins were also identified to be differentially expressed in subtypes of PCa 39 – 42 . Indeed, the dysregulation of protein levels is also mirrored by alterations of specific genes 43 .…”

Glycosylation: Rising Potential for Prostate Cancer Evaluation

“…Numerous studies have investigated whether a cancer-specific glycosylation pattern on PSA can be used to differentiate between BPH and PCa [75,133]. The most recent research on glycan composition in order to identify PCa and establish a prognosis has been conducted on diverse biological material, including serum [78], urine [77,136], expressed prostatic secretion urine [137], formalin-fixed, paraffin-embedded (FFPE) tissues [138,139], cell lines [124,140], and exosomes [141].…”

Quantitative Proteomics Analysis of FFPE Tumor Samples Reveals the Influences of NET-1 siRNA Nanoparticles and Sonodynamic Therapy on Tetraspanin Protein Involved in HCC

“…Most human tumor samples archived in hospitals for pathologic diagnosis are formalin-fixed paraffin-embedded (FFPE), which have been widely used in the long-term preservation of tissues and organs ( Bass et al, 2014 ). Besides, multifarious prescient technologies have been invented for transcriptomic ( Li et al, 2014 ), genomic ( Martelotto et al, 2017 ; Van Allen et al, 2014 ), proteomic, and protein ( Mantsiou et al, 2019 ; Iglesias-Gato et al, 2016 ) from FFPE samples. In 1991, FFPE tissues have been analyzed for protein using antibodies as the invention of the heat-induced antigen retrieval (HIAR) technique for immunohistochemistry (IHC) ( Shi et al, 1991 ).…”