Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 14, 2021; 27(42): 7324-7339
Published online Nov 14, 2021. doi: 10.3748/wjg.v27.i42.7324
Detailing the ultrastructure’s increase of prion protein in pancreatic adenocarcinoma
Matteo Bianchini, Maria Anita Giambelluca, Maria Concetta Scavuzzo, Gregorio Di Franco, Simone Guadagni, Matteo Palmeri, Niccolò Furbetta, Desirée Gianardi, Niccola Funel, Claudio Ricci, Raffaele Gaeta, Luca Emanuele Pollina, Alfredo Falcone, Caterina Vivaldi, Giulio Di Candio, Francesca Biagioni, Carla Letizia Busceti, Luca Morelli, Francesco Fornai
Matteo Bianchini, Gregorio Di Franco, Simone Guadagni, Matteo Palmeri, Niccolò Furbetta, Desirée Gianardi, Giulio Di Candio, Luca Morelli, General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy
Maria Anita Giambelluca, Maria Concetta Scavuzzo, Francesco Fornai, Human Anatomy, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy
Niccola Funel, Claudio Ricci, Raffaele Gaeta, Luca Emanuele Pollina, Division of Surgical Pathology, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa 56124, Italy
Alfredo Falcone, Caterina Vivaldi, Division of Medical Oncology, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy
Francesca Biagioni, Carla Letizia Busceti, Francesco Fornai, IRCCS Neuromed, Istituto Neurologico Mediterraneo, Pozzilli 86077, Italy
Luca Morelli, EndoCAS (Center for Computer Assisted Surgery), University of Pisa, Pisa 56124, Italy
Author contributions: Bianchini M and Giambelluca MA contributed equally to this work; Morelli L and Fornai F contributed equally to this work; Bianchini M, Giambelluca MA, Scavuzzo MC, Morelli L and Fornai F studied the conception and design; Bianchini M, Giambelluca MA, Scavuzzo MC, Di Franco G, Guadagni S, Palmeri M, Furbetta N, Gianardi D, Funel N, Ricci C, Gaeta R, Pollina LE, Falcone A, Vivaldi C and Di Candio G contributed to data acquisition; Morelli L and Fornai F are responsible for data analysis and interpretation, and revised the manuscript critically; Bianchini M, Giambelluca MA, Scavuzzo MC, Di Franco G, Guadagni S, Palmeri M, Furbetta N, Gianardi D, Funel N, Pollina LE, Falcone A, Vivaldi C, Di Candio C, Biagioni F and Busceti CL drafted the manuscript; all authors approved the final manuscript.
Supported by Tizzi Foundation and Arpa Foundation (www.fondazionearpa.it).
Institutional review board statement: The study was approved by Ethics committee of “Area Vasta Nord Ovest (CEAVNO)”.
Informed consent statement: All patients signed an informed consent to authorize the scientific use of the collected data.
Conflict-of-interest statement: The authors declare that they do not have any conflict of interest.
Data sharing statement: No additional data available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Luca Morelli, MD, Professor, General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via Paradisa 2, Pisa 56124, Italy. luca.morelli@unipi.it
Received: April 27, 2021
Peer-review started: April 27, 2021
First decision: June 13, 2021
Revised: June 14, 2021
Accepted: October 25, 2021
Article in press: October 25, 2021
Published online: November 14, 2021
Processing time: 196 Days and 11.6 Hours
Abstract
BACKGROUND

Recent evidences have shown a relationship between prion protein (PrPc) expression and pancreatic ductal adenocarcinoma (PDAC). Indeed, PrPc could be one of the markers explaining the aggressiveness of this tumor. However, studies investigating the specific compartmentalization of increased PrPc expression within PDAC cells are lacking, as well as a correlation between ultrastructural evidence, ultrastructural morphometry of PrPc protein and clinical data. These data, as well as the quantitative stoichiometry of this protein detected by immuno-gold, provide a significant advancement in understanding the biology of disease and the outcome of surgical resection.

AIM

To analyze quantitative stoichiometry and compartmentalization of PrPc in PDAC cells and to correlate its presence with prognostic data

METHODS

Between June 2018 and December 2020, samples from pancreatic tissues of 45 patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were collected. When the frozen section excluded a PDAC diagnosis, or the nodules were too small for adequate sampling, patients were ruled out from the present study. Western blotting was used to detect, quantify and compare the expression of PrPc in PDAC and control tissues, such as those of non-affected neighboring pancreatic tissue of the same patient. To quantify the increase of PrPc and to detect the subcellular compartmentalization of PrPc within PDAC cells, immuno-gold stoichiometry within specific cell compartments was analyzed with electron microscopy. Finally, an analysis of quantitative PrPc expression according to prognostic data, such as cancer stage, recurrence of the disease at 12 mo after surgery and recurrence during adjuvant chemotherapy was made.

RESULTS

The amount of PrPc within specimen from 38 out of 45 patients was determined by semi-quantitative analysis by using Western blotting, which indicates that PrPc increases almost three-fold in tumor pancreatic tissue compared with healthy pancreatic regions [242.41 ± 28.36 optical density (OD) vs 95 ± 17.40 OD, P < 0.0001]. Quantitative morphometry carried out by using immuno-gold detection at transmission electron microscopy confirms an increased PrPc expression in PDAC ductal cells of all patients and allows to detect a specific compartmentalization of PrPc within tumor cells. In particular, the number of immuno-gold particles of PrPc was significantly higher in PDAC cells respect to controls, when considering the whole cell (19.8 ± 0.79 particles vs 9.44 ± 0.45, P < 0.0001). Remarkably, considering PDAC cells, the increase of PrPc was higher in the nucleus than cytosol of tumor cells, which indicates a shift in PrPc compartmentalization within tumor cells. In fact, the increase of immuno-gold within nuclear compartment exceeds at large the augment of PrPc which was detected in the cytosol (nucleus: 12.88 ± 0.59 particles vs 5.12 ± 0.32, P < 0.0001; cytosol: 7.74. ± 0.44 particles vs 4.3 ± 0.24, P < 0.0001).

In order to analyze the prognostic impact of PrPc, we found a correlation between PrPc expression and cancer stage according to pathology results, with a significantly higher expression of PrPc for advanced stages. Moreover, 24 patients with a mean follow-up of 16.8 mo were considered. Immuno-blot analysis revealed a significantly higher expression of PrPc in patients with disease recurrence at 12 mo after radical surgery (360.71 ± 69.01 OD vs 170.23 ± 23.06 OD, P = 0.023), also in the subgroup of patients treated with adjuvant CT (368.36 ± 79.26 OD in the recurrence group vs 162.86 ± 24.16 OD, P = 0.028), which indicates a correlation with a higher chemo-resistance.

CONCLUSION

Expression of PrPc is significantly higher in PDAC cells compared with control, with the protein mainly placed in the nucleus. Preliminary clinical data confirm the correlation with a poorer prognosis.

Keywords: Pancreatic ductal adenocarcinoma; Prion protein; Western blotting; Electron microscopy; Cellular compartmentalization; Neuroinvasion

Core Tip: Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer and we are far away from understanding its biology. Recent in vitro evidence hypothesizes some role of cellular prion protein (PrPc) in PDAC carcinogenesis. We found that this protein is over-expressed in vivo within PDAC tissue of surgically resected patients, here we quantify the stoichiometric increase and provide evidence for a shift towards a nuclear compartmentalization. Such a protein amount is associated with poorer post-surgical prognosis and neuro-invasion. Thus, PrPc might be a key in the puzzled evidence of PDAC biology leading to better comprehend and cure such an aggressive disorder.