• PDAC
• prion protein
• alpha-synuclein
• aggressiveness
• neurotropism

SUMMARY
Background
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Patients with PDAC have dismal prognosis as the overall median survival is 6 months and the 5-year survival rate are less than 10%. Thus, identifying novel molecules and pathways which may serve as potential therapeutic targets to curb pancreatic cancer cell growth and/or metastasis is urgently needed, and understanding how cellular factors contribute to the tumorigenesis of PDAC is very important for the development of new treatment approaches.
Recently, several in vitro researches demonstrated that Prion Protein (PrPc) is expressed in a panel of human PDAC cell lines and it interacts with different pathways enhancing cellular growth, tumoral proliferation and invasion. PrPc is a cell surface glycoprotein quite ubiquitously expressed. Recent evidences obtained in vitro for a variety of malignancy indicate that the expression of PrPc is related with tumor stemness, invasiveness and resistance to chemotherapy. In vitro evidence in cell cultures showed the occurrence of PrPc in PDAC cells, while PrPc was absent in normal cell lines. However, no study so far investigated in vivo the expression of PrP in PDAC.
When dealing with PDAC biology, the occurrence of perineural infiltration (PNI) must be mentioned, since it is a peculiar biological behavior, deleterious for the prognosis of PDAC patients. Thus, a better insight into the mechanisms underlying neurotropism represents an important issue to understand the molecular mechanisms operating at the onset and spread of pancreatic cancer. This is supposed to foster and develop novel specific therapeutic strategies. The family of synucleins could be one of the biomarkers involved, since they have been recently involved in the mechanisms of oncogenesis, by selectively accelerating cellular processes leading to cancer. alpha-Synuclein (a-syn) in particular is a prionoid protein, which is involved in degeneration within the central nervous system (CNS), where it spreads from cell to cell according to a prion-like pattern when autophagy is altered, since α-syn is removed by autophagy. Since an alteration of autophagy machinery in PDAC occurs, it is expected that autophagy-dependent proteins may be altered consistently, but the role of α-syn in PDAC has never be investigated.
Thus, to address all these issues, the expression of PrPc and α-syn in PDAC and extra-PDAC tissue samples were measured with the aim to evaluate in vivo the presence of PrPc and a-syn in PDAC tissues, their role in PDAC biology and their possible clinical implication, as possible expression of a specifically worse biological behavior.

Matherials and methods
Tissue samples from patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were analyzed.
Western blotting and immunohistochemistry were used to detect, quantify and compare the expression of PrPc and a-syn in PDAC and control tissues, such as those of non-affected neighboring pancreatic tissue of the same patient. Immunohistochemistry was used also to evaluate the localization of PrPc and of CD155, a tumoral stem-cell marker. To quantify the increase of PrPc and a-syn and to detect their subcellular compartmentalization 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 after surgery and recurrence during adjuvant chemotherapy was made and the correlation between a-syn and the presence of perineural infiltration was analyzed.

Results
As regarding PrPc analysis, tissue samples from 45 patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were considered.
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]. Immunohistochemistry confirmed these findings, with higher linear staining of PrPc in PDAC ducts (127.145 ± 7.56 μm vs 75.21 ± 5.01 μm, p<0.0001).
PrPc and CD155 exactly overlapped in ductal tumoral cells, highlighting the possible relationship of PrPc with cancer stemness.
Quantitative morphometry carried out by using immuno-gold detection at transmission electron microscopy confirmed 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. In particular, PrPc expression at Western Blotting was 161.69 ± 63.92 OD in stage I, 173.25 ± 76.5 OD in stage II and 346.86 ± 55.26 OD in stage III (P = 0.0042).
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.
Concerning a-syn analysis, tumor (PDAC) and extra-tumor (extra-PDAC) samples from 20 patients affected by PDAC following pancreatic resections were collected. All patients were affected by moderately or poorly differentiated PDAC. The amount of a-syn was compared between tumor and extra-tumor specimen (sampled from non-affected neighboring pancreatic areas) by using in situ immuno-staining with peroxidase anti-a-syn immunohistochemistry, a-syn detection by using Western blotting and electron microscopy by using a-syn-conjugated immuno-gold particles. All the methods consistently indicate that each PDAC sample possesses higher amount of a-syn compared with extra-PDAC tissue. Moreover, the expression of a-syn was much higher in those PDAC samples from tumors with perineural infiltration compared with tumors without perineural infiltration. In fact, as measured by Western blotting, α-syn was higher in PDAC (2.1 ± 0.4 OD) compared with extra-PDAC areas (1.0 ± 0.3 OD, P=0.014). A significant difference between PDAC samples concerning the expression of a-syn was measured when comparing patients with or without perineural infiltration (2.64 ± 0.55 OD and 0.47 ± 0.12 OD, respectively P=0.035). Immuno-gold electron microscopy indicates higher α-syn particles in PDAC cells compared with cells from extra PDAC areas (107±6.1 and 18±4, respectively, P<0.00001).

Conclusion
Our results show that PrPc is markedly over-expressed in PDAC tissues compared with control, and that PrPc over-expression selectively occurs within ductal cells. Over-expression of PrPc overlaps with that of the staminal marker CD155 and this could be related to the role of PrPc in enhancing the emergence of the hallmarks of cancer in PDAC cells, promoting their aggressiveness. These data provide a step forward in the comprehension of PDAC biology. In fact, PrPc is likely to represent PDAC a marker of disease severity. The evidence of a peculiar nuclear compartmentalization of this protein in the cellular nuclei of PDAC cells is in line with in vitro data from literature showing over-expression of PRNP gene in PDAC cells, and suggests the presence of some, still unknown, molecular pathways triggered by PrPc in the nuclear compartment. This extends the influence of PrPc beyond its role as cell-membrane glycoprotein. Finally, PrPc expression seems to be associated with a greater risk of relapse after radical surgery and in shifting the cancer towards a phenotype with a higher chemo-resistance.
As for PrPc, our results indicate that also α-syn is markedly over-expressed within cells of PDAC tissues compared with extra-PDAC controls. The over-expression of α-syn significantly occurs in the presence of perineural invasion as assessed at pathological staging; this could be related to the role of α-syn in enhancing the emergence of a more aggressive behavior of cancer in PDAC cells, promoting their aggressiveness and neurotropism.
These data provide a step forward in the comprehension of PDAC biology, confirming that PrPc and α-syn are likely to represent markers of disease severity and determinant of PDAC biology and are a novelty in literature, as far as we know. Further studies are needed to validate these results and to investigate the molecular mechanism of PrPc and α-syn in PDAC pathogenesis and their potential clinical application.