General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy.
Human Anatomy, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa 56124, Italy.
World J Gastroenterol. 2021 Nov 14;27(42):7324-7339. doi: 10.3748/wjg.v27.i42.7324.
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.
To analyze quantitative stoichiometry and compartmentalization of PrPc in PDAC cells and to correlate its presence with prognostic data.
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.
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) 95 ± 17.40 OD, < 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 9.44 ± 0.45, 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 5.12 ± 0.32, 0.0001; cytosol: 7.74. ± 0.44 particles 4.3 ± 0.24, 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 170.23 ± 23.06 OD, = 0.023), also in the subgroup of patients treated with adjuvant CT (368.36 ± 79.26 OD in the recurrence group 162.86 ± 24.16 OD, = 0.028), which indicates a correlation with a higher chemo-resistance.
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.
最近的证据表明，朊病毒蛋白（PrPc）的表达与胰腺导管腺癌（PDAC）之间存在关系。事实上，PrPc 可能是解释这种肿瘤侵袭性的标志物之一。然而，目前缺乏研究探讨 PDAC 细胞内 PrPc 表达增加的具体分隔化，以及超微结构证据、PrPc 蛋白超微结构形态计量学与临床数据之间的相关性。这些数据以及免疫金检测到的该蛋白质的定量化学计量学，为理解疾病的生物学和手术切除的结果提供了重要进展。
分析 PDAC 细胞中 PrPc 的定量化学计量和分隔化，并将其与预后数据相关联。
2018 年 6 月至 2020 年 12 月，在我们机构对术前怀疑 PDAC 的胰腺切除患者收集胰腺组织样本。当冷冻切片排除 PDAC 诊断，或结节太小而无法进行充分取样时，患者将被排除在本研究之外。使用 Western 印迹法检测、定量和比较 PDAC 和对照组织（如同一患者的未受影响的相邻胰腺组织）中 PrPc 的表达。为了定量增加 PrPc 并检测 PDAC 细胞内 PrPc 的亚细胞分隔化，使用电子显微镜分析特定细胞区室中 PrPc 的免疫金化学计量。最后，根据癌症分期、手术后 12 个月疾病复发和辅助化疗期间复发等预后数据对 PrPc 的定量表达进行分析。
使用 Western 印迹法进行半定量分析，确定了 45 例患者中的 38 例样本中的 PrPc 量，这表明与健康胰腺区域相比，肿瘤胰腺组织中的 PrPc 增加了近三倍[242.41±28.36 光密度（OD）95±17.40 OD，<0.0001]。使用免疫金检测在透射电子显微镜下进行的定量形态计量学证实了所有患者 PDAC 导管细胞中 PrPc 的表达增加，并允许检测到肿瘤细胞内 PrPc 的特定分隔化。特别是，当考虑整个细胞时，PDAC 细胞中的 PrPc 免疫金颗粒数明显高于对照细胞[19.8±0.79 颗粒 9.44±0.45，<0.0001]。值得注意的是，在考虑 PDAC 细胞时，PrPc 在细胞核中的增加大于肿瘤细胞胞质中的增加，这表明肿瘤细胞内 PrPc 的分隔化发生了变化。事实上，核内免疫金的增加大大超过了在胞质中检测到的 PrPc 的增加（核：12.88±0.59 颗粒 5.12±0.32，<0.0001；胞质：7.74±0.44 颗粒 4.3±0.24，<0.0001）。为了分析 PrPc 的预后影响，我们发现了 PrPc 表达与病理结果所示癌症分期之间的相关性，晚期阶段的 PrPc 表达显著更高。此外，考虑了 24 名平均随访 16.8 个月的患者。免疫印迹分析显示，在根治性手术后 12 个月疾病复发的患者中，PrPc 的表达明显更高（360.71±69.01 OD 170.23±23.06 OD，=0.023），在接受辅助 CT 治疗的患者亚组中也是如此（复发组 368.36±79.26 OD 162.86±24.16 OD，=0.028），这表明与更高的化疗耐药性相关。
与对照相比，PDAC 细胞中的 PrPc 表达明显更高，该蛋白主要位于细胞核中。初步临床数据证实了与预后不良的相关性。
