Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

Abstract

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUN-EL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.

The Effects of Allicin, a Reactive Sulfur Species from Garlic, on a Selection of Mammalian Cell Lines

“…Therefore, we tested for apoptotic cells by using the YO-PRO-1 iodide fluorescent dye that can enter apoptotic cells but not non-apoptotic cells [63]. Interestingly, there were pronounced differences between rates of allicin-induced apoptosis between the various cell types.…”

Feasibility of a Modified Biopsy Needle for Irreversible Electroporation Ablation and Periprocedural Tissue Sampling

“…Unlike in thermal ablation techniques, IRE is known to take a period of time for completion of ongoing cellular necrosis, this may prevent early detection of cell death using H&E stains alone[24, 25]. This issue could be addressed with viability stains for immediate assessment of earlier stages of cellular death [16, 26, 27]. …”

“…While tissue may appear ablated on imaging, showing an 16 ablation zone with sufficient margin creation [6], imaging cannot rule out residual viable tissue, which can only be identified through pathology [8] using dedicated markers [26–28]. Core biopsy samples taken from the ablation zone may not only confirm that the target lesion was successfully met but may also indicate an incomplete ablation whenever there is evidence of viable tumor tissue in the biopsy sample.…”

Fluorescent dyes as a reliable tool in P2X7 receptor-associated pore studies

“…Moreover, in this specific example, scientists should consider use not only YO-PRO-1 but also another cationic dye, such as propidium iodide or ethidium bromide, because YO-PRO-1 becomes permeable to pro-apoptotic cells and may produces false-positive pore formation events. It is noteworthy such formation of large pores in plasma membrane are related to cell death (Idziorek et al 1995;Plantin-Carrenard et al 2003;Ortega-Ferrusola et al 2008;Fujisawa et al 2014). Nevertheless, there is evidence that even when researchers use different fluorescent dyes with the same charge, but different weight, distinct dye uptake patterns occur.…”