Herein, we report a simple procedure regarding the photodynamic therapy (PDT) treatment as a minimally invasive modality for treating superficial bladder cancer that utilizes a photosensitizer, light, and oxygen to generate cytotoxic reactive oxygen species (ROS). This study evaluates the histopathological and morphological changes induced by PDT in an ex vivo model of low-grade (LG) pTa non-muscle-invasive bladder cancer (NMIBC). We investigated the efficacy of exogenous protoporphyrin IX (PpIX) and Rose Bengal (RB) by incubating tissue samples (n = 30) with an oxygen-saturated solution of PpIX (1–3 mM) or RB (0.3–0.5 mM) for one hour. Since the criticism of using frozen tissue in research already exists, this framing explains how to mitigate those limitations. Thus, we use oxygen-saturated solutions PpIX and oxygen-saturated solutions of RB. We discussed a few aspects related to the use of frozen tissue in PDT. Frozen tissue preserves lipids critical for assessing membrane damage and maintains higher levels of metabolic markers like antioxidant molecules like glutathione and more likely lack factors such as metabolic activity, intact cell membranes, and oxygenation. It is critical to differentiate between “artifactual” changes and the “pathological” death of cells. Thus, we used histopathological microscopy observation typically used in daily clinical investigations to characterize cells before and after PDT. Following irradiation with the light dose of 72 J/cm2 (410 nm or 532 nm at 300 mW for 15 min), hematoxylin–eosin staining revealed concentration-dependent apoptotic changes, including chromatin condensation, pyknosis, and nuclear fragmentation. While both agents induced cell death, RB demonstrated faster and more intense cytotoxicity than PpIX. These findings provide microscopic evidence of PDT-induced tumor destruction and suggest that RB is a potent candidate for further preclinical evaluation. At 410 nm (deep blue/violet), light penetration in biological tissue is very shallow, typically only around 0.3 to 1 mm; therefore, in a 2 mm thick tissue sample, most of the light would be absorbed within the first millimeter, with minimal light reaching the full depth of tissues. In this protocol, the generated ROS is used to destroy tumor tissue by attacking the cellular microenvironment directly. This led to immediate membrane disruption and lipid peroxidation. The proof-of-concept is an early-stage study designed to verify that a PDT treatment is feasible, safe, and biologically active in an ex vivo model of LG pTa NMIBC.
Godlewski et al. (Fri,) studied this question.