Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy

Abstract

Summary The influence of photodynamic therapy (PDT) on vascular perfusion and the development of hypoxia was investigated in the murine RIF-I tumour. Image analysis was used to quantify changes in perfusion and hypoxia at 5 min after interstitial Photofrin-mediated PDT. The fluorescent stain Hoechst 33342 was used as an in vivo marker of functional vascular perfusion and the antibody anti-collagen type IV as a marker of the tumour vasculature. The percentage of total tumour vasculature that was perfused decreased to less than 30% of control values after PDT. For the lower light doses this decrease was more pronounced in the centre of the tumour. The observed reduction in vascular perfusion showed a good linear correlation (r = 0.98) with previously published tumour perfusion data obtained with the 86Rb extraction technique. The image analysis technique provides extra information concerning the localisation of (non)-perfused vessels. To detect hypoxic tumour areas in vivo, an immunohistochemical method was used employing NITP [7-(4'-(2-nitroimidazol-l-yl)-butyl)-theophylline]. A large increase in hypoxic areas was found for PDT-treated tumours. More than half the total tumour area was hypoxic after PDT, compared with <4% for control tumours. Our studies illustrate the potential of image analysis systems for monitoring the functional consequences of PDTmediated vascular damage early after treatment. This provides direct confirmation that the perfusion changes lead to tissue hypoxia, which has implications for the combined treatment of PDT with bioreductive drugs.

Reduction of tumour oxygenation during and after photodynamic therapy in vivo: effects of fluence rate

“…Consistent with the above observations, no differences were found in tumour perfusion after high-or low-fluence-rate PDT. In both cases, tumour perfusion progressively decreased with time after treatment as has been described by others (Roberts et al, 1994;van Geel et al, 1994van Geel et al, , 1996. However, marked differences were found in cutaneous perfusion.…”

Photodynamic therapy mediates the oxygen-independent activation of hypoxia-inducible factor 1α

“…Numerous reports have shown that PDT induces microvascular damage within treated tumors (3 -5) and that this leads to tumor hypoxia (4,11). Because hypoxia induces increased expression and stabilization of HIF-1a and activates the HIF-1 transcription factor, recent molecular studies have thus far investigated the effects of PDTinduced hypoxia on expression of proangiogenic factors and their relevance to treatment efficacy.…”

“…The oxygen levels gradually decreased due to PDT-induced photochemical oxygen consumption. However, the oxygen concentration in the medium surrounding the cells remained high throughout 4 http://rsb.info.nih.gov/ij/ the treatment fluence of 2.5 J cm À2 and never decreased below 90% of the air-saturated level. These experiments thus confirmed that transient hypoxia was not induced under these PDT irradiation conditions.…”

“…Briefly, the digitized images were analyzed using ImageJ (NIH) 4 and MATLAB (The MathWorks, Inc., Natick, MA). Quantifying the extent of GFP expression in these samples required various image processing and analysis steps.…”

“…Various immune responses to PDT have also been described (2, 6 -8). The therapy-induced decrease in blood flow leads to strong and persistent tumor tissue hypoxia (4). Further, because the PDT photochemistry consumes oxygen, oxygen consumption during the therapy can also create rapid and severe transient tissue hypoxia (9 -11).…”