Factors influencing tumor response to photodynamic therapy sensitized by intratumor administration of methylene blue

Abstract

We examined tumor response to methylene blue (MB)-mediated photodynamic therapy (PDT) in a murine tumor model. The goal was to investigate the effects of drug-light interval (DLI), injection vehicle, and fluence on tumor destruction. Fluorescence and reflectance spectroscopy informed our understanding. EMT6 tumor cells were implanted intradermally on the backs of female BALB/c mice and grown to ∼4-mm diameter. Mice were given a 35 µl, single site, intratumor injection of 500 µg/ml MB administered in either a water or a 5% ethanol-5% Cremophor-90% saline vehicle. PDT was begun either immediately or after a 1-hour DLI with a fluence rate of 60 mW/cm(2). Each animal received a fluence of 240 or 480 J/cm(2). Fluorescence and reflectance spectra were captured before and during irradiation. A protocol consisting of the Cremophor-based vehicle, 0 DLI, and a fluence of 480 J/cm(2) was the most effective, with a 55% cure rate as measured by no evidence of tumor 90 days after PDT. Use of the water vehicle with this fluence and DLI reduced the cure rate to 20%. Reducing the fluence to 240 J/cm(2) similarly reduced treatment efficacy with 0 and 1-hour DLIs. Univariate Cox proportional hazards analysis identified increased fluence, 0 versus 1-hour DLI, and the Cremophor versus water vehicle as highly significant independent predictors of long term tumor control (P < 0.01 in each case). Multivariate analysis with model selection revealed fluence and injection vehicle as the best predictors of survival hazards. Fluorescence spectroscopy in vivo showed that MB fluorescence decreased monotonically during a 2-hour dark interval but was restored by irradiation. Reflectance spectroscopy revealed that MB at this injected concentration attenuates the treatment beam significantly. Sensitizer delivery vehicle, drug-light interval, and fluence contribute significantly to the tumor response to MB-mediated PDT.