Research Article: ALA-PpIX mediated photodynamic therapy of malignant gliomas augmented by hypothermia

Date Published: July 31, 2017

Publisher: Public Library of Science

Author(s): Carl J. Fisher, Carolyn Niu, Warren Foltz, Yonghong Chen, Elena Sidorova-Darmos, James H. Eubanks, Lothar Lilge, Michael Hamblin.

http://doi.org/10.1371/journal.pone.0181654

Abstract

Malignant gliomas are highly invasive, difficult to treat, and account for 2% of cancer deaths worldwide. Glioblastoma Multiforme (GBM) comprises the most common and aggressive intracranial tumor. The study hypothesis is to investigate the modification of Photodynamic Therapy (PDT) efficacy by mild hypothermia leads to increased glioma cell kill while protecting normal neuronal structures.

Photosensitizer accumulation and PDT efficacy in vitro were quantified in various glioma cell lines, primary rat neurons, and astrocytes. In vivo studies were carried out in healthy brain and RG2 glioma of naïve Fischer rats. Hypothermia was induced at 1 hour pre- to 2 hours post-PDT, with ALA-PpIX accumulation and PDT treatments effects on tumor and normal brain PDT quantified using optical spectroscopy, histology, immunohistochemistry, MRI, and survival studies, respectively.

In vitro studies demonstrated significantly improved post-PDT survival in primary rat neuronal cells. Rat in vivo studies confirmed a neuroprotective effect to hypothermia following PpIX mediated PDT by T2 mapping at day 10, reflecting edema/inflammation volume reduction. Mild hypothermia increased PpIX fluorescence in tumors five-fold, and the median post-PDT rat survival time (8.5 days normothermia; 14 days hypothermia). Histology and immunohistochemistry show close to complete cellular protection in normal brain structures under hypothermia.

The benefits of hypothermia on both normal neuronal tissue as well as increased PpIX fluorescence and RG2 induced rat survival strongly suggest a role for hypothermia in photonics-based surgical techniques, and that a hypothermic intervention could lead to considerable patient outcome improvements.

Partial Text

Glioblastoma Multiforme (GBM), comprising the most common and aggressive adult intracranial malignancy, has presented with a constant increasing incidence rate at over 2% per year between 1970 and 2000 [1]. While the median survival has increased during this time, it is still reported at just 14–16 months following standard therapies including surgical removal, radiation, and chemotherapy[2]. Various therapies including image guided surgical resection[3–5], gamma knife surgery[6], intensity modulated ionizing radiation therapy[7] and brachytherapy[8], or adjuvant chemotherapy[9] are being investigated for GBM. Even with the most aggressive treatment plans, the patients’ benefit has extended to a few months of additional survival time[10]. Encouraging is that the fraction of long-term survivors is increasing, possibly due to the realization that 98% tumor resection is required to afford long-term benefit to the patient[5].

The University Health Network’s Animal Care Committee complying with regulations of the Canadian Council on Animal Care approved all procedures.

Survival assessment based on the Presto blue metabolic assay demonstrated that cultured primary rat neurons experienced significantly sparing of PDT-induced cell death when using ALA concentration [μM] LD50 as the PDT dose surrogate for constant radiant exposure. The normothermia LD50 was 68 μM whereas the hypothermia LD50 was 8000 μM or 2 orders of magnitude higher (p<0.05, Fig 1A). Factors which modify PpIX accumulation may lead to improved FGR and PDT efficacy. This study builds on a prior in vitro study demonstrating hypothermia (32–34°C) leading to higher PpIX concentrations in tumor cell lines, thus allowing for higher selectivity in FGR and a greater therapeutic index for PDT[20]. For glioma invading the normal brain, PDT selectivity cannot be provided by the fluence rate (ϕ), nor the oxygen gradient, as neither will vary across the size of the micro invasions. Thus, selectivity is provided only by the difference in PpIX accumulation in the tumor versus the normal host brain and the tissue’s intrinsic responsivity to the cytotoxic dose from PDT. The tissue responsivity is given by its PDT threshold value, T. When the PDT dose, given by the light dose, or fluence rate ϕ [mWcm-2] and the photosensitizer concentration [PpIX], exceeds a threshold value T, tissue destruction occurs. To achieve selective GBM destruction up to the clinically required depth, d, the following conditions need to be satisfied. TTumorP(0)[PpIX]Tumorϕ(d)http://doi.org/10.1371/journal.pone.0181654