Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model

Abstract

Background: The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7).

Methods: Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0-10 microg ml(-1)) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture.

Results: MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 microg ml(-1) mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0-3 microg ml(-1)), which caused neuron death equivalent to other cell types.

Conclusion: Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.

Figure 1

Representative micrographs showing (A) mTHPC fluorescence within the cell body of neurons (i and ii), satellite glia (iii) and MCF-7 cells (iv), but not detected in neurites (v). (B) Confocal micrograph projections confirming the presence of mTHPC fluorescence in neuronal cell body and satellite glia, but none detectable along the neurite. Meta-tetrahydroxyphenyl chlorin fluorescence is shown in red, β-III-tubulin immunoreactivity is shown in green, nuclei are blue. All scale bars are 20 μm. Confocal images are maximum intensity projections from 40 optical sections in a 14 μm z-series.

Figure 2

Reduction in neurite length due to mTHPC-mediated PDT. Dorsal root ganglion neurons were cultured on coverslips for 3 days, then treated with various concentrations of mTHPC and light, then maintained in culture for a further 24 h before fixation and quantification of the total length of all β-III-tubulin immunoreactive neurites. Data are means±s.e.m., N=4. (^**P<0.001 for treatments compared with no drug or light control, one-way ANOVA with Dunnett's multiple comparison post test).

Figure 3

Relative sensitivity of cells to mTHPC-mediated PDT. Cell death (neurons, satellite glia and MCF-7 cells) in the presence of various concentrations of mTHPC after light exposure (A) or in control samples with no light exposure (B). Differences in cell death in neurons compared with MCF-7 cells and satellite glia were present at concentrations above 2 μg ml^–1 mTHPC, with ∼50% cell death for MCF-7 cells and satellite glia at 4 μg ml^–1. Neuronal cell death after this treatment was significantly lower at ∼12%, which was not different from untreated neurons (P<0.001 using one-way ANOVA with Tukey's post test). Data are means±s.e.m. of at least nine replicates. Basal levels of % cell death using 0 μg ml^–1 mTHPC with light were 3.7±0.8, 4.3±1.3 and 7.6±1.0, and with no light were 3.1±1.0, 4.2±1.4 and 1.89±0.7, for neurons, satellite glia and MCF-7s, respectively.

Figure 4

Time course of neuronal and satellite glial death after mTHPC-mediated PDT. Cells in 3D gels were incubated with 4 μg ml^–1 mTHPC for 4 h, exposed to light (A) or used as no-light controls (B), then maintained in culture for various times before cell death analysis and immunodetection of neurons.

Figure 5

Relative sensitivity of cells to hypericin-mediated PDT. Cell death (satellite glia, neurons and MCF-7s) in the presence of various concentrations of hypericin after light exposure (A) or in control samples with no light exposure (B). Neurons showed a similar pattern of response as satellite glia and MCF-7 cells, with 50% cell death at ∼1–2 μg ml^–1. Data are means±s.e.m. of at least six replicates. The basal levels of % cell death using 0 μg ml^–1 hypericin with light were 7.9±1.1, 4.8±0.9 and 1.0±0.2, and with no light were 5.4±1.3, 8.0±2.8 and 1.6±0.7, for neurons, satellite glia and MCF-7s, respectively.

Figure 6

Neurons survive mTHPC-mediated PDT and produce new neurite outgrowth. After treatment with mTHPC-mediated PDT in 3D culture, neurons were grown in 2D in the presence of untreated satellite glia for 2 days. A similar number of neuronal cell bodies had neurites associated with them in the 3–4 μg ml^–1 mTHPC-mediated PDT samples as in the controls (A). There was a similar level of neurite length detected in controls and treated neurons, except for the samples that had received 10 μg ml^–1 mTHPC-mediated PDT, which showed a significant reduction in neurite length (^*P<0.05) (B).