Comparative Sensitivity of Microvascular Endothelial Cells, Fibroblasts and Tumor Cells after In Vitro Photodynamic Therapy with meso-Tetra-Hydroxyphenyl-Chlorin¶

Photochemistry and Photobiology, Sep/Oct 2004 by Triesscheijn, Martijn, Ruevekamp, Marjan, Aalders, Maurice, Baas, Paul, Stewart, Fiona A

ABSTRACT

The phototoxic effect of meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) on human micro vascular endothelial cells (hMVEC) was compared with that on human fibroblasts (BCT-27) and two human tumor cell lines (HMESO-1 and HNXOE). To examine the relationship between intrinsic phototoxicity and intracellular mTHPC content, we expressed cell survival as a function of cellular fluorescence. On the basis of total cell fluorescence, HNXOE tumor cells were the most sensitive and BCT-27 fibroblasts the most resistant, but these differences disappeared after correcting for cell volume. Endothelial cells were not intrinsically more sensitive to mTHPC-PDT than tumor cells or fibroblasts. Uptake of mTHPC in hMVEC increased linearly to at least 48 h, whereas drug uptake in the other cell lines reached a maximum by 24 h. No difference in drug uptake was seen between the cell lines during the first 24 h, but by 48 h hMVEC had a 1.8- to 2.8fold higher uptake than other cell lines. Endothelial cells showed a rapid apoptotic response after mTHPC-mediated PDT, whereas similar protocols gave a delayed apoptotic or neerotic like response in HNXOE. We conclude that endothelial cells are not intrinsically more sensitive than other cell types to mTHPC-mediated PDT but that continued drug uptake beyond 24 h may lead to higher intracellular drug levels and increased photosensitivity under certain conditions.

Abbreviations: BAEC, bovine aortic endothelial cells; CFA, colonyforming assay; CSLM, confocal scanning laser microscope; FCS, fetal calf serum; hMVEC, human microvascular endothelial cells; mTHPC, meso-tetra-hydroxyphenyl-chlorin; PDT, photodynamic therapy; SRB, sulphorodamine B.

INTRODUCTION

Photodynamic therapy (PDT) is an effective single modality treatment for small, superficial tumors (1-3). It can also be used as an adjuvant to debulking surgery for more advanced disease (4). PDT involves administration of a photosensitive drug, followed by local illumination of the tumor with light of the appropriate wavelength to activate the drug. Activation of the photosensitizer leads to the formation of free radicals and singlet oxygen that provoke tumor death, either via direct tumor cell kill or indirectly via vascular damage.

meso-Tetra-hydroxyphenyl-chlorin (mTHPC, trade name Foscan�) is a second-generation photosensitizer under investigation for several cancer types, including superficial head and neck cancers (5). Compared with first-generation photosensitizers such as Photofrin, mTHPC is 100 times more potent (drug dose � light dose) for equivalent cell killing. However, the mechanisms whereby mTHPC-mediated PDT results in such a strong response are not clearly understood.

To date, most clinical PDT protocols for mTHPC use a 4 day lime interval between drug administration and illumination of the tumor because the differential between photosensitizer levels in the tumor and normal tissue is maximal at this time interval (ratios of 3:6) (6). The tumor cells should therefore be preferentially sensitive to the direct cell killing effects of PDT at this time. However, indirect cell kill after immunological responses and vascular shutdown is also an important component of successful PDT (reviewed by Dolmans et al. [7]).

Both animal models (8,9) and clinical data (1,10) show that the optimal response to mTHPC-mediated PDT does not necessarily coincide with the maximal concentration of photosensilizer in the tumor. Shortening the interval between drug administration and illumination, and thereby changing the target from tumor cells to the vasculature, appears to give a greater tumor response to vivo PDT. Possible reasons for the apparent sensitivity of the tumor vasculature to PDT include: intrinsic sensitivity of the endothelial cells in the tumor; the presence of high local concentrations of both oxygen and photosensitizer in the blood vessels and the critical dependence of tumor growth on a vascular supply that is without reserve capacity.

In vitro photosensitivity using mTHPC has been described for a range of cell types (e.g. fibroblasts [11], leukemia cells [12] and cell lines derived from breast carcinoma [11], squamous cell carcinoma and colon adenocarcinoma [13,14]). A direct comparison of phototoxicity between endothelial and other cell types has not been made for this photosensitizer. Some studies using Photofrin, a first-generation photosensitizer, indicate that endothelial cells do have a greater intrinsic photosensitivity to PDT (15), whereas others report that this increased sensitivity reflects increased drug uptake (16,17). A direct comparison between the photosensitivity of endothelial cells, normal fibroblasts and tumor cells in relation to drug uptake and localization, should give insight into the relative importance of targeting the tumor cells or endothelial cells in therapeutic PDT.

The aim of this study was to determine the in vitro PDT efficacy of mTHPC on human microvascular endothelial cells (hMVEC) and its comparison with human fibroblasts (BCT-27) and two human tumor cell lines (HMESO-1, a mesothelioma-derived cell line and HNXOE, a squamous cell carcinoma-derived cell line). We also studied the intracellular uptake of mTHPC, the localization of the drug and the mechanism of cell death after PDT in two different cell types.