Biology contribution
Combining vascular and cellular targeting regimens enhances the efficacy of photodynamic therapy

https://doi.org/10.1016/j.ijrobp.2004.08.006Get rights and content

Purpose

Photodynamic therapy (PDT) can be designed to target either tumor vasculature or tumor cells by varying the drug-light interval. Photodynamic therapy treatments with different drug-light intervals can be combined to increase tumor response by targeting both tumor vasculature and tumor cells. The sequence of photosensitizer and light delivery can influence the effect of combined treatments.

Methods and materials

The R3327-MatLyLu rat prostate tumor model was used in this study. Photosensitizer verteporfin distribution was quantified by fluorescence microscopy. Tumor blood flow changes were monitored by laser-Doppler system and tumor hypoxia was quantified by the immunohistochemical staining for the hypoxic marker EF5. The therapeutic effects of PDT treatments were evaluated by the histologic examination and tumor regrowth assay.

Results

Fluorescence microscopic studies indicated that tumor localization of verteporfin changed from predominantly within the tumor vasculature at 15 min after injection, to being throughout the tumor parenchyma at 3 h after injection. Light treatment (50 J/cm2) at 15 min after verteporfin injection (0.25 mg/kg, i.v.) induced significant tumor vascular damage, as manifested by tumor blood flow reduction and increase in the tumor hypoxic fraction. In contrast, the vascular effect observed after the same light dose (50 J/cm2) delivered 3 h after administration of verteporfin (1 mg/kg, i.v.) was an initial acute decrease in blood flow, followed by recovery to the level of control. The EF5 staining revealed no significant increase in hypoxic fraction at 1 h after PDT using 3 h drug-light interval. The combination of 3-h interval PDT and 15-min interval PDT was more effective in inhibiting tumor growth than each individual PDT treatment. However, it was found that the combined treatment with the sequence of 3-h interval PDT before 15-min interval PDT led to a superior antitumor effect than the other combinative PDT treatments. Histologic studies confirmed that this combined treatment led to damage to both tumor vasculature and tumor cells. Importantly, the combined PDT treatment did not increase normal tissue damage and tissue recovered well at 60 days after treatment.

Conclusions

Our results suggest that targeting both tumor vascular and cellular compartments by combining a long-interval PDT with a short-interval PDT can be an effective and safe way to enhance PDT damage to tumor tissue.

Introduction

Photodynamic therapy (PDT) is an established cancer treatment modality in which a photosensitizer absorbs light and generates cytotoxic reactive oxygen species leading to cellular damage (1). It has been applied to both treatment of superficial tumors (such as cutaneous basal cell carcinoma and head and neck tumors) and to deeper tumors accessible by endoscopies (including esophageal and lung cancers) (2). In the United States, PDT with Photofrin has been approved for the treatment of Barrett's esophagus and endobronchial and esophageal carcinomas (3), and perhaps the most successful approval of PDT is with verteporfin for injection (Visudyne) to treat age-related macular degeneration (AMD) (4).

The mechanisms of how PDT causes tissue damage involve a complex interplay among the tissue, the photosensitizer, and light. To be photoactive, adequate amounts of photosensitizer and light must be deposited simultaneously in the target tissue with sufficient oxygen supply. The effectiveness of treatment can therefore be affected by any factors influencing the photosensitizer distribution, photobleaching, light delivery, blood flow, and oxygen distribution in the target tissue. For a particular tissue and a photosensitizer, the spatial distribution and compartmentalization of the photosensitizer change temporally after drug administration. The time between photosensitizer injection and light treatment (drug-light interval) is therefore a critical parameter in determining the site target and efficacy, mainly through drug distribution in different tissue compartments (5).

In general, a photosensitizer is confined within the tumor vasculature initially after injection and PDT that employs a short drug-light interval largely damages tumor vasculature (6, 7, 8, 9, 10, 11). This mechanism is mainly responsible for some of the more successful clinical implementations of PDT today, including AMD treatment with verterpofin (4) and prostate cancer treatment with TOOKAD (12, 13). In this vascular-targeting regime, it is hypothesized that photosensitizing effects occur when the drug is still largely localized within the blood vessels, thereby maximizing the singlet oxygen dose to the blood cells and endothelial cells (14). Flow stasis can then occur from a combination of vasoconstriction, endothelial cell damage and thrombosis. After vascular shutdown, tumor cells are largely killed by tissue ischemia (15, 16). Whereas a long drug-light interval allows free diffusion of the photosensitizer out into the tissue, to be accumulated into the tumor cellular compartment and consequent light irradiation generates more direct tumor cytotoxicity (5, 17, 18). In fact, our earlier studies have shown that direct cellular targeting can be achieved while preserving blood flow (19, 20).

While many studies have explored ways to maximize the therapeutic effect of PDT (21), recent efforts are more focused on utilizing targeting strategies that are directed at the tumor vasculature. However, it should be realized that neither vascular targeting nor cellular targeting PDT regime alone is perfect for tumor cell killing. Solely vascular targeting may be a good approach for purely vascular diseases such as AMD (22), yet it may not be enough for tumors because peripheral tumor vessels are shown to be somewhat resistant to both vascular-targeting agents (23) and PDT-induced vascular effects (12, 24, 25). Despite the extensive central tumor necrosis induced by vascular targeting PDT, tumor vessels/cells can regrow from the peripheral rim after treatment. The major problem for cellular-targeting PDT is that it suffers from complex issues such as heterogeneity of tumor microenvironment and inhomogeneous photosensitizer distribution. Additionally, tissue hypoxia has been identified as a major obstacle to direct targeting tumor cells by PDT (1, 5). It is generally believed that, compared with the well-perfused outer tumor region, the less-perfused tumor inner region is more hypoxic and therefore more resistant to radiotherapy, chemotherapy, and cellular targeting PDT as well (26). Inadequate photosensitizer delivery due to heterogeneous tumor perfusion, vascular permeability, and tumor interstitial pressure can also affect the effectiveness of cellular targeting PDT (26). Combination of tumor vascular and cellular targeting approaches can be a way to overcome the problem associated with each individual targeting strategy and to achieve maximal opportunity of tumor eradication (27).

Since the potential target of PDT can be effectively modified by using different drug-light intervals, it is hypothesized that the combination of a short interval PDT (vascular targeting) and a long interval PDT (cellular targeting) will damage both tumor vascular and cellular compartments. Such a combination may compensate each other and lead to an enhanced therapeutic effect. Specifically, we demonstrate in this study that a long drug-light interval PDT followed immediately by a short interval PDT is the best way to achieve both vascular and cellular targeting on tumor tissue.

Section snippets

Photosensitizer

Verteporfin (BPD, lipid-formulated benzoporphyrin derivative monoacid ring A) was obtained from QLT Inc. (Vancouver, British Columbia, Canada). A stock saline solution of verteporfin was reconstituted according to the manufacturer's instructions and stored at 4°C in the dark. It was injected i.v. 0.25 mg/kg at 15 min before light irradiation or 1.0 mg/kg at 3 h before light irradiation.

Animals and tumor model

Male Copenhagen rats (6–8 weeks old) obtained from Charles River Laboratories, Wilmington, MA) were used

Results

The fluorescence of verteporfin in the s.c. MatLyLu tumor at 15 min and 3 h after injection (i.v., 1 mg/kg) is shown in Fig. 2A. The perfused blood vessels in the same microscopic field were visualized by a fluorescent dye DiOC7(3). Tumor distribution of verteporfin at 15 min after injection was almost identical to the perfusion marker DiOC7(3), indicating the dominant intravascular localization with slight diffusion beyond the vascular boundaries. A distinct difference in verteporfin

Discussion

Photosensitizer pharmacokinetic distribution after administration provides an opportunity to alternate the primary target from tumor vasculature initially to parenchyma at longer times after injection. To enhance the therapeutic effect of PDT, a combined approach of PDT that targets both tumor vascular and cellular compartments was hypothesized and tested here. Verteporfin, an approved photosensitizer for the treatment of AMD (33), was selected in this study because it has a fast plasma

Acknowledgments

The authors thank QLT Inc. (Vancouver, Canada) for providing the verteporfin.

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