Reactive oxygen species explicit dosimetry (ROSED) for fractionated photofrin-mediated photodynamic therapy (PDT)

Abstract

Photodynamic therapy (PDT) is an established modality for cancer treatment and reactive oxygen species explicit dosimetry (ROSED), based on direct measurements of in-vivo light fluence (rate), in-vivo photofrin concentration, and tissue oxygenation concentration, has been proved to be an effective dosimetric quantity which can be used to predict PDT outcome. In this study, ROSED was performed for photofrin-mediated PDT for mice bearing radiation-induced fibrosacorma (RIF) tumor. PDT treatments were performed using single or fractionated illumination to a same total fluence of 135 Jcm⁻². The effects of light fractionation on the total reacted [ROS]_rx and treatment outcomes were evaluated.

1. INTRODUCTION

Fractionated illumination with a fixed dark interval has been proved to be able to significantly improve the treatment outcome of PDT both in previous studies [1, 2]. However, most of the previous fractionated studies were based on ALA, which might have a superficial distribution within the tumor and hence less effective. Photofrin-mediated PDT study has also showed increased effectiveness with light/dark fractions but several dark intervals were involved with different length of light/dark fractions [3]. For one fixed dark interval, 2 hour between two light fractions was found to produce better treatment outcomes. The improvement in treatment outcome of light fractionation could be attributed to the tumor oxygenation reaccumulating during the dark interval between the two light fractions [4]. To investigate the effect of fractionated illumination on treatment outcome, reactive oxygen species explicit dosimetry (ROSED) was performed for comparison between the total reacted reactive oxygen species, [ROS]_rx, and the treatment outcomes of different fractionated treatment schemes. To calculate the total [ROS]_rx generated by PDT processes, in-vivo light fluence (rate), in-vivo photosensitizer concentration and tumor oxygenation were measured during the treatment. ROSED has been recently developed to predict local tumor control and [ROS]_rx, as a dosimetric quantity, has been demonstrated to have a strong correlation with treatment outcome in preclinical PDT [5, 6].

In this study, both fractionated and continuous photofrin-mediated PDT was performed for mice bearing radiation-induced fibrosacorma (RIF) tumor. Fractionated treatment involves two light fractions of varying fluences separated by a 2-hour dark interval. The total light fluence of the two light fractions of varying fluences were kept the same, 135 Jcm⁻². Light fluence rate, photosensitiser concentrations, tumor oxygenation concentrations were measured during treatments to determine for [ROS]rx. In addition, cure index (CI) for short term (14 days after PDT) was calculated and compared between the single illumination and different light fractionation groups. Even though counted as cure in short term, there’s still chance for tumor regrowth beyond 14 days and hence the study was extended to observe the long-term effect of light fraction.

2. METHODS

2.1. Tumor model and PDT treatment conditions

The animal model involved in this study were female C3H mice (Charles River Laboratories, Kingston, NY) aged from 6 to 8 weeks. Radiation-induced fibrosarcoma (RIF) tumors were propagated on the mice’s shoulders via intradermal injection of 3 × 10⁵ cells per mouse. After injection, tumor volume was measured daily and treatment started when the diameter of tumor reached around 4–5 mm. Width, a and length, b, of tumors were measured with slide caliper on and after PDT, and tumor volume, V, was calculated using V = π × a² × b/6 [7]. Photofrin at a concentration of 5mg/kg was injected through tail 24 hours before treatment. And the treatment area was depilated with Nair (Church & Dwight Co., Inc., Ewing, New Jersey, US) prior to treatment. A 630-nm laser (Biolitec AG., A-1030, Vienna) was used for the superficial irradiation with a laser spot of 1-cm diameter on skin surface of the mice. A microlens fiber was coupled to the laser fiber for uniform irradiation covering the whole tumor. The in-air fluence rate was kept constant during treatment for all mice at 75 mWcm⁻². The total treatment time, excluding the 2-h dark interval, for all treatment schemes was 1800s and the total light fluence was 135 Jcm⁻². For continuous group, there were 7 mice. For the fractionated groups, the treatment time of the two light fractions were varied: (1) 7 mice with 400s and 1400s, (2) 5 mice with 600 and 1200s, and (3) 8 mice with 800s and 1000s, with intermediate 2-hour dark intervals. In addition to continuous and fractionated groups, 4 mice with RID tumor served as controlled group for comparison and cure index calculation. Animals used in this study were under the care of the University of Pennsylvania Laboratory Animal Resources and the study was approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

2.2. Measurements during treatment

During treatment, three quantities were measured: light fluence rate, oxygen concentration, and photofrin concentration. Light fluence rate was measured by an isotropic detector on tumor surface, connecting to a light dosimeter. The light dosimetry system was calibrated prior to treatment to ensure accuracy. The in vivo tissue oxygen partial pressure pO₂ was monitored throughout the illumination using a phosphorescence-based ³O₂ probe (OxyLite Pro, Oxford Optronix, Oxford, United Kingdom), with a bare-fiber-type probe (NX-BF/O/E, Oxford Optronix, Oxford, United Kingdom) inserted inside the tumor at a 3-mm depth from the skin surface (fig.1a). Tissue oxygen concentration [³O₂] was calculated by multiplying pO₂ by ³O₂ solubility in tissue, 1.295 μM/mmHg [9]. Photofrin concentration was calculated based on fluorescence spectra of tumor, obtained using a custom-made multifiber contact probe [8]. Measurements were taken before and after each illumination fraction (fig.1b).

Fig.1.

Experiment setup (a) tissue pO₂ measured with oxygen probe during treatment (b) A handheld multifiber contact probe was used to measure the photofrin concentration before and after PDT [10]

The measured light fluence rate ϕ, PS concentration [S₀], and tumor oxygenation [³O₂] were used to calculate the total reacted reactive oxygen species [ROS]_rx for each light fraction using equation,

$$\frac{d{[ROS]}_{rx}}{dt}=\xi \frac{\left[{}^3{O}_2\right]}{\left[{}^3{O}_2\right]+\beta }\varphi \left[S_0\right]$$

where the photophysical parameters ξ is 3.7 × 10⁻³ cm²S⁻¹mW⁻¹ and β is 11.9 μM.

3. RESULTS AND DISCUSSION

The photofrin concentration and oxygen concentration results for part of the mice are shown in fig.2, where the symbols represent the measured values. For all treatment schemes, the concentration drops to a lower value at the end of the illumination comparing to the initial concentration. But for the fractionated treatment, the concentration tend to restore during the 2-h dark interval, which may have a positive effect on treatment outcome.

Fig.2.

Photofrin (upper) and tissue oxygen (lower) concentration versus time during illumination for part of mice with various treatment conditions

The average tumor volumes for each treatment and controlled groups from day 0 to day 14 after PDT were plotted in fig.3. For comparison between different groups, the tumor volumes were normalized to average tumor volume on day 0. The measured and averaged tumor volumes were fitted to an exponential growth equation, V=A·exp(k·d), where A is the amplitude, k is the tumor regrowth rate and d is the number of days after PDT treatment. CI was calculated by using the following expression, CI = 1-k/k_control, where k is the tumour regrowth rate for mice in treatment groups and k_control is the tumor regrowth rate of the control mice without any illumination. For short-term study, mice with growth delay but no regrowth within 14 days are counted as a cure and its cure index is equal to 1.

Fig.3.

Exponential fitting of average tumor volumes of different treatment groups against time after PDT. Tumor volume (V) is calculated using V = π × a² × b/6.

The CI and total [ROS]_rx for each treatment group were presented in Fig.4 for comparison. It has been shown that the CI values of fractionated groups are higher than the continuous group, especially for the “600+1200s” and “800+1000s” groups. This shows that fractionated PDT was able to improve the treatment outcomes, comparing to non-fractionated PDT. However, this improvement is not obvious in 14-days because the continuous photofrin-mediated PDT group was sufficiently effective, around 80% of cure rate, in short term [6]. Further improvement beyond this will be restricted to be between 80% – 100%. The short-term improvement can also be observed in the 14-days Kaplan–Meier survival curves (Fig.5), where the treatment outcomes of all groups, fractionated and continuous, are similar. The total [ROS]_rx produced during PDT was evaluated but found no statistically significant difference between all treatment schemes studied for short-term results. The improvement of treatment efficacy of fractionated groups was also observed in the previous ALA-fractionated study, with no correlation to total [ROS]_rx [2].

Fig.4.

Comparison of cure index (left) and [ROS]_rx total obtained for different treatment schemes. CI was calculated by using the following expression, CI = 1-k/k_control.

Fig.5.

14-days Kaplan–Meier survival curves for Photofrin-mediated PDT for different groups.

The study was extended to 60 days to explore the long-term effect. The long-term results are shown in Fig.6. All PDT treatment groups exhibit improved survival compared to the control group. There are no significant difference for PDT treatment groups between continuous PDT treatment (1800s) and fractionated PDT treatments (400s+1400s, 600s+1200s), however the fractionated group “800s+1000s” shows significant improvement of survival comparing to the other PDT groups: The long-term survival rate has been increased from ~ (0 – 25)% to ~65%. Group “400s+1400s” has the worst treatment outcome since the initial tumor sizes in this group were too large so that the light was not able to penetrate through the whole tumors.

Fig.6.

Long-term Kaplan–Meier survival curves for Photofrin-mediated PDT for different groups. Group “800s+1000s” has significant better treatment outcome

Even though increased effectiveness of long-term fractionated illumination can be observed, it is not correlated to the total [ROS]_rx evaluated. The increased effectiveness suggests that a potential mechanism of differential cellular response to light fractionation. No corelation between the total [ROS]_rx produced and the treatment outcome of different treatment schemes suggests that the improvement is not due to the direct cell killings inflicted. The underlying mechanism is unknown, given that the total [ROS]_ex is almost the same. There are several possible reasons for the enhanced PDT efficacy [2, 3]: (1) immune response after PDT and (2) vascular response after PDT.

4. CONCLUSION AND FUTURE WORK

Fractionated and non-fractionated photofrin-mediated PDT and associated ROSED has been successfully performed on female mice with RIF tumor. The effect of light fractionation on the total [ROS]_rx and the treatment outcome for preclinical photofrin-mediated PDT was evaluated. In 14-days survival study (short term study), the cure index of the fractionated photofrin-mediated PDT is higher than the non-fractionated treatment, which shows that fractionated PDT has better efficacy than continuous PDT. The short-term results also show that the treatments with longer first light fraction tend to have better treatment outcome. In 60 days study, “800s + 1000s” treatment scheme shows substantially improvement in survival rate. All the other scheme shows similar results. However, The improvement in treatment efficacy in both short term or long term studies is not related to the total [ROS]rx.

Further optimization of the fractionated treatment schemes will be explored in the future, including longer first light fraction, different dark intervals and different light intensities. The treatment conditions will be optimized, aiming at further improvement of long-term survival of photofrin-mediated PDT.