Blood Flow Measurements Enable Optimization of Light Delivery for Personalized Photodynamic Therapy

Abstract

Fluence rate is an effector of photodynamic therapy (PDT) outcome. Lower light fluence rates can conserve tumor perfusion during some illumination protocols for PDT, but then treatment times are proportionally longer to deliver equivalent fluence. Likewise, higher fluence rates can shorten treatment time but may compromise treatment efficacy by inducing blood flow stasis during illumination. We developed blood-flow-informed PDT (BFI-PDT) to balance these effects. BFI-PDT uses real-time noninvasive monitoring of tumor blood flow to inform selection of irradiance, i.e., incident fluence rate, on the treated surface. BFI-PDT thus aims to conserve tumor perfusion during PDT while minimizing treatment time. Pre-clinical studies in murine tumors of radiation-induced fibrosarcoma (RIF) and a mesothelioma cell line (AB12) show that BFI-PDT preserves tumor blood flow during illumination better than standard PDT with continuous light delivery at high irradiance. Compared to standard high irradiance PDT, BFI-PDT maintains better tumor oxygenation during illumination and increases direct tumor cell kill in a manner consistent with known oxygen dependencies in PDT-mediated cytotoxicity. BFI-PDT promotes vascular shutdown after PDT, thereby depriving remaining tumor cells of oxygen and nutrients. Collectively, these benefits of BFI-PDT produce a significantly better therapeutic outcome than standard high irradiance PDT. Moreover, BFI-PDT requires ~40% less time on average to achieve outcomes that are modestly better than those with standard low irradiance treatment. This contribution introduces BFI-PDT as a platform for personalized light delivery in PDT, documents the design of a clinically-relevant instrument, and establishes the benefits of BFI-PDT with respect to treatment outcome and duration.

Keywords: Oxyphor; Photofrin®; blood flow monitoring; diffuse correlation spectroscopy; hemodynamic; light modulation; perfusion; phosphorescence quenching; photodynamic therapy; vascular response.

Figure 1

Schematic of blood-flow-informed photodynamic therapy (BFI-PDT) light delivery system.

Figure 2

Representative blood flow traces during standard light delivery to radiation-induced fibrosarcoma (RIF) tumors using (a) 150 mWcm⁻²-continuous; (b) 25 mWcm⁻²-continuous; and (c) 150 mWcm⁻² standard fractionated illumination with 30 second light-on/light-off intervals. Light delivery is shaded in red or yellow for illumination at 150 mWcm⁻² or 25 mWcm⁻², respectively. ► and ■ indicate the initiation and completion of light delivery, respectively. rBF_max(initial) and rBF_min(initial) are the maximum and minimum that define the initial peak and trough in relative blood flow (rBF) during photodynamic therapy (PDT). A dotted bracket on each plot represents the slope of rBF decrease, i.e., flow reduction rate. (d) Box plots of blood flow reduction rate for standard PDT treatments (open circles indicate means; n = 10–11 mice per group). Statistical differences between groups by Wilcoxon rank-sum tests with Holm-Bonferroni adjustment are indicated. * represents groups with statistically significant different flow reduction rates. (e) Kaplan-Meier survival curves for mice treated using standard PDT defined by tertiles of flow reduction rate (n = 10–11 mice per group). P < 0.001 for global Gehan Wilcoxon test of differences between irradiance levels; p < 0.001 for the 25.5–100% rBFmin⁻¹ group versus each lower group and p = 0.027 for 0–12.4% rBFmin⁻¹ versus 12.4–25.5% rBFmin⁻¹.

Figure 3

(a) Flow chart of the process for blood-flow-informed (BFI) light delivery. Irradiances are either 150 mWcm⁻² or 25 mWcm⁻² for BFI-Irrad PDT; and 150 mWcm⁻² or 0 mWcm⁻² for BFI-Frac photodynamic therapy (PDT). Representative blood flow traces for (b) blood-flow-informed irradiance light delivery (BFI-Irrad) and (c) blood-flow-informed fractionated light delivery (BFI-Frac) of radiation-induced fibrosarcoma (RIF) tumors. Light delivery is shaded in red or yellow for illumination at 150 mWcm⁻² or 25 mWcm⁻², respectively. ► and ■ indicate the initiation and completion of light delivery, respectively. rBF_max(initial) and rBF_min(global) are the respective first peak and global minimum of tumor blood flow during light delivery. Dashed red lines in each plot represents the slope of the decrease in rBF between rBF_max(initial) and the rBF_min(global), described as the overall flow reduction rate.

Figure 4

Tumor clonogenicity and oxygenation for standard photodynamic therapy (PDT) with 150 mWcm⁻²-continuous or BFI-PDT (BFI-Irradiance or BFI-Fractionated). (a) Timeline of tumor oxygen tension (pO₂) measurement and excision for clonogenic assay. Dark bands indicate periods of 1-min phosphorescence lifetime measurements of tumor pO₂; labels A-E indicate timepoints at which mice were euthanized for tumor excision. (b) Tumor clonogenicity for each treatment condition/timepoint, 6–7 mice per group. Controls (A) received 15 min of illumination with 150 mWcm⁻²-continuous in the absence of photosensitizer administration; this value was similar to that found for tumors unexposed to light and photosensitizer (log transformed value of 7.9 (0.87 ± 0.3 × 10⁸) clonogenic cells/g) (c) Tumor oxygenation for each treatment condition/timepoint, 5 mice per group. Baseline represents the overall pre-PDT tumor pO₂ for all mice in conditions A-E (n = 20 mice). A-E are post-PDT tumor pO₂ for each control or treatment scheme. For mice that received 150 mWcm⁻²-continuous illumination, pO₂ measurements were taken twice at different timepoints, B and C, post-PDT as indicated in (a). Statistical differences in log-transformed tumor clonogenicity were assessed using a one-way ANOVA and in tumor oxygenation by a mixed effects model to account for repeated measurements on the same animal. The mean for each dataset is indicated by open circles. * represents groups with statistically significant difference.

Figure 5

Kaplan Meier survival curves for radiation-induced fibrosarcoma (RIF)-bearing mice treated using (a) 150 mWcm⁻²-continuous, 25 mWcm⁻²-continuous, and standard 150 mWcm⁻²-fractionated illumination; (b) BFI-Irradiance and BFI-Fractionated illumination, and for comparison purposes, 150 mWcm⁻²-continuous is repeated as a solid line on panel b. (c) Kaplan Meier survival curves for murine mesothelioma tumors (AB12) treated using 150 mWcm⁻²-continuous, 25 mWcm⁻²-continuous and BFI-Irradiance illumination. n = 9–12 mice per group. Differences in the time-to-400 mm³ among groups were assessed using Gehan-Wilcoxon tests for comparisons to 150 mWcm⁻²-continuous for RIF tumors treated with (a) 25 mWcm⁻²-continuous (p = 0.031) and standard fractionated (p = 0.048) or (b) BFI-Irrad (p = 0.006) and BFI-Frac (p < 0.002), and for AB12 tumors (c) treated with 25 mWcm⁻²-continuous (p = 0.021) and BFI-Irrad (p = 0.007).