Hsing-Wen Wang

Noninvasive Monitoring of MurineTumor Blood Flow During and After PhotodynamicTherapy Provides Early Assessment of Therapeutic Efficacy

Purpose: To monitor tumor blood flow noninvasively during photodynamic therapy (PDT) and to correlate flow responses with therapeutic efficacy. Experimental Design: Diffuse correlation spectroscopy (DCS) was used to measure blood flow continuously in radiation-induced fibrosarcoma murine tumors during Photofrin (5 mg/kg)/PDT (75 mW/cm2, 135 J/cm2). Relative blood flow (rBF; i.e., normalized to preillumination values) was compared with tumor perfusion as determined by power Doppler ultrasound and was correlated with treatment durability, defined as the time of tumor growth to a volume of 400 mm3. Broadband diffuse reflectance spectroscopy concurrently quantified tumor hemoglobin oxygen saturation (SO2). Results: DCS and power Doppler ultrasound measured similar flow decreases in animals treated with identical protocols. DCS measurement of rBF during PDT revealed a series of PDT-induced peaks and declines dominated by an initial steep increase (average ± SE: 168.1 ± 39.5%) and subsequent decrease (59.2 ± 29.1%). The duration (interval time; range, 2.2-15.6 minutes) and slope (flow reduction rate; range, 4.4 -45.8% minute−1) of the decrease correlated significantly (P = 0.0001 and 0.0002, r2 = 0.79 and 0.67, respectively) with treatment durability. A positive, significant (P = 0.016, r2 = 0.50) association between interval time and time-to-400 mm3 was also detected in animals with depressed pre-PDT blood flow due to hydralazine administration. At 3 hours after PDT, rBF and SO2 were predictive (P ≤ 0.015) of treatment durability. Conclusion: These data suggest a role for DCS in real-time monitoring of PDT vascular response as an indicator of treatment efficacy.

Hemodynamic responses to antivascular therapy and ionizing radiation assessed by diffuse optical spectroscopies

Diffuse optical methods were used to monitor two different therapies in K1735 malignant mouse melanoma tumor models: anti-vascular therapy and radiation therapy. Anti-vascular therapy induced acute variation in hemodynamic parameters within an hour, and radiation therapy induced longitudinal changes within 2 weeks. During anti-vascular therapy, the drug Combretastatin A-4 3-O-Phosphate (CA4P, 2.5 mg/200 mul PBS/mouse) significantly decreased tissue blood flow (65%) and blood oxygenation (38%) one hour after injection. In the longitudinal study, single-fraction ionizing radiation (12 Gy x 1) induced significant reduction of tissue blood flow (36%) and blood oxygenation (24%) 14 days after radiation. The results correlated well with contrast enhanced ultrasound, tumor histology, and a nitroimidazole hypoxia marker (EF5). The research provides further evidence that noninvasive diffuse optical spectroscopies can be useful tools for monitoring cancer therapy in vivo.

Increasing Damage to Tumor Blood Vessels during Motexafin Lutetium-PDT through Use of Low Fluence Rate

Abstract Photodynamic therapy (PDT) with low light fluence rate has rarely been studied in protocols that use short drug–light intervals and thus deliver illumination while plasma concentrations of photosensitizer are high, creating a prominent vascular response. In this study, the effects of light fluence rate on PDT response were investigated using motexafin lutetium (10 mg/kg) in combination with 730 nm light and a 180-min drug–light interval. At 180 min, the plasma level of photosensitizer was 5.7 ng/&mgr;l compared to 3.1 ng/mg in RIF tumor, and PDT-mediated vascular effects were confirmed by a spasmodic decrease in blood flow during illumination. Light delivery at 25 mW/cm2 significantly improved long-term tumor responses over that at 75 mW/cm2. This effect could not be attributed to oxygen conservation at low fluence rate, because 25 mW/cm2 PDT provided little benefit to tumor hemoglobin oxygen saturation. However, 25 mW/cm2 PDT did prolong the duration of ischemic insult during illumination and was correspondingly associated with greater decreases in perfusion immediately after PDT, followed by smaller increases in total hemoglobin concentration in the hours after PDT. Increases in blood volume suggest blood pooling from suboptimal vascular damage; thus the smaller increases after 25 mW/cm2 PDT provide evidence of more widespread vascular damage, which was accompanied by greater decreases in clonogenic survival. Further study of low fluence rate as a means to improve responses to PDT under conditions designed to predominantly damage vasculature is warranted.

Effect of Photosensitizer Dose on Fluence Rate Responses to Photodynamic Therapy

Photodynamic therapy (PDT) regimens that conserve tumor oxygenation are typically more efficacious, but require longer treatment times. This makes them clinically unfavorable. In this report, the inverse pairing of fluence rate and photosensitizer dose is investigated as a means of controlling oxygen depletion and benefiting therapeutic response to PDT under conditions of constant treatment time. Studies were performed for Photofrin‐PDT of radiation‐induced fibrosarcoma tumors over fluence rate and drug dose ranges of 25–225 mW cm−2 and 2.5–10 mg kg−1, respectively, for 30 min of treatment. Tumor response was similar among all inverse regimens tested, and, in general, tumor hemoglobin oxygen saturation (SO2) was well conserved during PDT, although the highest fluence rate regimen (225 mW × 2.5 mg) did lead to a modest but significant reduction in SO2. Regardless, significant direct tumor cell kill (>1 log) was detected during 225 mW × 2.5 mg PDT, and minimal normal tissue toxicity was found. PDT effect on tumor oxygenation was highly associated with tumor response at 225 mW × 2.5 mg, as well as in all other regimens tested. These data suggest that high fluence rate PDT can be carried out under oxygen‐conserving, efficacious conditions at low photosensitizer dose. Clinical confirmation and application of these results will be possible through use of minimally invasive oxygen and photosensitizer monitoring technologies, which are currently under development.

Quantitative comparison of tissue oxygen and motexafin lutetium uptake by ex vivo and noninvasive in vivo techniques in patients with intraperitoneal carcinomatosis

Near-infrared diffuse reflectance spectroscopy (DRS) has been used to noninvasively monitor optical properties during photodynamic therapy (PDT). This technique has been extensively validated in tissue phantoms; however, validation in patients has been limited. This pilot study compares blood oxygenation and photosensitizer tissue uptake measured by multiwavelength DRS with ex vivo assays of the hypoxia marker, 2-(2-nitroimida-zol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5), and the photosensitizer (motexafin lutetium, MLu) from tissues at the same tumor site of three tumors in two patients with intra-abdominal cancers. Similar in vivo and ex vivo measurements of MLu concentration are carried out in murine radiation-induced fibrosarcoma (RIF) tumors (n=9). The selection of optimal DRS wavelength range and source-detector separations is discussed and implemented, and the association between in vivo and ex vivo measurements is examined. The results demonstrate a negative correlation between blood oxygen saturation (StO(2)) and EF5 binding, consistent with published relationships between EF5 binding and electrode measured pO(2), and between electrode measured pO(2) and StO(2). A tight correspondence is observed between in vivo DRS and ex vivo measured MLu concentration in the RIF tumors; similar data are positively correlated in the human intraperitoneal tumors. These results further demonstrate the potential of in vivo DRS measurements in clinical PDT.

Noninvasive monitoring hemodynamic responses in RIF tumors during and after PDT

Changes in blood flow and oxygenation during and after PDT provide information about tumor vessel and cellular damage. The characterization of these changes may improve our understanding of PDT mechanisms and help predict treatment efficacy. We have designed a hybrid system that can non-invasively measure in vivo hemodynamic changes and provide independent information about tumor oxygenation and blood flow. Diffuse correlation spectroscopy (DCS) monitors blood flow by measuring the optical phase shifts caused by moving blood cells, while diffuse photon density wave (DPDW) spectroscopy measures tissue absorption and scattering. When mounted on a camera, our unique probe allows non-contact measurements that avoid compressing the tumor and altering blood flow. An optical filter mounted in front of the camera lens cut off light below 650nm, which allowed monitoring of blood flow during PDT. The utility of the hybrid system was demonstrated by monitoring the hemodynamic changes during and after PDT in mice bearing the experimental radiation-induced fibrosarcoma (RIF). For the first time, we non-invasively and continually monitored the in vivo flow changes during PDT. Relative oxygen consumption was calculated using flow values measured by DCS and oxygenation measured by a broadband absorption spectrometer. During PDT an initial rapid increase in blood flow was found, followed by a decrease and slow recovery. After PDT, substantial and continued reductions in blood saturation, blood flow and oxygen consumption were found after 3 hours, suggesting that permanent damage to tumor cells and blood vessels had occurred. The comparison of flow values after PDT as measured by DCS and by Power Doppler ultrasound (CWFA) demonstrated that both techniques non-invasively detected similar global changes in tumor blood flow or perfusion after PDT.

In vivo measurements of penetration depth, oxygenation, and drug concentration using broadband absorption spectroscopy in human tissues before and after photodynamic therapy

Photodynamic therapy (PDT) employs a combination of photosensitizing chemical, light, and oxygen Knowledge of tissue optical properties, including absorption (μa) and reduce scattering coefficients (μs’), makes possible to derive blood oxygen saturation, light penetration depth, and drug concentration, which are important to ensure PDT treatment efficacy at the specific wavelengths. We have developed an absorption spectroscopy system to measure μa and μs’ in the spectral range 600-800nm using a contact linear probe with a source fiber and multiple source-detector separation distances less than 1 cm. The μa and μs’ were recovered based on diffusion approximations of the photon transport equation. We measured tissue optical properties among various organs of patients with intraperitoneal malignancies for an on-going Phase II PDT protocol. The results from 12 patients showed various effective penetration depth from site to site and from organ to organ. The percentage oxygen saturation (%StO2) are similar before and after PDT. Before PDT, meff (mean (standard deviation) (number of patients)) in cm-1 at 630nm are 2.4 (0.2) (12) in small bowel, 2.2(0.4) (9) in large bowel, 4.2(2.7) (7) in tumor, 3.3 (0.3) (10) in peritoneum, 2.7 (0.3) (11) in skin, and 10.1 (0.6) (10) in liver. %StO2 is 60-80% for most organs but 30-40% for tumor.

Association of in-vivo and ex-vivo measurements of tissue oxygenation and photosensitizer concentration in patients with intraperitoneal caracinomatosis

We have measured tissue oxygenation and drug concentration using ex-vivo methods and in-vivo diffuse reflectance spectroscopy. The correlation of two methods is reported.

Rapid in-vivo measurement of optical and physiological properties in human intraperitoneal tissues before and after photodynamic therapy

We have measured tissue optical properties during a Phase II clinical trial to treat peritoneal carcinomatosis. Physiological properties before and after PDT will be reported.

Tumor oxygenation measured by non-insavive broadband diffuse reflectance spectroscopy predicts photodynamic therapy outcome

Photodynamic therapy (PDT) requires oxygen to damage tumor. We demonstrated that the fractional change in tumor oxygenation, measrued by diffuse reflectance spectroscopy, shortly after versus before PDT significantly correlates with time-to-regrowth of the same tumor.