Simple Photodynamic Therapy Dose Models Fail to Predict the Survival of MLL Cells After HPPH–PDT In Vitro

Fluorescence photobleaching, photodynamic therapy (PDT) oxygen consumption and clonogenic cell survival were investigated during 2-(1-hexyloxethyl)-2-devinyl pyropheophoribde-a (HPPH) PDT of MAT-LyLu cells in vitro . Cells were incubated with HPPH concentrations of 0.24, 1.2, 3.6 or 12 μ m for 4 h and then treated with 650 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment and photobleaching was quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony forming assay. Intracellular fluorescence lifetime measurements were also performed at each incubation concentration. The photobleaching kinetics did not follow first- or second-order kinetics and the fluorescence lifetime was similar for all intracellular concentrations. As the intracellular concentration of drug was increased, the amount of singlet oxygen and the absorbed quanta per cell required to achieve the same cell kill increased. Singlet oxygen dose was calculated using one- and two-compartment models of HPPH intracellular distribution. It was found that a two-compartment model, in which a PDT-sensitive binding site saturates at low concentrations, accounts for the observed photobleaching, oxygen consumption and cell survival.     

Calculation of singlet oxygen dose from photosensitizer fluorescence and photobleaching during mTHPC photodynamic therapy of MLL cells

Predicting the therapeutic outcome of photodynamic therapy (PDT) requires knowledge of the amount of cytoxic species generated. An implicit approach to assessing PDT efficacy has been proposed where changes in photosensitizer (PS) fluorescence during treatment are used to predict treatment outcome. To investigate this, in vitro experiments were performed in which Mat-LyLu cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 652 nm light. PS concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was monitored during treatment and, at selected fluence levels, cell viability was determined using a colony-formation assay. Singlet oxygen dose was calculated using four different models and was compared with cell survival. For the dose metric based on singlet oxygen-mediated PS photobleaching, a universal relationship between cell survival and singlet oxygen dose was found for all treatment parameters. Analysis of the concentration dependence of bleaching suggests that the lifetime of singlet oxygen within the cell is 0.05-0.25 micros. Generation of about 9 x 10(8) molecules of singlet oxygen per cell reduces the surviving fraction by 1/e.     

Investigation of photobleaching of hypocrellin B in non-polar organic solvent and in liposome suspension

Hypocrellin B (HB) is a natural pigment with a promising application in the photodynamic therapy (PDT) for anticancer treatment. The photobleaching of HB in non-polar organic solvents and in liposomes in aqueous solution were investigated by the measurements of absorption spectra, quenching experiments and determination of photoproducts. Control experiments indicated that the sensitizer, oxygen and light were all essential for the photobleaching of HB, which suggested that it was mainly self-sensitized photooxidation. The illumination of HB with visible light in aerobic non-polar solvent generated singlet oxygen efficiently [Phi(1O(2))=0.76] which then attacked the sensitizer HB with formation of an endoperoxide product. The endoperoxide of HB was unstable at room temperature and underwent predominantly loss of singlet oxygen with regeneration of parent HB. The singlet oxygen released from the endoperoxide of HB was detected with chemical trapping experiments. When HB was embedded in EPC liposomes, no endoperoxide product and no singlet oxygen release from the photobleaching process of HB were detected. The quenching experiments indicated that the singlet oxygen mechanism (type II) played an important role in the non-polar solvent and the free radical mechanism (type I) was predominant in liposomal aqueous solution for the photobleaching of HB.     

Photobleaching-based dosimetry predicts deposited dose in ALA-PpIX PDT of rodent esophagus

An improved method to estimate dose to esophageal tissue was investigated in the setting of photodynamic therapy with aminolevulinic acid-induced protoporphyrin IX (PpIX) treatment. A model of treatment-induced edema in the esophagus mucosa proved to be a well controlled and useful way to test the dosimetry model, and the light from the treatment laser together with the PpIX fluorescence intensity could be quantified reliably in real time. Dosimetry calculations based upon the detected fluorescence and bleaching kinetics were used to calculate the "effective" dose to the tissue, and a correlation was shown to exist between this metric and the edema induced in the esophagus. The difference between animals with no detectable treatment effect and those with significant edema was predictable based upon the dose calculation. The underlying assumption in the interpretation of the data is that rapid photobleaching of PpIX occurs when there is ample oxygen supply, and this bleaching is not present when oxygen is limited. This leads to the prediction that integration of the light and drug dose, in intervals where appreciable photobleaching occurs, should provide a prediction of the relative dose of singlet oxygen produced. This detection system and rodent model can be used for prospective dosimetry studies that focus on optimization of esophageal PDT.     

Monitoring photoproduct formation and photobleaching by fluorescence spectroscopy has the potential to improve PDT dosimetry with a verteporfin-like photosensitizer

In current clinical practice, photodynamic therapy (PDT) is carried out with prescribed drug doses and light doses as well as fixed drug-light intervals and illumination fluence rates. This approach can result in undesirable treatment outcomes of either overtreatment or undertreatment because of biological variations between different lesions and patients. In this study, we explore the possibility of improving PDT dosimetry by monitoring drug photobleaching and photoproduct formation. The study involved 60 mice receiving the same drug dose of a novel verteporfin-like photosensitizer, QLT0074, at 0.3 mg/kg body weight, followed by different light doses of 5, 10, 20, 30, 40 or 50 J/cm2 at 686 nm and a fluence rate of 70 mW/cm2. Photobleaching and photoproduct formation were measured simultaneously, using fluorescence spectroscopy. A ratio technique for data processing was introduced to reliably detect the photoproduct formed by PDT on mouse skin in vivo. The study showed that the QLT0074 photoproduct is stable and can be reliably quantified. Three new parameters, photoproduct score (PPS), photobleaching score (PBS) and percentage photobleaching score (PBS%), were introduced and tested together with the conventional dosimetry parameter, light dose, for performance on predicting PDT-induced outcome, skin necrosis. The statistical analysis of experimental results was performed with an ordinal logistic regression model. We demonstrated that both PPS and PBS improved the prediction of skin necrosis dramatically compared to light dose. PPS was identified as the best single parameter for predicting the PDT outcome.     

Photobleaching Kinetics and Effect of Solvent in the Photophysical Properties of Indocyanine Green for Photodynamic Therapy

Indocyanine green is an attractive molecule for photodynamic therapy due to its near infrared absorption, resulting in a higher tissue penetration. However, its quantum yields of the triplet and singlet state have been reported to be low and then, reactive oxygen species are unlikely to be formed. Aiming to understand the ICG role in photodynamic response, its photobleaching behavior in solution has been studied under distinct conditions of CW laser irradiation at 780 and 808 nm, oxygen saturations and solvents. Sensitizer bleaching and photoproduct formation were measured by absorption spectroscopy and analyzed using the PDT bleaching macroscopic model to extract physical parameters. ICG photobleaching occurs even at lower oxygen concentrations, indicating that the molecule presents more than one way of degradation. Photoproducts were produced even in solution of less than 4 % oxygen saturation for both solvents and excitation wavelengths. Also, the amplitude of absorption related to J-dimers was increased during irradiation, but only in 50 % PBS solution. The formation of photoproducts was enhanced in the presence of J-type dimers under low oxygen concentration, and the quantum yields of triplet and singlet states were one order of magnitude and two times higher, respectively, when compared to ICG in distilled H₂O.     

Photodynamic actinometry using microspheres: concept, development and responsivity

Photodynamic therapy (PDT) relies on three main ingredients, oxygen, light and photoactivating compounds, although the PDT response is definitively contingent on the site and level of reactive oxygen species (ROS) generation. This study describes the development of a novel, fluorescent-based actinometer microsphere system as a means of discerning spatially resolved dosimetry of total fluence and ROS production. Providing a high resolution, localized, in situ measurement of fluence and ROS generation is critical for developing in vivo PDT protocols. Alginate-poly-L-lysine-alginate microspheres were produced using ionotropic gelation of sodium alginate droplets, ranging from 80 to 200 microm in diameter, incorporating two dyes, ADS680WS (ADS) and Rhodophyta-phycoerythrin (RPE), attached to the spheres' inside and outside layers, respectively. To test the responsivity and dynamic range of RPE for ROS detection, the production of ROS was initiated either chemically using increasing concentrations of potassium perchromate or photochemically using aluminum tetrasulphonated phthalocyanine. The generation of singlet oxygen was confirmed by phosphorescence at 1270 nm. The resulting photodegradation and decrease in fluorescence of RPE was found to correlate with increased perchromate or PDT treatment fluence, respectively. This effect was independent of pH (6.5-8) and could be inhibited using sodium azide. RPE was not susceptible to photobleaching with light alone (670 nm; 150 Jcm(-2)). ADS, which absorbs light between 600 and 750 nm, showed a direct correlation between radiant exposure (670 nm; 0-100 Jcm(-2)) and diminished fluorescence. Photobleaching was independent of irradiance (10-40 mW cm(-2)). We propose that actinometer microspheres may provide a means for obtaining high spatial resolution information regarding delivered PDT dose within model systems during investigational PDT development and dosimetric information for clinical extracorporeal PDT as in the case of ex vivo bone marrow purging.     

Modeling of a type II photofrin-mediated photodynamic therapy process in a heterogeneous tissue phantom

We present a quantitative framework to model a Type II photodynamic therapy (PDT) process in the time domain in which a set of rate equations are solved to describe molecular reactions. Calculation of steady-state light distributions using a Monte Carlo method in a heterogeneous tissue phantom model demonstrates that the photon density differs significantly in a superficial tumor of only 3 mm thickness. The time dependences of the photosensitizer, oxygen and intracellular unoxidized receptor concentrations were obtained and monotonic decreases in the concentrations of the ground-state photosensitizer and receptor were observed. By defining respective decay times, we quantitatively studied the effects of photon density, drug dose and oxygen concentration on photobleaching and cytotoxicity of a photofrin-mediated PDT process. Comparison of the dependences of the receptor decay time on photon density and drug dose at different concentrations of oxygen clearly shows an oxygen threshold under which the receptor concentration remains constant or PDT exhibits no cytotoxicity. Furthermore, the dependence of the photosensitizer and receptor decay times on the drug dose and photon density suggests the possibility of PDT improvement by maximizing cytotoxicity in a tumor with optimized light and drug doses. We also discuss the utility of this model toward the understanding of clinical PDT treatment of chest wall recurrence of breast carcinoma.     

Measurement of Intracellular Oxygen Concentration During Photodynamic Therapy in vitro

A technique is introduced that monitors the depletion of intracellular ground state oxygen concentration ([³O₂]) during photodynamic therapy of Mat‐LyLu cell monolayers and cell suspensions. The photosensitizer Pd(II) meso‐tetra(4‐carboxyphenyl)porphine (PdT790) is used to manipulate and indicate intracellular [³O₂] in both of the in vitro models. The Stern–Volmer relationship for PdT790 phosphorescence was characterized in suspensions by flowing nitrogen over the suspension while short pulses of 405 nm light were used to excite the sensitizer. The bleaching of sensitizer and the oxygen consumption rate were also measured during continuous exposure of the cell suspension to the 405 nm laser. Photodynamic therapy (PDT) was conducted in both cell suspensions and in cell monolayers under different treatment conditions while the phosphorescence signal was acquired. The intracellular [³O₂] during PDT was calculated by using the measured Stern–Volmer relationship and correcting for sensitizer photobleaching. In addition, the amount of oxygen that was consumed during the treatments was calculated. It was found that even at large oxygen consumption rates, cells remain well oxygenated during PDT of cell suspensions. For monolayer treatments, it was found that intracellular [³O₂] is rapidly depleted over the course of PDT.     

Effects of fluence rate on cell survival and photobleaching in meta-tetra-(hydroxyphenyl)chlorin-photosensitized Colo 26 multicell tumor spheroids

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm-2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 +/- 2.2 mM in this system.     

Porphyrin bleaching and PDT-induced spectral changes are irradiance dependent in ALA-sensitized normal rat skin in vivo

Photobleaching kinetics of aminolevulinic acid-induced protoporphyrin IX (PpIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for changes in excitation wavelength optical properties using diffuse reflectance measured at the excitation wavelength. Loss of PpIX fluorescence was monitored during photodynamic therapy (PDT) performed using 514 nm irradiation. Bleaching in response to irradiances of 1, 5 and 100 mW cm-2 was evaluated. The results demonstrate an irradiance dependence to the rate of photobleaching vs irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence. The kinetics for the accumulation of the primary fluorescent photoproduct of PpIX also exhibit an irradiance dependence, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction mechanism in vivo, and they provide spectroscopic evidence that PDT delivered at low irradiance deposits greater photodynamic dose for a given irradiation fluence. We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.     

Fluorescence Photobleaching of ALA-induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced dam-age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo-bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo-bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm², 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced dam-age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo-bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.     

血浆溶液中竹红菌乙素和2-牛磺酸修饰的竹红菌乙素的光漂白

光动力学疗法是应用光敏剂受激光激发后对靶体产生光化学作用来治疗病变。光漂白是光动力学治疗过程中普遍现象，在光动力疗法治疗血管类疾病中，光敏剂与血浆中的生物分子相互作用及其在血管中的光漂白行为直接关系到治疗效果。本文考察了HB和THB与血浆的作用和在血浆溶液中的光漂白过程，研究表明在富氧条件下，以单重态氧漂白为主；在有血浆生物分子溶液中光产物与水溶液中的光产物不同。研究表明光敏剂的结构和氧化电位导致了它们不同的光漂白机制，HB和THB与生物分子的相互作用加速了它们光漂白并影响了光产物。     

Benzophenothiazine and Benzoporphyrin Derivative Combination Phototherapy Effectively Eradicates Large Murine Sarcomas

Abstract— The tumoricidal effects of photochemotherapy with two photosensitizers, 5-ethylamino-9-diethylaminobenzo[ a ] phenothiazinium chloride (EtNBS) and benzoporphyrin derivative monoacid ring A (BPD-MA), were evaluated separately and in combination against the EMT-6 fibrosarcoma implanted subcutaneously in BALB/c mice. Animals carrying tumors 8-10 mm in diameter were divided into eight different groups (∼20/group) and subjected to various photoirradiation and drug conditions. The tumor response to photodynamic therapy (PDT) was measured as the mean tumor wet weight 2 weeks post-PDT. The combination treatment with 5.25 mg/kg EtNBS and 2.5 mg/kg BPD-MA followed by photoirradiation with 100 J/cm² at 652 nm and then by 100 J/cm² at 690 nm resulted in a 95% reduction in the average tumor weights compared to controls (no light, no drugs) with 76% of the mice being tumor free 2 weeks post-PDT. Because treatment with EtNBS or BPD-MA at twice the light dose and drug concentration resulted in either no significant reduction in tumor weights or increased the lethality of treatment, respectively, the data suggest that the enhanced PDT effect observed with the combination of drugs is synergistic rather than additive. Histology of tumors 24 h post-PDT with the combination of drugs showed nearly complete destruction of the tumor mass with little or no damage to the vasculature and no extravasation of red blood cells. There was no damage to the normal skin adjacent to the tumor. Fluorescence microscopy of EMT-6 cells incubated in vitro with the two photosensitizers revealed that they were localized to different intracellular compartments. The fluorescence pattern from frozen tumor tissue slices following the in vivo administration of the photosensitizers indicated a greater intracellular localization for EtNBS vs BPD-MA.     

Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin

Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is one of the most potent photosensitizers currently available for clinical photodynamic therapy (PDT). However the reason or reasons for its high photodynamic efficacy remain(s) unresolved. To investigate the PDT efficacy of mTHPC vs Photofrin we use the knowledge of photophysical parameters extracted from the analysis of oxygen electrode measurements in spheroids to compute and compare their respective singlet oxygen (1O2) dose depositions. The electrode measurements indirectly report the bleaching kinetics of mTHPC and indicate that its photobleaching mechanism is consistent with 1O2-mediated reactions. mTHPC's photodegradation via 1O2 reactions is confirmed by a more direct evaluation of the spatially resolved fluorescence in confocal sections of intact spheroids during irradiation. The PDT efficacy comparisons establish that mTHPC's enhanced potency may be accounted for completely on the basis of its ability to sequester tightly in cells and its photophysical properties, in particular its higher extinction coefficient at a redshifted wavelength. We extend the efficacy comparison to include the influence of hemoglobin absorption of PDT treatment light and show that incorporating the influence of wavelength-dependent light attenuation in tissue further contributes to significantly higher efficacy for mTHPC- vs Photofrin-PDT.     

Skin Necrosis due to Photodynamic Action of Benzoporphyrin Depends on Circulating Rather than Tissue Drug Levels: Implications for Control of Photodynamic Therapy

In an ideal world, photodynamic therapy (PDT) of abnormal tissue would reliably spare the surrounding normal tissue. Normal tissue responses set the limits for light and drug dosimetry. The threshold fluence for necrosis (TFN) was measured in normal skin following intravenous infusion with a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) Verteporfin as a function of drug dose (0.25-2.0 mg/kg), wavelength of irradiation (458 and 690 nm) and time interval (0–5h) between drug administration and irradiation. The BPD-MA levels were measured in plasma and skin tissue to elucidate the relationship between TFN, drug kinetics and biodistribution. The PDT response of normal skin was highly reproducible. The TFN for 458 and 690 nm wavelengths was nearly identical and the estimated quantum efficiency for skin response was equal at these two wavelengths. Skin phototoxicity, quantified in terms of 1/ TFN, closely correlated with the plasma pharmacokinetics rather than the tissue pharmacokinetics and was quadratically dependent on the plasma drug concentration regardless of the administered drug dose or time interval between drug and light exposure. This study strongly suggests that noninvasive measurements of the circulating drug level at the time of light treatment will be important for setting optimal light dosimetry for PDT with liposomal BPD-MA, a vascular photosensitizer.     

Photobleaching of hypericin bound to human serum albumin,cultured adenocarcinoma cells and nude mice skin

Hypericin is a promising photosensitizer for photodynamic therapy (PDT) characterized by a high yield of singlet oxygen. Photobleaching of hypericin has been studied by means of absorption and fluorescence spectroscopy in different biological systems: in human serum albumin solution, in cultured human adenocarcinoma WiDr cells and in the skin of nude mice. Prolonged exposure to light (up to 95 min, 100 mW/cm2) of wavelength around 596 nm induced fluence-dependent photobleaching of hypericin in all studied systems. The photobleaching was not oxygen dependent, and singlet oxygen probably played no significant role. Emission bands in the spectral regions 420-560 nm and above 600 nm characterize the photoproducts formed. An emission band at 615-635 nm was observed after irradiation of cells incubated with hypericin or of mouse skin in vivo but not in albumin solution. The excitation spectrum of these products resembled that of hypericin. Hypericin appears to be more photostable than most sensitizers used in PDT, including mTHPC and Photofrin.     

The influence of oxygen depletion and photosensitizer triplet-state dynamics during photodynamic therapy on accurate singlet oxygen luminescence monitoring and analysis of treatment dose response

To date, singlet oxygen ((1)O(2)) luminescence (SOL) detection was predictive of photodynamic therapy (PDT) treatment responses both in vitro and in vivo, but accurate quantification is challenging. In particular, the early and strongest part of the time-resolved signal (500-2000ns) is difficult to separate from confounding sources of luminescence and system noise, and so is normally gated out. However, the signal dynamics change with oxygen depletion during PDT, so that this time gating biases the (1)O(2) measurements. Here, the impact of gating was investigated in detail, determining the rate constants from SOL and direct pO(2) measurements during meso-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated PDT of cells in vitro under well-controlled conditions. With these data as input, numerical simulations were used to examine PDT and SOL dynamics, and the influence of various time gates on cumulative SOL signals. It is shown that gating can underestimate the SOL at early treatment time points by ～40% and underestimate the cumulative SOL signal by 20-25%, representing significant errors. In vitro studies with both mTHPC and aminolevulinic acid-photosensitizer protoporphyrin IX demonstrate that rigorous analysis of SOL signal kinetics is then crucial in order to use SOL as an accurate and quantitative PDT dose metric.     

A Comparison of Dose Metrics to Predict Local Tumor Control for Photofrin‐mediated Photodynamic Therapy

This preclinical study examines light fluence, photodynamic therapy (PDT) dose and “apparent reacted singlet oxygen,” [¹O₂]_rx, to predict local control rate (LCR) for Photofrin‐mediated PDT of radiation‐induced fibrosarcoma (RIF) tumors. Mice bearing RIF tumors were treated with in‐air fluences (50–250 J cm^?2) and in‐air fluence rates (50–150 mW cm^?2) at Photofrin dosages of 5 and 15 mg kg^?1 and a drug‐light interval of 24 h using a 630‐nm, 1‐cm‐diameter collimated laser. A macroscopic model was used to calculate [¹O₂]_rx and PDT dose based on in vivo explicit dosimetry of the drug concentration, light fluence and tissue optical properties. PDT dose and [¹O₂]_rx were defined as a temporal integral of drug concentration and fluence rate, and singlet oxygen concentration consumed divided by the singlet oxygen lifetime, respectively. LCR was stratified for different dose metrics for 74 mice (66 + 8 control). Complete tumor control at 14 days was observed for [¹O₂]_rx ≥ 1.1 mm or PDT dose ≥1200 μm J cm^?2 but cannot be predicted with fluence alone. LCR increases with increasing [¹O₂]_rx and PDT dose but is not well correlated with fluence. Comparing dosimetric quantities, [¹O₂]_rx outperformed both PDT dose and fluence in predicting tumor response and correlating with LCR.