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

^{1}

^{2}

^{3}

^{4}

^{5}

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Tumor Models/PDT

^{5}RIF or 1 × 10

^{6}AB12 cells were injected intradermally over the right shoulder or the flank of the mice, respectively. The animals were entered in studies ~one week later with tumor diameters of ~5 mm. The treatment area was depilated (Nair hair removal lotion, Church & Dwight Co., Inc., Ewing, NJ, USA), and Photofrin

^{®}was injected 20–24 h before illumination (tail vein, 5 mg/kg) to allow for its accumulation in tumor tissues [41]. Photofrin

^{®}distributes to both malignant cells and blood vessels in tumors, and with longer incubations it localizes mainly to organelle membranes such as the mitochondria, endoplasmic reticulum and Golgi complex [42,43].

^{−2}, at a high irradiance of 150 mWcm

^{−2}, a low irradiance of 25 mWcm

^{−2}, or a combination of these irradiances as a function of the treatment scheme. It should be noted that external beam PDT induces no to minimal tissue heating (2–3 °C) at irradiances ≤150 mWcm

^{−2}[44]. Mice were anesthetized using ~1.5% isoflurane, while a heating pad maintained body temperature.

^{3}(i.e., time-to-400mm

^{3}). Tumor volume (V) was calculated as V = π/6 × width

^{2}× length. An absence of tumor burden at 90 days after PDT was defined as a complete response. Animal studies were approved by the IACUC of the University of Pennsylvania and animal facilities accredited by AAALAC under protocol #803526 (approved on 3/24/2011, latest renewal 2/12/2020).

#### 2.2. Diffuse Correlation Spectroscopy

#### 2.2.1. DCS Instrumentation

#### 2.2.2. Tumor Blood Flow Monitoring

_{i}) measured at time t (i.e., BF

_{i}(t)) to the baseline flow measurements (BF

_{i}(0)); $rBF\left(t\right)=B{F}_{i}\left(t\right)/B{F}_{i}\left(0\right)\times 100\%$. The percent change in rBF per minute (i.e., %rBFmin

^{−1}) or the slope of rBF was computed in real time by fitting a linear regression model to 72 rBF readings (~3.5 min of data). These parameters provided the source data for defining BFI-PDT light delivery and for in situ decisions about how light irradiances should be varied. In practice, DCS is susceptible to motion artifacts due to sudden movements. Random spikes in blood flow may occur, for example, due to occasional deep breathing or twitching of a muscle during light delivery. Therefore, to avoid false triggers of irradiance changes, the rBF data train was smoothed with a window of 10 data points (~30 s).

#### 2.3. Illumination Schemes

^{−2}. For each scheme, 10–12 mice were treated.

- 150 mWcm
^{−2}-continuous: Continuous illumination at high irradiance of 150 mWcm^{−2}for 15 min. - 25 mWcm
^{−2}-continuous: Continuous illumination at low irradiance of 25 mWcm^{−2}for 90 min. - 150 mWcm
^{−2}-fractionated: 150 mWcm^{−2}in equal intermittent intervals of 30 s light-on and 30 s light-off for a total of 30 min. - Blood-flow-informed-irradiance (BFI-Irrad): Continuous illumination was initially 150 mWcm
^{−2}, but illumination was cyclically decreased to 25 mWcm^{−2}and returned to 150 mWcm^{−2}in response to the blood flow monitoring parameters. Treatment time was adjusted to deliver a total fluence of 135 Jcm^{−2}(between 15 and 90 min). - Blood-flow-informed-fractionated (BFI-Frac): Fractionated illumination was initiated at 150 mWcm
^{−2}, but illumination was intermittently discontinued (light-off, 0 mWcm^{−2}) in response to blood flow monitoring. Treatment time was adjusted to deliver a total fluence of 135 Jcm^{−2}, which was reached within 90 min in the current investigations. Note, this guidance platform requires a maximum treatment time to be established irrespective of whether or not a total fluence of 135 Jcm^{−2}is achieved because the light can remain “off” for extended periods of time if blood flow recovery is slow.

#### 2.4. In Vivo/In Vitro Clonogenic Assay

_{2}) colonies were fixed, stained (2.5 mg/mL methylene blue in 30% alcohol), and counted. The number of clonogenic cells per gram was calculated as the number of cells per gram of tumor multiplied by the ratio of the number of colonies to the number of cells plated.

#### 2.5. Tumor Oxygenation Measurements

_{max}= 523 nm), and the phosphorescence was detected using an avalanche photodiode (APD) through a long-pass filter (710 nm). Phosphorescence lifetime oximetry permits absolute measurement of tumor oxygen partial pressure. The technique is based on variations in the probe phosphorescence decay time due to quenching of the probe triplet state by oxygen. The measurements are thus unaffected by the concentration of the probe, the excitation light intensity, and signal collection efficiency.

#### 2.6. Statistical Analysis

_{2}were analyzed using a mixed effects model. Median time-to-400 mm

^{3}was estimated using the Kaplan-Meier method. In order to weight earlier failure times more strongly, differences in the time-to-400 mm

^{3}among groups were assessed using Gehan-Wilcoxon tests [50]. The proportion of animals with a complete response was estimated and the exact 95% confidence interval (CI) reported. The association between regrowth and either treatment group or flow reduction rate was assessed using a Cox model. Statistical analyses were carried out in R (3.6.1) with the package survminer for pairwise comparisons of the time-to-event data. The family-wise error rate was maintained at 0.05 using either a Holm-Bonferroni correction for multiplicity (flow reduction rate, pO

_{2}, or time-to-400 mm

^{3}) or Tukey’s Honest Significance Difference (clonogenicity, ΔrBF).

## 3. Results

#### 3.1. Irradiance Alters Tumor Blood Flow During PDT

^{−2}in red), (b) low irradiance-continuous (25 mWcm

^{−2}in yellow) and (c) high irradiance-fractionated illumination (150 mWcm

^{−2}light-on intervals in red).

_{max(initial)}and rBF

_{min(initial)}, defined as the respective maximum and minimum flow associated with this first rise and fall (Figure 2a–c). A flow reduction rate was calculated for each mouse as the slope of the line segment between rBF

_{max(initial)}and rBF

_{min(initial)}(depicted by the dotted line). During continuous irradiance at 25 mWcm

^{−2}the median flow reduction rate was 9.3 %rBFmin

^{−1}, a value significantly smaller than the median flow reduction rate of 23 %rBFmin

^{−1}for 150 mWcm

^{−2}-continuous (p < 0.001) and 25 %rBFmin

^{−1}for 150 mWcm

^{−2}-fractionated (p = 0.0016) (Figure 2d and Table 1).

^{3}with groups defined by tertiles of flow-reduction. As in previous studies [11], larger flow reduction rates were associated with worse treatment outcome (p < 0.001 ). In a Cox model, the instantaneous ‘risk’ of regrowth to 400 mm

^{3}increased by a factor of 2.1 (95% confidence interval, 1.6, 2.7) per 10% increase in flow reduction rate (p < 0.001). In a Cox model including only the treatment groups, there was a significant association with time time-to-400 mm

^{3}(p = 0.05), but once flow reduction rate was added to the model, treatment group was no longer statistically significant (p = 0.87). This suggests that a considerable portion of the treatment effect was mediated through flow reduction rate.

#### 3.2. BFI-PDT Alters Flow Dynamics during Illumination Compared to Standard PDT

^{−2}or flow-conserving 25 mWcm

^{−2}. BFI-Frac employed either an irradiance of 150 mWcm

^{−2}, or when the laser was paused, an irradiance of 0 mWcm

^{−2}.

^{−2}. Irradiance was attenuated to 25 mWcm

^{−2}for BFI-Irrad (exemplified in Figure 3b), or to 0 mWcm

^{−2}for BFI-Frac (exemplified in Figure 3c), when the reduction in rBF during PDT was more rapid than a cutoff value of 10% per minute (i.e., when the “time-derivative” of rBF(t) was less than or equal to −10% rBFmin

^{−1}). This cutoff value was selected based on our previous work [11] where we observed rapid tumor regrowth after PDT (time-to-400 mm

^{3}of <10 days) when flow reduction rate was higher than 10 %rBFmin

^{−1}, and substantial delay in tumor regrowth when flow reduction rate was less than or equal to 10 %rBFmin

^{−1}. If rBF(t) recovered to a value above its level during the baseline period (rBF

_{baseline}), then the irradiance returned to 150 mWcm

^{−2}. At each subsequent instance of a flow decrease of more than 10% rBFmin

^{−1}, the irradiance was again attenuated to 25 mWcm

^{−2}(BFI-Irrad) or 0 mWcm

^{−2}(BFI-Frac), and then, the irradiance was returned to 150 mWcm

^{−2}when rBF(t) recovered to above rBF

_{baseline.}Light was delivered until a total fluence of 135 Jcm

^{−2}was reached, or, in the case of BFI-Frac, until a maximum treatment time of 90 min (including light-off time). Figure S1a,b additionally illustrates how temporal fluctuations in rBF (Figure S1a) associate with the slope of its change (Figure S1b); BFI-PDT attenuated the irradiance of light delivery when this slope decreased at a rate greater than 10% rBFmin

^{−1}.

_{min(initial)}for standard PDT generally represents the global minimum in rBF (rBF

_{min(global)}) for the entire treatment. By contrast, during BFI-PDT, modulation of light delivery interrupts and reverses PDT-induced decreases in rBF. Consequently, rBF

_{min(global)}rarely corresponds with rBF

_{min(initial)}for BFI-PDT. The rBF

_{min(global)}during BFI-PDT often occurs later during treatment, i.e., after illumination has been attenuated or paused at least once per the rules of the BFI platform. Furthermore, maximum rBF during standard PDT is generally the first peak in rBF after the start of illumination (i.e., rBF

_{max(initial)}), which differs from BFI-PDT because the BFI platform promotes blood flow recovery that may lead to subsequent peaks higher than rBF

_{max(initial)}. These differences in blood flow trends during BFI-PDT versus standard PDT provide hemodynamic evidence of a modified vascular response during illumination.

#### 3.3. BFI-PDT Decreases Blood Flow Reduction Rate during Light Delivery, While Shortening Treatment Time

_{max(initial)}and rBF

_{min(global)}, as depicted by the red dotted lines in Figure 3b,c. Median overall flow reduction rate for BFI-Irrad was 5.2% rBFmin

^{−1}and for BFI-Frac was 10% rBFmin

^{−1}. These values were compared to those of standard conditions. Overall flow reduction rates for BFI-Irrad were lower than those for each of the standard PDT conditions with continuous illumination (p ≤ 0.001). Overall flow reduction rates for BFI-Frac were lower than for 150 mWcm

^{−}

**using either continuous or fractionated light (p < 0.001 in each case) but did not differ from 25 mWcm**

^{2}^{−}

**continuous (p = 0.63).**

^{2}^{−2}at 25 mWcm

^{−}

**-continuous. Median treatment length for BFI-Irrad was 53 min, about 59% of the time required for 25 mWcm**

^{2}^{−}

**-continuous. During BFI-Irrad, total fluence was divided about equally between 150 mWcm**

^{2}^{−2}and 25 mWcm

^{−2}, corresponding to ~15% of the illumination time at 150 mWcm

^{−2}and ~85% of the time at 25 mWcm

^{−2}. With a median treatment time of 61 min, BFI-Frac took twice as long as for treatment with standard 150 mW/cm

^{2}-fractionated (30 min), but less time than treatment with 25 mWcm

^{−}

**-continuous (90 min). Unlike BFI-Irrad, BFI-Frac incorporates light-off periods. For BFI-Frac, there is no pre-specified maximum time for completion of treatment; if rBF remains below the pre-PDT baseline value for extensive periods, the desired fluence cannot be delivered in a reasonable time. For these reasons, BFI-Irrad is more clinically relevant than BFI-Frac.**

^{2}#### 3.4. BFI-PDT Alters Mechanisms of PDT Effect on Vascular Damage

^{−2}-continuous (mean ΔrBF of −46%; p < 0.001 and 150 mWcm

^{−}

**-fractionated (−32%; p = 0.018) both resulted in substantially more damage to tumor vasculature than 150 mWcm**

^{2}^{−2}-continuous (+25%). For BFI-PDT, both BFI-Irrad (mean −31%; p = 0.016) and BFI-Frac (−44%; p = 0.002) led to more vascular damage after PDT than 150 mWcm

^{−}

**-continuous. In contrast, each BFI-PDT scheme resulted in similar mean ΔrBF compared to 25 mWcm**

^{2}^{−}

**-continuous or 150 mWcm**

^{2}^{−}

**-fractionated PDT (p > 0.05 in each comparison). Thus, PDT produces more vascular damage than the comparative standard treatment with 150 mWcm**

^{2}^{−2}-continuous.

^{−2}-continuous did not reduce tumor clonogenicity. The number of clonogenic cells/g (mean ± SD) was 1.16 ± 0.35 × 10

^{8}for controls and 1.06 ± 0.63 × 10

^{8}at the conclusion PDT (timepoint B). Even when tumors were left for an additional 45 min (a time lapse of 60 min from the start of illumination; timepoint C), treatment with 150 mWcm

^{−2}-continuous did not yield cytotoxicity (mean = 1.61 ± 1.4 × 10

^{8}). In contrast, compared to controls, both BFI-Irrad and BFI-Frac dramatically reduced the number of clonogenic cells/g to 0.18 ± 0.15 × 10

^{8}(p < 0.001) and 0.13 ± 0.11 × 10

^{8}(p < 0.001), respectively, for tumor assayed immediately upon treatment conclusion (timepoints D and E for BFI-Irrad and BFI-Frac, respectively). Because treatment with BFI-Irrad and BFI-Frac required 53 to 61 min, tumor clonogenicity was compared to that at 60 min after the start of 150 mWcm

^{−}

**-continuous (timepoint C). At this timepoint, both BFI-Irrad (p < 0.001) and BFI-Frac (p < 0.001) created an additional ~1 log**

^{2}_{10}of cell kill compared to 150 mWcm

^{−}

**-continuous. Thus, BFI-PDT was consistently more cytotoxic to tumor cells than 150 mWcm**

^{2}^{−}

**-continuous standard PDT.**

^{2}_{2}declined in all experimental groups immediately after illumination (p < 0.001 for each group). At the conclusion of 150 mWcm

^{−2}-continuous illumination, mean tumor oxygenation decreased sharply to a mean of 13.3 mmHg, in contrast to more modest declines to means of 26.7 mmHg and 25.5 mmHg respectively for BFI-Irrad and BFI-Frac (p < 001 for each compared to 150 mW cm

^{−2}-continuous). Interestingly, tumor oxygenation recovered over the 45 min after 150 mWcm

^{−2}-continuous to a mean pO

_{2}of 26.2 mmHg at timepoint C (p < 0.001 compared to timepoint B). This reoxygenation likely reflected recovery of tumor blood flow after PDT, as shown in Figure S2a. The acute hypoxia induced by illumination with 150 mWcm

^{−2}-continuous could therefore limit direct cytotoxicity to tumor cells, and spare tumor vasculature.

#### 3.5. BFI-PDT Improves Therapeutic Outcome

^{−2}-continuous, the median time-to-400 mm

^{3}was 11.0 days compared to 19.6 days for 25 mWcm

^{−2}-continuous (p = 0.031 versus 150 mWcm

^{−2}-continuous) and 18 days for 150 mWcm

^{−2}-fractionated (p = 0.048 versus 150 mWcm

^{−2}-continuous (Figure 5a and Table S1)). No animals at 150 mWcm

^{−2}-continuous achieved a complete response compared to 30% (95% confidence interval (CI) of 7–65%) at 25 mWcm

^{−2}-continuous and 22% (95% CI of 2–48%) at 150 mWcm

^{−2}-fractionated. Importantly, however, standard fractionation was also associated with morbidity that was not found with either of the continuous treatment schemes. Fractionation promoted high levels of edema and culminated in mortality within several days of PDT for 25% (95% CI of 5–57%) of animals.

^{−2}-continuous at inducing a tumor response. For BFI-Irrad, the median time-to-400 mm

^{3}was 29 days (p = 0.006 versus 150 mWcm

^{−2}-continuous) and 40% (95% CI of 12–74.5%) of animals exhibited a complete response (Figure 5b and Table S1). No differences in tumor response were observed for BFI-Irrad versus 25 mWcm

^{−2}-continuous (p = 0.88), but the required treatment time was ~40% shorter for BFI-Irrad than 25 mWcm

^{−2}-continuous (see Table 1).

^{3}was 39 days and 56% (95% CI of 18–91%) achieved a complete response (p < 0.002 versus 150 mWcm

^{−2}-continuous). No differences in tumor response were observed for BFI-Frac versus 25 mWcm

^{−2}-continuous (p = 0.45). BFI-Frac appeared less acutely toxic than standard 150 mWcm

^{−2}-fractionated illumination. With BFI-Frac, 10% (95% CI of 0–45%) of animals experienced a non-acute death (>1 week after PDT), compared to 25% (95% CI of 5–57%) acute deaths within a week of standard 150 mWcm

^{−2}-fractionation.

^{−2}-continuous, 89% (95% CI of 52–100%) for 25 mWcm

^{−2}-continuous and in 100% (95% CI of 69–100%) for BFI-Irrad. The median survival time was 20 days for 150 mWcm

^{−2}-continuous and exceeded 90 days for both 25 mWcm

^{−2}-continuous (p = 0.021) and BFI-Irrad (p = 0.007). Survival times did not differ significantly for 25 mWcm

^{−2}-continuous versus BFI-Irrad (p = 0.29). The median length of treatment with BFI-Irrad was ~60% shorter than that required for 25 mWcm

^{−2}-continuous. Table 2 summarizes illumination duration and other treatment parameters for AB12 tumors. The median overall flow reduction rate for BFI-Irrad was significantly slower at 6% rBFmin

^{−1}compared to 21% rBFmin

^{−1}for 150 mWcm

^{−2}-continuous (p = 0.004) or 13% rBFmin

^{−1}at 25 mWcm

^{−2}-continuous (p = 0.04). BFI-Irrad also produced more vascular shutdown at 1 h after PDT (−59% ΔrBF) compared to 150 mWcm

^{−2}-continuous (−11% ΔrBF) (p = 0.001). Thus, BFI-Irrad PDT produced similar benefit to tumor vascular damage, complete response, and treatment length in both RIF and AB12 tumors.

## 4. Discussion

^{−2}to be more effective than 150 mWcm

^{−2}; however, treatment to a therapeutically relevant fluence of 135 Jcm

^{−2}requires substantially longer time at 25 mWcm

^{−2}(90 min) than at 150 mWcm

^{−2}(15 min). Initially upon illumination, tumor blood flow decreased rapidly at high irradiance, in contrast to more gradual changes at lower irradiance. Rapid decreases could promote hypoxia limiting the therapeutic effect. From these observations, we posited that an interactive approach to light delivery could be guided in real-time by tumor blood flow, utilizing high irradiance during periods of stable blood flow to provide time-efficient light delivery, coupled with low irradiance during periods of declining blood flow to facilitate its recovery. Indeed, results demonstrate that when compared to high irradiance treatment, BFI-PDT interrupts the PDT-induced decrease in blood flow during illumination, conserves tumor oxygenation, increases direct tumor cytotoxicity, and promotes vascular damage after treatment. The tumor damage inflicted by BFI-PDT contributes to significant improvement in long-term therapeutic outcome compared to high irradiance treatment.

^{−2}-continuous, both immediately after illumination and ~60 min after the start of illumination. Both BFI-PDT conditions preserve tumor oxygenation during treatment compared to 150 mWcm

^{−2}-continuous. Maintenance of tumor blood flow during light delivery appears to ensure better supply of oxygen, which, in turn, could contribute to production of more cytotoxic ROS and greater tumor damage. Lower irradiance or light-off cycles during BFI-PDT may also reduce photochemical oxygen consumption, further preserving tumor oxygenation during these treatments.

^{−2}illumination was divided into equal 30- second light-on and light-off intervals, producing treatment outcome similar to that achieved by 25 mWcm

^{−2}-continuous. However, the benefit of fractionated illumination has been inconsistently demonstrated, with others failing to reveal a treatment benefit [56]. These discrepancies may reflect the lack of an informed approach for fractionation. Choice of irradiance, frequency of light fractions and duration of each pause could each affect treatment efficacy. Moreover, the tumor control provided by fractionated illumination in the present study was accompanied by morbidity, resulting in a 25% acute death rate. This is not unexpected, as others have reported high fluence rate illumination could lead to significant morbidity from the inflammation that it may induce [57,58]. In this study, BFI-Frac guided the insertion of pauses in illumination after rapid decreases in blood flow, allowing treated tissue to re-perfuse before resuming illumination. The observed complete response rate was better in BFI-Frac compared to standard fractionated PDT, and importantly, BFI-Frac did not lead to any acute morbidity. BFI-Frac had one delayed morbidity (4 weeks post-PDT) of unknown cause, whereas no morbidities of any kind were associated with BFI-Irrad.

## 5. Conclusions

## Supplementary Materials

^{−2}-continuous; (b) 25 mWcm

^{−2}-continuous; (c) standard 150 mWcm

^{−2}- fractionated illumination; (d) BFI-Irrad; (e) BFI-Frac., Table S1: Summary of median (95% confidence interval) percent of complete response and time-to-400 mm

^{3}for treatments of RIF tumors using each of the standard and BFI-PDT illumination schemes., Table S2: Summary of median (95% confidence interval) percent of complete response and time-to-400 mm

^{3}for treatments of AB12 tumors using 150 mWcm

^{−2}-continuous, 25 mWcm

^{−2}-continuous and BFI-Irrad illumination schemes.

## Author Contributions

## Funding

## Conflicts of Interest

## References

- Thong, P.; Lee, K.; Toh, H.J.; Dong, J.; Tee, C.S.; Low, K.P.; Chang, P.H.; Bhuvaneswari, R.; Tan, N.C.; Soo, K.C. Early assessment of tumor response to photodynamic therapy using combined diffuse optical and diffuse correlation spectroscopy to predict treatment outcome. Oncotarget
**2017**, 8, 19902–19913. [Google Scholar] [CrossRef] [PubMed][Green Version] - Mroz, P.; Hashmi, J.T.; Huang, Y.Y.; Lange, N.; Hamblin, M.R. Stimulation of anti-tumor immunity by photodynamic therapy. Expert Rev. Clin. Immunol.
**2011**, 7, 75–91. [Google Scholar] [CrossRef] [PubMed][Green Version] - Fingar, V.H.; Wieman, T.J.; Wiehle, S.A.; Cerrito, P.B. The role of microvascular damage in photodynamic therapy: The effect of treatment on vessel constriction, permeability, and leukocyte adhesion. Cancer Res.
**1992**, 52, 4914–4921. [Google Scholar] - Busch, T.M. Local physiological changes during photodynamic therapy. Lasers Surg. Med.
**2006**, 38, 494–499. [Google Scholar] [CrossRef] [PubMed] - Khurana, M.; Moriyama, E.H.; Mariampillai, A.; Wilson, B.C. Intravital high-resolution optical imaging of individual vessel response to photodynamic treatment. J. Biomed. Opt.
**2008**, 13, 040502. [Google Scholar] [CrossRef][Green Version] - Wang, W.; Moriyama, L.T.; Bagnato, V.S. Photodynamic therapy induced vascular damage: An overview of experimental PDT. Laser Phys. Lett.
**2013**, 10, 023001. [Google Scholar] [CrossRef] - Rohrbach, D.J.; Tracy, E.C.; Walker, J.; Baumann, H.; Sunar, U. Blood flow dynamics during local photoreaction in a head and neck tumor model. Front. Phys.
**2015**, 3, 13. [Google Scholar] [CrossRef][Green Version] - Yu, G.; Durduran, T.; Zhou, C.; Zhu, T.C.; Finlay, J.C.; Busch, T.M.; Malkowicz, S.B.; Hahn, S.M.; Yodh, A.G. Real-time in situ monitoring of human prostate photodynamic therapy with diffuse light. Photochem. Photobiol.
**2006**, 82, 1279–1284. [Google Scholar] [CrossRef] [PubMed] - Becker, T.L.; Paquette, A.D.; Keymel, K.R.; Henderson, B.W.; Sunar, U. Monitoring blood flow responses during topical ALA-PDT. Biomed. Opt. Express
**2010**, 2, 123–130. [Google Scholar] [CrossRef] [PubMed] - Busch, T.M.; Xing, X.; Yu, G.; Yodh, A.; Wileyto, E.P.; Wang, H.W.; Durduran, T.; Zhu, T.C.; Wang, K.K. Fluence rate-dependent intratumor heterogeneity in physiologic and cytotoxic responses to Photofrin photodynamic therapy. Photochem. Photobiol. Sci.
**2009**, 8, 1683–1693. [Google Scholar] [CrossRef][Green Version] - Yu, G.; Durduran, T.; Zhou, C.; Wang, H.W.; Putt, M.E.; Saunders, H.M.; Sehgal, C.M.; Glatstein, E.; Yodh, A.G.; Busch, T.M. Noninvasive monitoring of murine tumor blood flow during and after photodynamic therapy provides early assessment of therapeutic efficacy. Clin. Cancer Res.
**2005**, 11, 3543–3552. [Google Scholar] [CrossRef][Green Version] - Busch, T.M.; Wileyto, E.P.; Emanuele, M.J.; Del Piero, F.; Marconato, L.; Glatstein, E.; Koch, C.J. Photodynamic Therapy Creates Fluence Rate-dependent Gradients in the Intratumoral Spatial Distribution of Oxygen. Cancer Res.
**2002**, 62, 7273. [Google Scholar] [PubMed] - Henderson, B.W.; Busch, T.M.; Snyder, J.W. Fluence rate as a modulator of PDT mechanisms. Lasers Surg. Med.
**2006**, 38, 489–493. [Google Scholar] [CrossRef] [PubMed] - Angell-Petersen, E.; Spetalen, S.; Madsen, S.J.; Sun, C.-H.; Peng, Q.; Carper, S.W.; Sioud, M.; Hirschberg, H. Influence of light fluence rate on the effects of photodynamic therapy in an orthotopic rat glioma model. J. Neurosurg.
**2006**, 104, 109–117. [Google Scholar] [CrossRef] [PubMed] - Rizvi, I.; Anbil, S.; Alagic, N.; Celli, J.; Zheng, L.Z.; Palanisami, A.; Glidden, M.D.; Pogue, B.W.; Hasan, T. PDT dose parameters impact tumoricidal durability and cell death pathways in a 3D ovarian cancer model. Photochem. Photobiol.
**2013**, 89, 942–952. [Google Scholar] [CrossRef] - Foster, T.H.; Hartley, D.F.; Nichols, M.G.; Hilf, R. Fluence rate effects in photodynamic therapy of multicell tumor spheroids. Cancer Res.
**1993**, 53, 1249–1254. [Google Scholar] - Guo, H.W.; Lin, L.T.; Chen, P.H.; Ho, M.H.; Huang, W.T.; Lee, Y.J.; Chiou, S.H.; Hsieh, Y.S.; Dong, C.Y.; Wang, H.W. Low-fluence rate, long duration photodynamic therapy in glioma mouse model using organic light emitting diode (OLED). Photodiagnosis Photodyn. Ther.
**2015**, 12, 504–510. [Google Scholar] [CrossRef] - Seshadri, M.; Bellnier, D.A.; Vaughan, L.A.; Spernyak, J.A.; Mazurchuk, R.; Foster, T.H.; Henderson, B.W. Light delivery over extended time periods enhances the effectiveness of photodynamic therapy. Clin. Cancer Res.
**2008**, 14, 2796–2805. [Google Scholar] [CrossRef][Green Version] - Busch, T.M.; Wang, H.W.; Wileyto, E.P.; Yu, G.; Bunte, R.M. Increasing damage to tumor blood vessels during motexafin lutetium-PDT through use of low fluence rate. Radiat. Res.
**2010**, 174, 331–340. [Google Scholar] [CrossRef][Green Version] - Sitnik, T.M.; Hampton, J.A.; Henderson, B.W. Reduction of tumour oxygenation during and after photodynamic therapy in vivo: Effects of fluence rate. Br. J. Cancer
**1998**, 77, 1386–1394. [Google Scholar] [CrossRef] - Iinuma, S.; Schomacker, K.T.; Wagnieres, G.; Rajadhyaksha, M.; Bamberg, M.; Momma, T.; Hasan, T. In vivo fluence rate and fractionation effects on tumor response and photobleaching: Photodynamic therapy with two photosensitizers in an orthotopic rat tumor model. Cancer Res.
**1999**, 59, 6164–6170. [Google Scholar] [PubMed] - de Bruijn, H.S.; Brooks, S.; van der Ploeg-van den Heuvel, A.; Ten Hagen, T.L.; de Haas, E.R.; Robinson, D.J. Light Fractionation Significantly Increases the Efficacy of Photodynamic Therapy Using BF-200 ALA in Normal Mouse Skin. PLoS ONE
**2016**, 11, e0148850. [Google Scholar] [CrossRef] [PubMed][Green Version] - de Bruijn, H.S.; van der Veen, N.; Robinson, D.J.; Star, W.M. Improvement of systemic 5-aminolevulinic acid-based photodynamic therapy in vivo using light fractionation with a 75-min interval. Cancer Res.
**1999**, 59, 901–904. [Google Scholar] [PubMed] - de Haas, E.R.; Kruijt, B.; Sterenborg, H.J.; Martino Neumann, H.A.; Robinson, D.J. Fractionated illumination significantly improves the response of superficial basal cell carcinoma to aminolevulinic acid photodynamic therapy. J. Investig. Dermatol.
**2006**, 126, 2679–2686. [Google Scholar] [CrossRef][Green Version] - de Vijlder, H.C.; Sterenborg, H.J.; Neumann, H.A.; Robinson, D.J.; de Haas, E.R. Light fractionation significantly improves the response of superficial basal cell carcinoma to aminolaevulinic acid photodynamic therapy: Five-year follow-up of a randomized, prospective trial. Acta Derm. Venereol.
**2012**, 92, 641–647. [Google Scholar] [CrossRef][Green Version] - van der Veen, N.; Hebeda, K.M.; de Bruijn, H.S.; Star, W.M. Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination. Photochem. Photobiol.
**1999**, 70, 921–929. [Google Scholar] [CrossRef] - Pogue, B.W.; Hasan, T. A theoretical study of light fractionation and dose-rate effects in photodynamic therapy. Radiat. Res.
**1997**, 147, 551–559. [Google Scholar] [CrossRef] - Muller, S.; Walt, H.; Dobler-Girdziunaite, D.; Fiedler, D.; Haller, U. Enhanced photodynamic effects using fractionated laser light. J. Photochem. Photobiol. B
**1998**, 42, 67–70. [Google Scholar] [CrossRef] - Estevez, J.P.; Ascencio, M.; Colin, P.; Farine, M.O.; Collinet, P.; Mordon, S. Continuous or fractionated photodynamic therapy? Comparison of three PDT schemes for ovarian peritoneal micrometastasis treatment in a rat model. Photodiagnosis Photodyn. Ther.
**2010**, 7, 251–257. [Google Scholar] [CrossRef] - Ascencio, M.; Estevez, J.P.; Delemer, M.; Farine, M.O.; Collinet, P.; Mordon, S. Comparison of continuous and fractionated illumination during hexaminolaevulinate-photodynamic therapy. Photodiagnosis Photodyn. Ther.
**2008**, 5, 210–216. [Google Scholar] [CrossRef] - Xiao, Z.; Halls, S.; Dickey, D.; Tulip, J.; Moore, R.B. Fractionated versus standard continuous light delivery in interstitial photodynamic therapy of dunning prostate carcinomas. Clin. Cancer Res.
**2007**, 13, 7496–7505. [Google Scholar] [CrossRef][Green Version] - Durduran, T.; Yodh, A.G. Diffuse correlation spectroscopy for non-invasive, micro-vascular cerebral blood flow measurement. Neuroimage
**2014**, 85, 51–63. [Google Scholar] [CrossRef][Green Version] - Favilla, C.G.; Mesquita, R.C.; Mullen, M.; Durduran, T.; Lu, X.; Kim, M.N.; Minkoff, D.L.; Kasner, S.E.; Greenberg, J.H.; Yodh, A.G.; et al. Optical bedside monitoring of cerebral blood flow in acute ischemic stroke patients during head-of-bed manipulation. Stroke
**2014**, 45, 1269–1274. [Google Scholar] [CrossRef][Green Version] - Busch, D.R.; Rusin, C.G.; Miller-Hance, W.; Kibler, K.; Baker, W.B.; Heinle, J.S.; Fraser, C.D.; Yodh, A.G.; Licht, D.J.; Brady, K.M. Continuous cerebral hemodynamic measurement during deep hypothermic circulatory arrest. Biomed. Opt. Express
**2016**, 7, 3461–3470. [Google Scholar] [CrossRef] - Shang, Y.; Gurley, K.; Yu, G. Diffuse Correlation Spectroscopy (DCS) for Assessment of Tissue Blood Flow in Skeletal Muscle: Recent Progress. Anat. Physiol.
**2013**, 3, 128. [Google Scholar] - Yu, G.; Floyd, T.F.; Durduran, T.; Zhou, C.; Wang, J.; Detre, J.A.; Yodh, A.G. Validation of diffuse correlation spectroscopy for muscle blood flow with concurrent arterial spin labeled perfusion MRI. Opt. Express
**2007**, 15, 1064–1075. [Google Scholar] [CrossRef] - Cochran, J.M.; Chung, S.H.; Leproux, A.; Baker, W.B.; Busch, D.R.; DeMichele, A.M.; Tchou, J.; Tromberg, B.J.; Yodh, A.G. Longitudinal optical monitoring of blood flow in breast tumors during neoadjuvant chemotherapy. Phys. Med. Biol.
**2017**, 62, 4637–4653. [Google Scholar] [CrossRef] - Dong, L.; Kudrimoti, M.; Cheng, R.; Shang, Y.; Johnson, E.L.; Stevens, S.D.; Shelton, B.J.; Yu, G. Noninvasive diffuse optical monitoring of head and neck tumor blood flow and oxygenation during radiation delivery. Biomed. Opt. Express
**2012**, 3, 259–272. [Google Scholar] [CrossRef][Green Version] - Mesquita, R.C.; Han, S.W.; Miller, J.; Schenkel, S.S.; Pole, A.; Esipova, T.V.; Vinogradov, S.A.; Putt, M.E.; Yodh, A.G.; Busch, T.M. Tumor blood flow differs between mouse strains: Consequences for vasoresponse to photodynamic therapy. PLoS ONE
**2012**, 7, e37322. [Google Scholar] [CrossRef][Green Version] - Ong, Y.H.; Dimofte, A.; Kim, M.M.; Finlay, J.C.; Sheng, T.; Singhal, S.; Cengel, K.A.; Yodh, A.G.; Busch, T.M.; Zhu, T.C. Reactive Oxygen Species Explicit Dosimetry for Photofrin-mediated Pleural Photodynamic Therapy. Photochem. Photobiol.
**2020**, 96, 340–348. [Google Scholar] [CrossRef] - Li, L.B.; Luo, R.C. Effect of drug-light interval on the mode of action of Photofrin photodynamic therapy in a mouse tumor model. Lasers Med. Sci.
**2009**, 24, 597–603. [Google Scholar] [CrossRef] - Allison, R.R.; Moghissi, K. Photodynamic Therapy (PDT): PDT Mechanisms. Clin. Endosc.
**2013**, 46, 24–29. [Google Scholar] [CrossRef] - Hsieh, Y.J.; Yu, J.S.; Lyu, P.C. Characterization of photodynamic therapy responses elicited in A431 cells containing intracellular organelle-localized photofrin. J. Cell. Biochem.
**2010**, 111, 821–833. [Google Scholar] [CrossRef] - Shafirstein, G.; Bellnier, D.A.; Oakley, E.; Hamilton, S.; Habitzruther, M.; Tworek, L.; Hutson, A.; Spernyak, J.A.; Sexton, S.; Curtin, L.; et al. Irradiance controls photodynamic efficacy and tissue heating in experimental tumours: Implication for interstitial PDT of locally advanced cancer. Br. J. Cancer
**2018**, 119, 1191–1199. [Google Scholar] [CrossRef] - Buckley, E.M.; Parthasarathy, A.B.; Grant, P.E.; Yodh, A.G.; Franceschini, M.A. Diffuse correlation spectroscopy for measurement of cerebral blood flow: Future prospects. Neurophotonics
**2014**, 1, 011009. [Google Scholar] [CrossRef][Green Version] - Durduran, T.; Choe, R.; Baker, W.B.; Yodh, A.G. Diffuse optics for tissue monitoring and tomography. Rep. Prog. Phys.
**2010**, 73, 076701. [Google Scholar] [CrossRef][Green Version] - Wang, H.-W.; Rickter, E.; Yuan, M.; Wileyto, E.P.; Glatstein, E.; Yodh, A.; Busch*, T.M. Effect of Photosensitizer Dose on Fluence Rate Responses to Photodynamic Therapy. Photochem. Photobiol.
**2007**, 83, 1040–1048. [Google Scholar] [CrossRef] - Esipova, T.V.; Karagodov, A.; Miller, J.; Wilson, D.F.; Busch, T.M.; Vinogradov, S.A. Two New “Protected” Oxyphors for Biological Oximetry: Properties and Application in Tumor Imaging. Anal. Chem.
**2011**, 83, 8756–8765. [Google Scholar] [CrossRef][Green Version] - Gallagher-Colombo, S.M.; Miller, J.; Cengel, K.A.; Putt, M.E.; Vinogradov, S.A.; Busch, T.M. Erlotinib Pretreatment Improves Photodynamic Therapy of Non–Small Cell Lung Carcinoma Xenografts via Multiple Mechanisms. Cancer Res.
**2015**, 75, 3118–3126. [Google Scholar] [CrossRef][Green Version] - Harrington, D.P.; Fleming, T.R. A Class of Rank Test Procedures for Censored Survival Data. Biometrika
**1982**, 69, 553–566. [Google Scholar] [CrossRef] - Maas, A.L.; Carter, S.L.; Wileyto, E.P.; Miller, J.; Yuan, M.; Yu, G.; Durham, A.C.; Busch, T.M. Tumor Vascular Microenvironment Determines Responsiveness to Photodynamic Therapy. Cancer Res.
**2012**, 72, 2079. [Google Scholar] [CrossRef][Green Version] - Engbrecht, B.W.; Menon, C.; Kachur, A.V.; Hahn, S.M.; Fraker, D.L. Photofrin-mediated Photodynamic Therapy Induces Vascular Occlusion and Apoptosis in a Human Sarcoma Xenograft Model. Cancer Res.
**1999**, 59, 4334. [Google Scholar] [PubMed] - Sitnik, T.M.; Henderson, B.W. The effect of fluence rate on tumor and normal tissue responses to photodynamic therapy. Photochem. Photobiol.
**1998**, 67, 462–466. [Google Scholar] [CrossRef] - Feins, R.H.; Hilf, R.; Ross, H.; Gibson, S.L. Photodynamic therapy for human malignant mesothelioma in the nude mouse. J. Surg. Res.
**1990**, 49, 311–314. [Google Scholar] [CrossRef] - Gibson, S.L.; Foster, T.H.; Feins, R.H.; Raubertas, R.F.; Fallon, M.A.; Hilf, R. Effects of photodynamic therapy on xenografts of human mesothelioma and rat mammary carcinoma in nude mice. Br. J. Cancer
**1994**, 69, 473–481. [Google Scholar] [CrossRef][Green Version] - Babilas, P.; Schacht, V.; Liebsch, G.; Wolfbeis, O.S.; Landthaler, M.; Szeimies, R.M.; Abels, C. Effects of light fractionation and different fluence rates on photodynamic therapy with 5-aminolaevulinic acid in vivo. Br. J. Cancer
**2003**, 88, 1462–1469. [Google Scholar] [CrossRef][Green Version] - Francois, A.; Salvadori, A.; Bressenot, A.; Bezdetnaya, L.; Guillemin, F.; D’Hallewin, M.A. How to avoid local side effects of bladder photodynamic therapy: Impact of the fluence rate. J. Urol.
**2013**, 190, 731–736. [Google Scholar] [CrossRef] - Tetard, M.C.; Vermandel, M.; Leroy, H.A.; Leroux, B.; Maurage, C.A.; Lejeune, J.P.; Mordon, S.; Reyns, N. Interstitial 5-ALA photodynamic therapy and glioblastoma: Preclinical model development and preliminary results. Photodiagnosis Photodyn. Ther.
**2016**, 13, 218–224. [Google Scholar] [CrossRef][Green Version]

**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 (pO

_{2}) measurement and excision for clonogenic assay. Dark bands indicate periods of 1-min phosphorescence lifetime measurements of tumor pO

_{2}; 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 × 10

^{8}) clonogenic cells/g) (

**c**) Tumor oxygenation for each treatment condition/timepoint, 5 mice per group. Baseline represents the overall pre-PDT tumor pO

_{2}for all mice in conditions A-E (n = 20 mice). A-E are post-PDT tumor pO

_{2}for each control or treatment scheme. For mice that received 150 mWcm

^{−2}-continuous illumination, pO

_{2}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 mm

^{3}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).

**Table 1.**For RIF tumors, flow reduction rate (overall flow reduction rate for BFI-Irrad and BFI-Frac), treatment length (segregated by time spent at each irradiance), and ΔrBF at 1 h after illumination with standard or BFI-PDT.

Type of PDT | Group | Flow Reduction Rate (%rBFmin^{−1})Median (IQR) | Treatment Length in mMminutes Median (IQR) | ΔrBF(%) at 1 h after PDT Mean (SD) | |||
---|---|---|---|---|---|---|---|

150 mWcm^{−2} | 25 mWcm^{−2} | 0 mWcm^{−2} | Total | ||||

Standard | 150 mWcm continuous^{−2} | 23.0 (21.4, 49.8) | 15 | 0 | 0 | 15 | 24.8 (67.3) |

25 mWcm^{−2} continuous | 9.3 (7.8, 11.6) (p < 0.001 ^{1}) | 0 | 90 | 0 | 90 | −46.2 (23.7) (p = 0.001 ^{1}) | |

150 mWcm fractionated^{−2} | 25.0 (14.9 34,6) (p = 0.002 ^{2}) | 15 | 0 | 15 | 30 | −32.2 (31.3) (p = 0.018 ^{1}) | |

Blood-flow informed | BFI-Irrad | 5.2 (4.6, 5.4) (p < 0.001 ^{1,3}, p = 0.001^{2}) | 8 (7, 9) | 45 (39, 50) | 0 | 53 (48, 57) | −31.4 (19.7) (p = 0.016 ^{1}) |

BFI-Frac | 10.0 (6.7, 10.2) (p < 0.001 ^{1,3}) | 15 | 0 | 46 (34, 55) | 61 (49, 70) | −43.6 (29.0) (p = 0.002 ^{1}) |

^{1}for comparison to 150 mWcm

^{−2}-continuous;

^{2}for comparison to 25 mWcm

^{−2}-continuous;

^{3}for comparison to 150 mWcm

^{−2}-fractionated. RIF: radiation-induced fibrosarcoma; PDT: photodynamic therapy; rBF: relative blood flow; BFI-Irrad: blood-flow-informed irradiance; BFI-Frac: blood-flow-informed fractionated; IQR: interquartile range; SD: standard deviation.

**Table 2.**For AB12 tumors, flow reduction rate (overall flow reduction rate for BFI-Irrad), treatment length (segregated by time spent at each irradiance), and ΔrBF at 1 h after PDT with 150 mWcm

^{−2}-continuous, 25 mWcm

^{−2}-continuous or BFI-Irrad.

Group | Flow Reduction Rate (%rBFmin^{−1})Median (IQR) | Treatment Length in Minutes Median (IQR) | ΔrBF(%) at 1 h after PDT Mean (SD) | |||
---|---|---|---|---|---|---|

150 mWcm^{−2} | 25 mWcm^{−2} | 0 mWcm^{−2} | Total | |||

150 mWcm^{−}^{2}-continuous | 20.7 (15.5, 32.6) | 15 | 0 | 0 | 15 | −10.7 (21.6) |

25 mWcm^{−}^{2}-continuous | 13.4 (11.9, 16.8) | 0 | 90 | 0 | 90 | −56.6 (18.6) (p = 0.001 ^{1}) |

BFI-Irrad | 6.0 (3.7, 12.1) (p = 0.004 ^{1}, p = 0.04 ^{2}) | 11 (9, 14) | 27 (9, 36) | 0 | 37 (23, 45) | −58.5 (15.3) (p = 0.001 ^{1}) |

^{1}for comparison to 150 mWcm

^{−2}-continuous;

^{2}for comparison to 25 mWcm

^{−2}-continuous. AB12: mouse mesothelioma cell line; PDT: photodynamic therapy; rBF: relative blood flow; BFI-Irrad: blood-flow-informed irradiance; BFI-Frac: blood-flow-informed fractionated; IQR: interquartile range; SD: standard deviation.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Ong, Y.H.; Miller, J.; Yuan, M.; Chandra, M.; El Khatib, M.; Vinogradov, S.A.; Putt, M.E.; Zhu, T.C.; Cengel, K.A.; Yodh, A.G.; Busch, T.M. Blood Flow Measurements Enable Optimization of Light Delivery for Personalized Photodynamic Therapy. *Cancers* **2020**, *12*, 1584.
https://doi.org/10.3390/cancers12061584

**AMA Style**

Ong YH, Miller J, Yuan M, Chandra M, El Khatib M, Vinogradov SA, Putt ME, Zhu TC, Cengel KA, Yodh AG, Busch TM. Blood Flow Measurements Enable Optimization of Light Delivery for Personalized Photodynamic Therapy. *Cancers*. 2020; 12(6):1584.
https://doi.org/10.3390/cancers12061584

**Chicago/Turabian Style**

Ong, Yi Hong, Joann Miller, Min Yuan, Malavika Chandra, Mirna El Khatib, Sergei A. Vinogradov, Mary E. Putt, Timothy C. Zhu, Keith A. Cengel, Arjun G. Yodh, and Theresa M. Busch. 2020. "Blood Flow Measurements Enable Optimization of Light Delivery for Personalized Photodynamic Therapy" *Cancers* 12, no. 6: 1584.
https://doi.org/10.3390/cancers12061584