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 μ l 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). 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Introduction
Knowledge of tumor hemodynamic properties such as blood oxygenation and blood fow can inform tumor therapy and permit optimization of individual treatment protocols [1][2][3]. Thus noninvasive, repetitive measurements of these parameters are attractive in the clinical context. Radiation, for example, is an important nonsurgical tool for treatment of tumors for which tumor oxygen status affects therapy outcome [4,5]; in this case higher radiation doses are needed to kill hypoxic tumor cells compared to well-oxygenated cells [1,6,7]. In a different vein, tumor responses to drug therapies depend on adequate blood fow to replenish tissue oxygen and for effcient drug delivery [8,9]; in some cases anti-vascular drug therapy has even been used as an adjuvant strategy for radiation therapy, enhancing radiation effcacy by modifying tumor vasculature and consequently, tumor blood fow and oxygenation [10-12].
Repetitive measurements of blood fow and oxygenation are also valuable for pre-clinical animal studies that, for example, facilitate further understanding of antivascular agents and their translation to the clinic. In this study we focus on the potential of diffuse optics for frequent assessment of tissue hemodynamics in pre-clinical models. In particular, we investigate tumor blood fow and blood oxygenation changes induced by an anti-vascular drug, Combretastatin A-4 3-O-Phosphate (CA4P), and by radiation. We employ two noninvasive optical methods for these studies: diffuse correlation spectroscopy (DCS) and diffuse refectance spectroscopy (DRS).
Diffuse correlation spectroscopy (DCS) measures the temporal fuctuations of light transmitted through tissue and enables us to assess dynamical changes of tumor blood fow by detecting the motion of blood cells. To  . Diffuse refectance spectroscopy (DRS) is a more traditional near-infrared (NIR) spectroscopy approach that uses the average attenuation and phase shift of scattered light to derive information about blood oxygen saturation; it has recently been applied in therapy monitoring [20, 26-28].
Herein we report on measurements of hemodynamics induced by CA4P and by the radiation in K1735 malignant melanoma tumor models. Our results clearly demonstrate CA4P induced rapid shutdown of tumor blood fow and tumor hypoxia. On the other hand, radiation effects were delayed; signifcant reductions in both blood fow and oxygenation occurred during the second week. The research further validates and corroborates the utility of the diffuse optical methodologies in a useful context.

Animal and tumor model
All animal experiments were approved by the University of Pennsylvania Animal Care and Use Committee. Six-to 8-week-old C3H/HeN female mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) and were maintained in micro-isolator cages under sterile conditions. The K1735 melanoma tumor cell lines were cultured and injected subcutaneously into anesthetized mice. Measurements were made when tumors reached a diameter of 6-10 mm, generally 2-3 weeks after cell inoculation. During diffuse optical measurements mice were anesthetized with an isofurane and air mixture.
For antivascular therapy study, 9 and 5 mice were used for DCS and DRS measurements, respectively. Optical measurements were carried out ∼5 minutes before CA4P injection to provide a baseline and after 1 hour to provide post injection information. DCS was continuously employed during the 1 hour period. At least 2 repeated measurements were obtained for DCS and 10 repeated measurements were carried out for DRS. For radiation therapy experiments, the frst group of mice were irradiated using a Philips X-ray generator operating at 225 kV, 17 mA, and using a 0.2 μm Cu flter at a dose rate of 3.4 Gy/min. The mice bodies were shielded with lead except for the tumor-bearing hind limb. Tumors were irradiated with 12 Gy in a single fraction (N 8). Optical measurements were carried out prior to radiation to provide a baseline, and then were carried out again at 7-days and 14-days post-radiation. Control tumors (N 8), which did not receive radiation and grew more quickly than the radiated ones, were measured at baseline, 5-days and 12-days after baseline measurements. Minimum 5 repeated measurements were carried out for each group by positioning the optical probe on different tumor locations. Therefore, the averages presented in this paper represent the mean obtained from a total of (N animals ) × (N repeated measurements ) data sets for both studies.

Contrast-enhanced ultrasound imaging
To corroborate the optical blood fow measurements, representative tumors (N 2) in each group were also imaged using contrast-enhanced Doppler ultrasound with a Philips ATL 5000 ultrasound scanner (Philips ATL, Bothell, WA) [23,29]. The ultrasound measurements were carried out just prior to the optical measurements. Initial scanning of each tumor was performed in grayscale ultrasound mode to defne the boundary of the tumor mass. Mice were then injected with one hundred micro-liters of micro-bubble ultrasound contrast agent (Optison, Amersham, Princeton, NJ) via tail vein catheter. The area of contrast enhancement denoted perfused regions in the tumor. Tissue regions with blood fow are color coded [30].

Tumor histology and immunohistochemistry
Tumor histology analysis were performed using Hematoxylin and Eosin (H&E) staining. All histological tumor section specimens were viewed under a Nikon E600 Eclipse (Nikon, Melville, NY) equipped with a krypton-argon laser and optical flters for visualization of the dyes FITC (fuorescein isothiocyanate), Texas-red and cyanine-3 by fuorescence. Images were acquired by a charged-coupled device (CCD) camera (Roper Scientifc, Trenton, NJ). Nitroimidazole (EF5) staining was performed 5 hours post-CA4P injection to determine tumor cell hypoxia. Immunofuorescent analysis of EF5 in tumor sections were performed as previously described [29,31], using a cyanine 3-conjugated anti-EF5 monoclonal antibody. In the case of intracellular hypoxia, EF5 binding occurs and hypoxic tumor cells stain reddish.

Diffuse optical spectroscopies
DCS was used to monitor blood fow. The details of the DCS method and further background information can be found elsewhere [14, [32][33][34][35][36]. Briefy, the electric feld temporal autocorrelation function of the scattered light depends on the motion of the red blood cells. Diffusing photons that scatter from moving blood cells experience phase shifts which cause the intensity of the diffusing light to fuctuate in time. The fuctuations are more rapid for faster moving blood cells, or for tissues with greater numbers of moving blood cells. To insure deeper penetration, the DCS light source wavelength is typically chosen in the near-infrared range. Therefore, one can derive information about deep tissue blood fow from measurements of temporal fuctuations impressed upon the transmitted near-infrared light [34]. Our custom built DCS instrument consists of a long coherence length (> 50 m) laser (Crysta Laser, Nevada) operating at 785 nm, an optical switch (Dicon Fiberoptics, California), single photon-counting fast avalanche photodiodes (Perkin-Elmer, Canada), and a non-contact probe [23] wherein source and detector fbers were located in the image plane of a regular SLR camera, permitting continuous non-contact measurements with source-detector separations ranging from 1 to 4 mm. Photodetector outputs are fed into a correlator board (Correlator.com, New Jersey) that computes light intensity temporal autocorrelation functions that are recorded by a computer [24].
From light intensity temporal autocorrelation functions, one readily extracts the data needed to derive normalized electric feld (E(r,t)) autocorrelation function, g 1 (r, τ) = G 1 (r, τ)/ < E * (r,t)E(r,t) >, where G 1 (r, τ) =< E(r,t)E * (r,t + τ) > is the electric feld autocorrelation function [37]. It has been shown that G 1 (r, τ) satisfes the correlation diffusion equation in highly scattering media [34,35,38] and it has been shown empirically in several physiological settings that the extracted parameters αD B characterize the temporal fuctuations of the medium due to scatterer motions such as blood fow [14, 15, 18, 25]. Here α is a factor representing the probability that a scattering event in tissue is from a moving scatterer (α is generally proportional to tissue blood volume fraction) and D B is an "effective" diffusion coeffcient for the blood cells. In this paper we report relative blood fow, rBF, to describe blood Noninvasive tissue oxygen saturation is assessed using a broadband diffuse refectance spectrometer. The original design principle of our in vivo spectrometer is due to Wilson [39]; it consists of a tungsten halogen lamp (Cuda Fiberoptics), dispersion system (monochromator, Acton Reseach), and liquid nitrogen cooled charged coupled device (CCD, Roper Scientifc) camera [40,41]. Light is delivered to tissue with a single source fber and diffuse light is collected using multiple detector fbers arranged in a linear array. The concentrations of deoxy-, oxy-hemoglobin (C Hb ,C HbO 2 ) are extracted using multi-wavelength and multi-source/detector separation (ρ 1.2, 2.4, 3, 4) information. A multi-wavelength ftting algorithm was applied to directly extract C Hb , C HbO 2 assuming absorption is coming from oxy-and deoxy-hemoglobin, μ a ∑ i ε i C i (i Hb, HbO 2 ). Here ε is the extinction coeffcient of a given chromophore at a given wavelength obtained from literature [42]. It is also assumed that scattering parameter follows the Mie-type behavior in NIR spectral window, μ s Aλ −B . Here A and B are related to scatterer size and concentration and they were also ftted in the multi-wavelength ftting algorithm. After obtaining the hemoglobin concentrations, one can derive tissue oxygen saturation (StO 2 ) as StO 2 = [C HbO 2 /(C HbO 2 + C Hb )] 100.

Statistical analysis
All measured values are presented as the mean ± standard error (SE). Percent changes were determined individually for each mouse, based on pretreatment values. Statistical analyses were performed using Matlab (Mathwork, Inc.). Paired comparisons were performed by using Wilcoxon rank sum test to identify trends and substantial changes. Differences were considered signifcant for p ≤ 0.05.

Combretastatin induces significant blood flow Reduction
A representative example of DCS blood fow kinetics is shown in Figure 1(A). The data represent the single animal bulk tumor response averaged over multiple source-detector pairs before drug injection (baseline) and up to 1 hour after drug injection. The data are presented in percent units with 100% implying no change. It is clearly seen that blood fow decreased substantially following a transient initial fow increase. After 1 hour an ∼60% decrease in blood fow was observed. Figure 1(B) summarizes the average response of 9 such mice measurements, i.e. predrug (baseline) and 1 hour after drug injection. The average decrease in blood fow after 1 hour is 65 ± 12% (p < 0.001).
Power Doppler ultrasound images of the same tumor ( Fig. 1(C), (D)) show the effects of CA4P on vasculature. Yellow pixels denote perfused blood vessels of the tumor. It is clear that K1735 tumors were uniformly perfused with no evidence of avascular regions, suggesting that blood vessel growth kept up with tumor growth [29] (Fig. 1(C)). After injection of the drug, much of the vasculature was destroyed and blood perfusion reduced ( Fig. 1(D)). Histological examination of tumor sections also reveals the blood fow reduction; luminal thrombi and occluded blood vessels were seen after 5 hours in addition to the presence of intraluminal collection of packed red blood cells or "blood cell lakes" (Fig. 2).
The biological reason for an initial increase in tumor blood fow during the frst 10 minutes is not well understood. This effect, however, was observed consistently across all mice. Perhaps this early increase is due to an increase in heart rate induced by stress during drug injection. Moreover, it has been reported that the induction of anesthesia can induce 10 to 20 minutes physiological perturbation [29]. It is clearly seen that after treatment, vessel occlusion occurs, blood vessels become congested and cells coagulated, forming "blood cell lakes" (B and C).

Combretastatin induces significant blood oxygen saturation reduction
The changes of blood fow (rBF) were accompanied by changes in blood oxygen saturation (StO 2 ). Our data from 5 mice show that mean blood oxygen saturation decreased signifcantly (p < 0.005) after one hour (Fig. 3(A)) from 47 ± 7% to 29 ± 7%. Control mice showed no EF5 binding [31] (Fig. 3(B)), but substantial binding (shown in red) was found in treated tumors, again confrming that hypoxia is induced by the drug (Fig. 3(C)). These data demonstrate good correlation between intravascular oxygen status and intracellular oxygenation in this tumor

Radiation induced significant blood flow reduction
Figure 4(A) shows average blood fow for radiated mice (N 8) before radiation, 7-days after and 14 -days after radiation. Irradiated K1735 tumors after 7 days were not statistically different (p ≥ 0.05), but a clear decrease in blood fow was observed after 14 days (p < 0.005). The control group did not exhibit a statistically signifcant trend (p ≥ 0.05). In total, these data demonstrate radiation induced vascular destruction and blood fow reduction in 14 days.

Radiation decreased blood oxygen saturation
Radiation treated and control groups did not exhibit signifcant changes in tissue blood oxygen saturation (StO 2 ) within 7 days (p ≥ 0.05). However, evidently the signifcant blood fow falloff produced a signifcant average StO 2 reduction (p < 0.005, N 8) 14 days post radiation as shown in Figure 4(B). StO 2 variation in the control group was not statistically signifcant (p ≥ 0.05). These observations are consistent with a previous study of EF5 binding that demonstrated tumor hypoxia was induced in treated mice after 2 weeks in the same tumor types [43].

Discussion
In this study, we examined the effects of antivascular and radiation therapies on tumor hemodynamics using a murine tumor model. K1735 melanoma tumors are well-suited for these studies since initially these tumors are well perfused and well oxygenated [29,43]. Ultrasound Doppler micro-bubble injection confrmed K1735 tumors have relatively homogeneous vascularity throughout the whole tumor with very few regions of necrosis. Both the antivascular agent CA4P and radiation therapy induced vascular destruction, causing reduced blood fow and tissue oxygenation, and creating more regions of ineffcient blood perfusion and hypoxia. The CA4P hemodynamic response was acute: Blood fow was reduced approximately 65% within the hour and tissue oxygenation was concurrently reduced by ∼ 38%. Blood fow reduction due to CA4P has also been observed by other modalities. A study using superfcial laser Doppler measurements showed a 73% decrease in tumor perfusion after 1 hour in a CH3 mouse mammary carcinoma model [44]. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) [45] and Doppler ultrasound [46] showed signifcant blood   [48,49]. Compared to CA4P, radiation effects were more longitudinal: a single dose of radiation (12 Gy) induced a signifcant decrease in blood fow (36%, N=8) and oxygenation (24%, N=8) 2weeks-post-radiation, but did not signifcantly modify these hemodynamic parameters 1-weekpost-radiation. Moreover, it was previously observed in a similar study that 12 Gy of radiation retarded K1735 tumor growth and induced thrombosis, hemorrhage and hypoxia 2-weeks-postradiation [43].
The average penetration depth of diffused photons in tissue is approximately one-third to onehalf of the source-detector separation; thus our signal originates from ∼1-3 mm depth below the tissue surface. In clinical applications, sometimes as much as 2 -3 cm depth of penetration has been achieved [19,27]. We have initiated chemo-radiation therapy monitoring of head and neck cancer patients with superfcial neck nodes [20] and chemotherapy monitoring of breast cancer patients [22]. These preliminary investigations suggest diffuse-optics-based therapy monitoring is feasible and may have clinical promise.
Diffuse optical techniques have unique advantages compared to other imaging modalities (e.g. MRI, PET and ultrasound), including low-cost, non-invasiveness and small size. Moreover, there has been signifcant recent interest in using antivascular therapy as an adjuvant to radiation therapy in order to develop more effective cancer therapies. The diffuse optical methods may play an important role in assessing acute and delayed responses in such newly developing combined therapy strategies.

Conclusion
In this work we have shown that tumor responses to an antivascular drug, CA4P, and to radiation can be assessed by noninvasively using diffuse optical spectroscopies in a pre-clinical mouse model. Although the temporal responses to CA4P and radiation were quite different, both CA4P and radiation were measured to induce signifcant blood fow and oxygenation reduction. The combined blood fow and blood oxygen saturation information may be valuable for understanding and assessing the working mechanism of antivascular drugs and radiation in both pre-clinical studies and clinical trials. Eventually, diffuse optical methods hold potential for evaluation of drug effcacy and prognosis of concurrent radiation and antivascular therapy. Such experiments should be a next step.