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induced by cyclophosphamide on breast cancer xenografts using diffuse optics. Regine Choe1, Ki Won Jung1, Hyun Jin Kim1, Ashley R. Proctor1, Daniel K.
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Biomedical Optics 2014 © OSA 2014

Quantification of early hemodynamic changes induced by cyclophosphamide on breast cancer xenografts using diffuse optics Regine Choe1 , Ki Won Jung1 , Hyun Jin Kim1 , Ashley R. Proctor1 , Daniel K. Byun1 , Parisa Farzam2 , Kelley S. Madden1 , Turgut Durduran2 , Edward B. Brown1 2 ICFO-

1 Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA Institut de Ci`encies Fot`oniques, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain Regine [email protected]

Abstract: A significant difference between a treated and a control group of mice with breast cancer were observed in hemodynamic parameters quantified with diffuse optics at day 3 after cyclophosphamide treatment. OCIS codes: (170.3660), (170.3890), (170.6480), (290.4210)

1.

Introduction

In the USA, 10-20% of over 230,000 new cases of invasive breast cancer will have locally advanced breast cancer (a large primary lesion or a cancer which has spread to the chestwall, the breast skin or to the lymph nodes, but not to other organs). Of these patients who receive neoadjuvant chemotherapy (NAC), around 20% will not respond to this pre-surgical therapy [1, 2]. Patient response is variable and unpredictable due to inter- and intra-subject tumor heterogeneities [3]. Currently, primary tumor volume change through palpation or conventional imaging modalities is used as the indication of NAC response. However, these morphological changes may take up to 2-4 months to be detectable, and these methods can be hampered by therapy-induced fibrosis. Thus, there is a strong need for imaging modalities that can identify non-responders quickly to modify/optimize treatment option to avoid unnecessary side effects of ineffective therapies. Diffuse optical method is a non-invasive technique that quantifies blood flow, total hemoglobin concentration, tissue blood oxygenation, water, lipid concentrations and tissue scattering simultaneously, using multiple near-infrared (NIR) light sources (650 - 1000 nm) [4]. Most importantly, diffuse optical methods are suitable for frequent monitoring because they do not use ionizing radiation, with the added benefit that these methods are inexpensive, and portable. To date, many researchers including us have demonstrated that diffuse optical methods are sensitive to changes induced by breast cancer therapies and have the potential to predict therapeutic efficacy [5]. In a preliminary clinical study using diffuse optical methods, we observed that a human subject with an acute increase in blood flow and decrease in total hemoglobin concentration at 1-2 days after the first chemotherapy exhibited better pathological outcomes than the other subject with slow increase in blood flow and total hemoglobin concentration [5]. Even though chemotherapy has been employed to treat breast cancer for many decades, not much attention has been given to the quantification of hemodynamic responses due to chemotherapy. This could be due to the limited availability of non-invasive techniques that provide quantitative hemodynamic information of deep-tissue, without any contrast agent in both preclinical and clinical settings. Recently, Vishwanath et al. have demonstrated the ability of broadband optical spectroscopy for longitudinal monitoring of tumors under a radiation therapy [6] and a chemotherapy using Doxorubicin (brand name: Adriamycin) [7]. They have demonstrated the temporal kinetics of hemoglobin related parameters (and sometimes tissue scattering) were significantly different between treated and control groups. Here, we report early changes in optically-measured hemodynamic parameters from a breast cancer preclinical model undergoing a chemotherapy using cyclophosphamide. Cyclophosphamide is a cytotoxic anticancer agent that works by interfering with DNA replication: it is typically administered to breast cancer patients undergoing chemotherapy as a part of Adriamycin-Cyclophosphamide (AC) cocktail in the clinic. Early hemodynamic changes due to cyclophosphamide have not been characterized in preclinical setting. In addition, we employed a hybrid diffuse optics system that (1) provides blood flow via diffuse correlation spectroscopy in addition to other hemodynamic/tissue parameters available by diffuse optical spectroscopy (DOS), and (2) quantifies water concentration with great fidelity by improved wavelength sensitivity in 900-1000 nm of the DOS detection.

BS3A.10.pdf

2.

Biomedical Optics 2014 © OSA 2014

Materials and Methods

Instrumentation: This study utilizes two types of diffuse optical instruments: 1) a diffuse optical spectroscopy (DOS) and 2) a diffuse correlation spectroscopy (DCS). DOS consists of a broadband whitelight source (250 W Quartz Tungsten Halogen lamp, Newport), and a detection unit with a spectrometer (Acton Insight, Princeton Instruments, Inc) and a CCD camera (PIXIS 400, Princeton Instrument; 1340×400 pixels). DCS comprises a 786 nm long-coherence laser (Crysta Laser), and a detection unit consisting of a four-channel photon-count avalanche photodetector (PerkinElmer) and a four-channel hardware correlator (Correlator.com). A custom-made probe (Fiberoptic Systems, Inc) enables near-simultaneous temporal and spatial measurements of DOS and DCS [8]. DOS measurements were done at seven source-detector (SD) separations (1.87, 2.55, 2.89, 3.25, 3.60, 3.94 and 4.28 mm) and DCS were done at four source-detector separations (2.55, 2.89, 3.25 and 3.94 mm). Animal model and measurement protocol: All experimental protocols were approved by the University Committee on Animal Resources (UCAR) of University of Rochester. 5-6 weeks old female BALB/cByJ mouse (Jackson Laboratory) was used for this study. Each mouse was injected with a murine breast cancer cell line 4T1 in a mammary fat-pad at the concentration of 1×105 cells/100 µ L. After the tumor cells were inoculated, the tumor size was measured with a digital caliper in horizontal width and vertical length on the days when the diffuse optical measurements were performed. The size then assessed in terms of area based on the formula π ab/4, where a and b are the horizontal width and the vertical length respectively. When the tumor size reached approximately 40 mm2 in terms of its area (i.e., the effective diameter of ∼7 mm), the in vivo mouse experiment was initiated. On the first day (Day 0) of the experiment the baseline signals were measured on the center of the tumor using diffuse optical instruments under Isoflurane as the anesthesia. Measurements were repeated three times on the same location by lifting the probe and placing it back. After the baseline measurements were finished, ten mice were divided into two groups, the control and the treatment groups (i.e., 5 mice/group). While the control group received 200 µ L of saline via intraperitoneal injection, the treatment group received cyclophosphamide at the dosage rate of 200 mg/bodyweight in kg [9] dissolved in 200 µ L of saline via intraperitoneal injection as well. Diffuse optical measurements were repeated on Days 1, 3, 7, 9, and 11 as well as tumor size measurements. Data Analysis: For both DOS and DCS, an analytic solution of the corresponding diffusion equation in the homogeneous semi-infinite geometry was utilized for each case. From DOS measurements, oxy- and deoxy-hemoglobin, water, and lipid concentrations (CHbO2 , CHb , CH2 O , and Clipid respectively), and tissue scattering coefficients µs′ (λ ) (where λ was the wavelength) were quantified using a multi-spectral method using multiple SD separations [4, 10]. Then, total hemoglobin concentration (T HC = CHbO2 + CHb ) and blood oxygen saturation (StO2 = CHbO2 /T HC) were extracted. To derive the tissue blood flow index, BFI, the measured DCS temporal light intensity autocorrelation functions were fit to the analytic solution for each SD separation [4]. Relative blood flow (rBF) was computed by normalizing BFI with respect to the first time point (i.e., Day 0). 3.

Results

Figure 1 shows temporal changes of selected hemodynamic parameters along with the tumor growth. For hemodynamic parameters, mean value was computed by averaging 3 repeat measurements for each mouse at each time point. Then mean values were averaged over all the mice in the group (N=5) and assigned at each time point. Thus, the error bar indicates the inter-subject variation. With the treatment, tumor growth was delayed (Figure 1(a)). At day 3, T HC, StO2 and rBF were significantly higher in the treated group than in the control group. However, the temporal changes became indistinguishable between groups after day 7. These early changes may be attributed to transient blood vessel renormalization observed in various anti-angiogentic treatment [11]. References 1. P. Rastogi, S. J. Anderson, H. D. Bear, C. E. Geyer, M. S. Kahlenberg, A. Robidoux, R. G. Margolese, J. L. Hoehn, V. G. Vogel, S. R. Dakhil, D. Tamkus, K. M. King, E. R. Pajon, M. J. Wright, J. Robert, S. Paik, E. P. Mamounas, and N. Wolmark, “Preoperative chemotherapy: updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27,” J. Clin. Onc. 26, 778–785 (2008). 2. A. S. Caudle, A. M. Gonzalez-Angulo, K. K. Hunt, P. Liu, L. Pusztai, W. F. Symmans, H. M. Kuerer, E. A. Mittendorf, G. N. Hortobagyi, and F. Meric-Bernstam, “Predictors of tumor progression during neoadjuvant chemotherapy in breast cancer,” J. Clin. Onc. 28, 1821–1828 (2010). 3. E. G. E. de Vries, T. H. O. Munnink, M. A. T. M. van Vugt, and W. B. Nagengast, “Toward molecular imagingdriven drug development in oncology,” Cancer Discovery pp. 25–28 (2011).

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Biomedical Optics 2014 © OSA 2014

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Fig. 1. (a) Tumor growth, (b) T HC changes over time, (c) StO2 changes over time, (d) Changes of rBF at 3.25 mm SD separation over time

4. T. Durduran, R. Choe, W. B. Baker, and A. G. Yodh, “Diffuse optical spectroscopy and tomography for tissue monitoring and imaging,” Rep. Prog. Phys. 73, 076,701 (2010). 5. R. Choe and T. Durduran, “Diffuse optical monitoring of the neoadjuvant breast cancer therapy,” IEEE J. Sel. Top. Quantum Electron. 18, 1367–1386 (2012). 6. K. Vishwanath, D. Klein, K. Chang, T. Schroeder, M. W. Dewhirst, and N. Ramanujam, “Quantitative optical spectroscopy can identify long-term local tumor control in irradiated murine head and neck xenografts,” J. Biomed. Opt. 14, 054,051 (2009). 7. K. Vishwanath, H. Yuan, W. T. Barry, M. W. Dewhirst, and N. Ramanujam, “Using optical spectroscopy to longitudinally monitor physiological changes within solid tumors,” Neoplasia 11, 889–900 (2009). 8. P. Farzam and T. Durduran, “Design of a broadband near infrared spectroscopy (nirs) and diffuse correlation spectroscopy (dcs) device with a self-calibrated probe for experimental oncology,” in “European Conferences on Biomedical Optics,” , vol. 20th (Munich, Germany, 2011), vol. 20th, pp. 8088–41. 9. R. J. Viola, J. M. Provenzale, F. Li, C. Y. Li, H. Yuan, J. Tashjian, and M. W. Dewhirst, “In vivo bioluminescence imaging monitoring of hypoxia-inducible factor 1alpha, a promoter that protects cells, in response to chemotherapy,” Am. J. Roentgenol. 191, 1779–1784 (2008). 10. E. L. Hull, M. G. Nichols, and T. H. Foster, “Localization of luminescent inhomogeneities in turbid media with spatially resolved measurements of cw diffuse luminescence emittance,” Appl. Opt. 37, 2755–2565 (1998). 11. R. K. Jain, “Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy,” Science 307, 58–62 (2005).