Monitoring Responses to Treatment With High-Frequency Ultrasound In Vivo: Assessing Response to Radiation vs. Photodynamic Therapy in Melanoma Xenograft Tumors
High-frequency ultrasound (HFUS) is a novel method to detect apoptotic cell death based on changes in cell morphology and consequently the acoustic properties of cell ensembles and tissues. Photodynamic therapy (PDT) is a rapidly evolving cancer treatment modality and apoptosis has been shown to be a dominant form of cell death following PDT. In this study, we evaluated the use of HFUS to evaluate tumor responses to radiation treatment and PDT in vivo.
Materials/Methods
Solid tumours were grown in SCID mice using a malignant human melanoma cell line (HTB-67). For PDT experiments, tumours were treated with 110J/cm2 of 633 nm laser light, 24 hours following exposure to 10 mg/kg of Photofrin given i.p. Tumors were examined by 26 and 40 MHz HFUS prior to treatment and at different times after administration of PDT. For radiation therapy experiments, tumors were treated with 0, 2, 4 or 8 Gy of 100 kV photons in a single fraction. US data collection consisted of acquiring tumor images in addition to spectroscopic data for quantitative analyses of backscattered (BS) US. Animals were sacrificed immediately after analysis and tumor was excised for histopathologic analysis. Histological sections were examined by H&E and TUNEL staining for apoptosis. US images and corresponding spectroscopic data were analyzed and tested for correlation with histopathological findings.
Results
We observed a time-dependant increase in ultrasound BS after treatment. Increases in backscatter findings correlated with morphological findings indicating increases in apoptotic cell death, which peaked at 24 h after PDT. We observed 0%, 1.48%, 3.2%, 26.8%, 52.5% and 34% apoptosis in terms of cross-sectional tumor area from histopathology after 1 h, 3 h, 6 h, 12 h, 24 h and 48 h of PDT, respectively. These correspond to 0.5%, 5.4%, 9.07%, 16.07%, 16.23% and 13.1% increases in the logarithmically uncorrected mean intensity in ultrasonograms obtained at 26 MHz. In the spectroscopic analysis of regions of interest we detected a similar pattern of change in the spectral slope and mid-band fit (MBF) of the best fit line. The spectral slope was 0.20, 0.26, 0.29, 0.37, 0.43, and 0.41 at the mentioned times. MBF was −34.7 dBr at 1 h and increased to the range of −31 between 12–24 h and decreased to −33.1 dBr at 48 h. The findings with 40 MHz-pulsed HFUS followed the same trend. These findings were closely correlated to the changes in the nuclear size in the irradiated area. In contast, there were minimal apoptosis and detectable differences in the spectral parameters in both the control and orthovoltage irradiated melanoma tumors consistent with radioresistance.
Conclusions
spectroscopic US analyses can detect time-dependent, photodynamic-induced cell death. In our study, we observed a close correlation between PDT-induced apoptosis cell death and changes in mean intensity of ultrasonographic BS along with changes in spectral slope and MBF in spectroscopic analysis. At the same time, we could not observe any significant change in HFUS parameters with radiation treatment, consistent with radio-resistance of Melanoma cells to radiation. These findings will provide a foundation for future investigations regarding the use of US in cancer patients to individualize the treatment based on their response to specific intervention.
1Toronto Sunnybrook Cancer Centre, University of Toronto, Toronto, ON, Canada
2Department of Biophysics, Ryerson University, Toronto, ON, Canada
Author Disclosure: B. Banihashemi, None; W. Chu, None; A. Giles, None; B. Debeljevic, None; R. Vlad, None; M. Kolios, None; G. Czarnota, None.
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