Technical note: Noninvasive monitoring of normal tissue radiation damage using spectral quantitative ultrasound spectroscopy

Abstract

Background: While radiation therapy (RT) is a critical component of breast cancer therapy and is known to decrease overall local recurrence rates, recent studies have shown that normal tissue radiation damage may increase recurrence risk. Fibrosis is a well-known consequence of RT, but the specific sequence of molecular and mechanical changes induced by RT remains poorly understood.

Purpose: To improve cancer therapy outcomes, there is a need to understand the role of the irradiated tissue microenvironment in tumor recurrence. This study seeks to evaluate the use of spectral quantitative ultrasound (spectral QUS) for real time determination of the normal tissue characteristic radiation response and to correlate these results to molecular features in irradiated tissues.

Methods: Murine mammary fat pads (MFPs) were irradiated to 20 Gy, and spectral QUS was used to analyze tissue physical properties pre-irradiation as well as at 1, 5, and 10 days post-irradiation. Tissues were processed for scanning electron microscopy imaging as well as histological and immunohistochemical staining to evaluate morphology and structure.

Results: Tissue morphological and structural changes were observed non-invasively following radiation using mid-band fit (MBF), spectral slope (SS), and spectral intercept (SI) measurements obtained from spectral QUS. Statistically significant shifts in MBF and SI indicate structural tissue changes in real time, which matched histological observations. Radiation damage was indicated by increased adipose tissue density and extracellular matrix (ECM) deposition.

Conclusions: Our findings demonstrate the potential of using spectral QUS to noninvasively evaluate normal tissue changes resulting from radiation damage. This supports further pre-clinical studies to determine how the tissue microenvironment and physical properties change in response to therapy, which may be important for improving treatment strategies.

Keywords: noninvasive imaging; normal tissue radiation response; quantitative ultrasound spectroscopy.

Figure 1.. Spectral QUS measurements reveal longitudinal tissue property changes following irradiation of mouse MFPs.

(A) Experimental timeline. Mouse mammary fat pads (MFPs) were irradiated to 20 Gy. Ultrasound imaging was conducted immediately before irradiation and 1, 5, and 10 days post-irradiation. (B) B-mode images of MFPs pre-irradiation (baseline) and days 1 and 5 post-irradiation in control and irradiated animals. Scale bar is 1 mm. Histology was performed 10 days post-irradiation to correlate spectral QUS measurements with radiation response. Percent changes in mid-band fit (C), spectral slope (D), and spectral intercept (E) across 10 days for control and irradiated groups. Error bars show standard error with ***p<0.001 as determined by one-way ANOVA analysis between the control and irradiated groups.

Figure 2.. Adipocyte morphology change following irradiation.

(A) Representative hematoxylin and eosin (H&E) staining of control (n=5) and irradiated MFPs (n=5). (B) Perilipin (red) and nuclear (blue) staining of control and irradiated MFPs demonstrate an increase in adipocyte density in irradiated MFPs. Quantification of (C) adipocyte count per field and (D) average adipocyte size. Error bars represent standard error with *p < 0.05 and **p < 0.01 as determined by a two-tailed unpaired t-test. Scale bar is 100 μm.

Figure 3.. Visualizing tissue properties following irradiation of MFPs shows an increase in ECM deposition.

Scanning electron microscopy was used to visualize the MFP in (A) unirradiated and (B) irradiated mouse tissues. White arrows indicate ECM deposition, and black stars denote adipocytes. An increase in ECM deposition, fiber disorder, and a decrease in adipocyte size were observed in irradiated MFPs. Scale bar is 50 μm.