PET/CT imaging of c-Myc transgenic mice identifies the genotoxic N-nitroso-diethylamine as carcinogen in a short-term cancer bioassay

Abstract

Background: More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay.

Methodology/principal findings: μCT and ¹⁸F-FDG μPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced μCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis.

Conclusions/significance: The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgenic animal models may possibly enable short-term cancer bioassays to significantly improve hazard identification and follow-up examinations of different organs by non-invasive methods.

Figure 1. Contrast-enhanced CT and ¹⁸F-FDG-PET of the liver of an NDEA-treated c-Myc transgenic mouse.

Axial slices of a CT (A) and an FDG-PET scan (B) as well as coronal slices of a CT scan (C) and macroscopic view (D) of an explanted liver of an NDEA treated mouse at the age of 7 months. When compared to the normal liver parenchyma tumors are hypodens at CT as the uptake of the liver specific contrast agent is reduced. PET scans reveal tumor lesions by an increased tracer uptake due to enhanced glucose metabolism. Because of the advanced stage of disease tumors of different size had merged together. K = kidney, L = lung, S = spine, Sp = spleen, T = tumor.

Figure 2. Time dependent changes in tumor growth of NDEA-treated c-Myc transgenic mice.

(A) The organ and tumor volume was assessed by CT and by histopathology. Depicted is the percentage tumor volume at different stages measured either by histopathology (light gray) or by contrast enhanced CT imaging (dark gray). The mean diameter of lesions (B) and the total number of lesions (C) are shown. *p<0.05. **p<0.01.

Figure 3. Contrast-enhanced CT, ¹⁸F-FDG-PET and fused images of c-Myc transgenic mice treated with NDEA at the age of 5.5 and 7 months.

(A) CT image demonstrates a single tumor lesion (A1) in a 5.5 months old mouse without increased ¹⁸F-FDG uptake (A2). The fused CT and PET image is depicted in A3. (B) At the age of 7 months expansive tumor growth (B1) as well as an increased tracer uptake in hepatocellular carcinoma (B2) is observed. The fused CT and PET image is depicted in B3. G = gallbladder, K = kidney, L = liver, S = spine, St = stomach, T = tumor.

Figure 4. ¹⁸F-FDG uptake to determine the tumor-to-non-tumor ratios in tumors of different size.

Tumor-to-non-tumor ratio, determined by PET glucose imaging, was significantly increased in lesions >10 mm. *p<0.05. **p<0.01.

Figure 5. Fused μPET/μCT images of the liver of c-Myc transgenic mice treated with physiological saline, BHT or NDEA.

Depicted are the liver morphology as determined by CT (A1, B1,C1), the glucose metabolism (A2, B2, C2) and fused PET and CT scans (A3, B3, C3) of transgenic animals treated with either physiological saline (A), with BHT (B) or with NDEA (C) at the age of 8.5 months. Note, after treatment with NDEA expansive tumor growth with large increase of liver weight and compression and displacement of adjacent organs was observed. Here, the lesions showed an increased 18F-FDG uptake. In contrast, in corresponding control animals treated with physiological saline no liver lesions were observed. After treatment with BHT small hypodens lesions are noticed, but PET did not show an increased 18F-FDG uptake. K = kidney, L = liver, S = spine, Sp = spleen, St = stomach, T = tumor.

Figure 6. Histopathology of the liver of c-Myc treated transgenic animals.

(A) Diffuse liver cell dysplasia of physiological saline ( = vehicle) treated transgenic mice at (A1) 50- and (A2) 200-fold magnification. (B) Large cell dysplasia of various degrees in BHT treated animals at (B1) 50- and (B2) 200-fold magnification. (C) Hepatocellular carcinoma of a transgenic mouse treated with the genotoxic carcinogen NDEA at (C1) 50 and (C2) 200-fold magnification.

Figure 7. CT and histopathology of the lung of NDEA treated c-Myc transgenic mice.

(A) Normal lung parenchyma of a vehicle treated control animal as shown by CT (A1) and by histopathology (A2). CT of NDEA treated animals at the age of 8.5 months. Depicted are lung nodules of different size (red arrows). Histopathology evidenced those lung nodules as metastasis of a poorly differentiated HCC (B2) and as well adenocarcinoma of the lung (C2).