Novel esophageal squamous cell carcinoma bone metastatic clone isolated by scintigraphy, X ray and micro PET/CT

Abstract

Aim: To establish a Chinese esophageal squamous cell carcinoma (ESCC) cell line with high bone metastasis potency using (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) micro-pinhole scintigraphy, X ray and micro-positron emission tomography/computed tomography (PET/CT) for exploring the mechanism of occurrence and development in esophageal cancer.

Methods: The cells came from a BALB/c nu/nu immunodeficient mouse, and oncogenic tumor tissue was from a surgical specimen from a 61-year-old male patient with ESCC. The cell growth curve was mapped and analysis of chromosome karyotype was performed. Approximately 1 × 10⁶ oncogenic cells were injected into the left cardiac ventricle of immunodeficient mice. The bone metastatic lesions of tumor-bearing mice were detected by (99m)Tc-MDP scintigraphy, micro-PET/CT and X-ray, and were resected from the mice under deep anesthesia. The bone metastatic cells in the lesions were used for culture and for repeated intracardiac inoculation. This in vivo/in vitro experimental metastasis study was repeated for four cycles. All of the suspicious bone sites were confirmed by pathology. Real-time polymerase chain reaction was used to compare the gene expression in the parental cells and in the bone metastatic clone.

Results: The surgical specimen was implanted subcutaneously in immunodeficient mice and the tumorigenesis rate was 100%. First-passage oncogenic cells were named CEK-Sq-1. The chromosome karyotype analysis of the cell line was hypotriploid. The bone metastasis rate went from 20% with the first-passage oncogenic cells via intracardiac inoculation to 90% after four cycles. The established bone metastasis clone named CEK-Sq-1BM had a high potential to metastasize in bone, including mandible, humerus, thoracic and lumbar vertebrae, scapula and femur. The bone metastasis lesions were successfully detected by micro-pinhole bone scintigraphy, micro-PET/CT, and X-ray. The sensitivity, specificity and accuracy of the micro-pinhole scintigraphy, X-ray, and micro-PET/CT imaging examinations were: 89.66%/32%/80%, 88.2%/100%/89.2%, and 88.75%/77.5%/87.5%, respectively. Some gene expression difference was found between parental and bone metastasis cells.

Conclusion: This newly established Chinese ESCC cell line and animal model may provide a useful tool for the study of the pathogenesis and development of esophageal carcinoma.

Keywords: Bone metastasis; Cell line; Esophageal squamous cell carcinoma; Molecular imaging; Real-time polymerase chain reaction.

Figure 1

Morphology of human esophageal squamous cell carcinoma cell line CEK-Sq-1. A: First passage oncogenic cells (× 100); B: After four cycles, the bone-seeking subline CEK-Sq-1BM was harvested.

Figure 2

Growth curve of the CEK-Sq-1 cell line at passages 7, 24 and 40.

Figure 3

Chromosomal imbalances of CEK-Sq-1 shows the DNA over-representation on chromosomes 1-3, 7, 8, 12, 14, 16–19, X and Y.

Figure 4

Whole-body X-ray and micro-pinhole bone scintigraphy images of experimental bone metastasis in mice. A: X-ray image, upper arrow indicates the hot spot of the left humeral osteoblastic metastatic lesion, and the lower arrow indicates the cold spot of the left scapular osteolytic metastatic lesion and circle around the osteogenic reaction; B: Micro-pinhole bone scintigraphy images of the same mouse in the POST position. The osteoblastic metastatic lesion (upper arrow) showed greater accumulation of ^99mTc-methylene diphosphonate (⁹⁹mTc-MDP), and the osteolytic lesion (lower arrow) combined with osteogenic reaction showed less; C: The arrow indicates a mandibular lesion in another mouse with accumulation of ⁹⁹mTc-MDP.

Figure 5

¹⁸F-FLT micro-PET/CT images of CEK-Sq-1MB mouse. A: Arrow indicates ¹⁸F-FLT uptake positivity in the cells (2 × 10⁶) injected subcutaneously into the left lateral subcutis of the mouse for 3 wk; B: Arrows indicate the mandibular and thoracic vertebral metastatic lesions of the cells after intracardiac injection in mice for 4 wk.

Figure 6

Histological features of the the mandibular (A) (hematoxylin and eosin, ×100) and lumbar vertebral (B) (hematoxylin and eosin, × 200) metastatic lesions.

Figure 7

Expression changes of genes determined in CEK-Sq-1BM cells compared with CEK-Sq-1 cells.