[Establishment of a novel chinese human lung adenocarcinoma cell line CPA-Yang3 and its real bone metastasis clone CPA-Yang3BM in immunodeficient mice]

Abstract

Background and objective: The recurrence and metastasis of lung cancer is a tough problem worldwide. The aim of this study is to establish a novel Chinese lung adenocarcinoma cell line and its real bone-seeking clone sub-line for exploring the molecular mechanism of lung cancer metastasis.

Methods: The cells came from the pleural effusion of a sixty-five years old female patient with lung adenocarcinoma and supraclavicular lymph node metastases. The gene expression was detected by real-time quantitative PCR. Intracardiac injection of the cells into nude mice was performed and in vivo imaging was obtained by bone scintigraphy and conventional radiography. Bone metastases were determined on bone scintigraphy and then the lesions were resected under deep anesthesia for bone metastasis cancer cell culture. The process was repeated for four cycles to obtain a real bone-seeking clone.

Results: The tumorigenesis rate started at 4th passage in immunodeficient mice via subcutaneously and as well as later passages. Approximately 1×10⁶ cancer cells were injected into left cardiac ventricle of immunodeficient mice resulted bone metastasis sites were successfully revealed by bone scintigraphy and pathological diagnosis, the mandible (100%), scapula (33%), humerus (50%), vertebral column (50%), femur (66.7%) and accompanied invasion with other organs, the adrenal gland (17%), pulmonary (33%), liver (50%), submaxillary gland (33%) in the mice after inoculation two-three weeks. The chromosome karyotype analysis of the cells was subdiploid. Quantitative real-time PCR was used to examined and compared with SPC-A-1 lung adenocarcinoma, ESM1, VEGF-C, IL-6, IL-8, AR, SVIL, FN1 genes were overexpress. The novel cell was named CPA-Yang3. The femur metastasis cell was repeated in vivo-in vitro-in vivo with three cycles and harvested a real bone metastasis clone. It was named CPA-Yang3BM.

Conclusions: Tne characteristics of novel strain CPAYang3 is a highly metastasis cell line of Chinese lung adenocarcinoma and CPA-Yang3BM is a real bone-seeking clone.

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第二代CPA-Yang3细胞相差显微镜图(A：×100；B：×200) The CPA-Yang3 cells in second passage under the contrast phase microscope (A: ×100; B: ×200)

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CPA-Yang3细胞生长曲线 The cell growth curve of CPA-Yang

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染色体核型分析图 Chromosomal instability in CPA-Yang3

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RT-PCR法比较CPA-Yang3与SPC-A-1部分基因ESM1、VEGF-c、IL-6、IL-8、AR、SVIL和FN1的表达水平 Real-time PCR was performed on CPA-Yang3 compare with SPC-A-1 to evaluate the expression changes of genes

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微孔针孔核素骨显像检测小鼠骨转移（后前位）。A：箭头所指左右下颌骨和左肱骨、右肩胛骨转移；B：上箭头示胸椎转移；C：箭头所指两膝关节骨转移。 The pinhole bone scintigraphy detected bone metastasis in vivo mice. A: Arrows show jaw bones, left humerus and right scapula metastases; B: Arrows show thoracic vertebrae metastasis; C: Arrows show knee joints metastases.

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图 5鼠的X线片（后前位） The X ray of Fig 5 mouse

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图 5鼠在核素骨显像、X线拍片后解剖时照片 The photo of Fig 5 mouse was bled and then sacrificed after bone scan and X ray detection

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高骨转移亚代细胞CPA-Yang3BM（×100）相差显微镜图 Bone-seeking clone CPA-Yang3BM under the contrast phase microscope (×100)

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CPA-Yang3经左心室接种后引发小鼠多脏器转移病理(HE, ×100)。A：肩胛骨转移；B：胸椎转移；C：颌下腺转移；D：股骨转移；E：肾上腺转移；F：肺转移。 Histological features of multi-organ metastases in the mouse with CPA-Yang3 (HE, ×100). A: scapula metastasis; B: thoracic vertebrae metastasis; C: submaxillary gland metastasis; D: femur metastasis; E: adrenal metastasis; F: lung metastasis.