[Establishing the nude mice bone metastasis model of lung adenocarcinoma and applying MicroCT into the observation]

Abstract

Background and objective: 50%-70% of patients with advanced lung cancer will develop bone metastases. The aim of this study is to establish the nude mice bone metastasis model of lung adenocarcinoma using A549, H1299, SPC-A-1 and XL-2, all of which own different invasion and migration abilities in vitro and supervise the bone metastases by MicroCT.

Methods: fifty BALB/C-nu/nu nude mice were grouped into five groups on average randomly. Cells of the four cell lines were injected into the left cardiac ventricle of mice in the four experimental groups (0.2 mL/mouse) respectively; meanwhile, mice in the control group were injected with normal saline (0.2 mL/mouse) in the same manner. Periodical radiological examination was carried out to supervise the variation of the mice since the second week after injection. When mice in each group became thin obviously, end the experiment of this group. Before the end, pathological sections of bone tissues were made. We classified the bone metastatic sites into axial skeleton and limb bone, in order to compare the metastatic rates of these two different parts. The bone metastatic abilities of the four cell lines was statistically analyzed by comparing the average time cost in the appearance of bone metastases and the percentage of bone metastases among the experimental groups.

Results: Different metastatic sites which had been identified both by MicroCT and pathological sections appeared in each group of the four experimental groups. By contrast, no metastasis was observed in the control group. The percentage of cancer metastasizing to axial skeleton was remarkably higher than the percentage of tumor metastasizing to the limb bone in each experimental group, which was consistent with the clinical regularity and characteristics of skeletal metastases with lung cancer. Thus, the model has been established triumphantly. However, there were no statistical differences in the average time consumed and skeletal metastatic rate among the four experimental groups.

Conclusions: The disruption in the bone can be clearly detected by MicroCT, which is benefit to supervise the osseous metastasis. We successfully developed the nude mice bone metastasis model of lung adenocarcinoma, which will pave the way for exploring novel prevention and therapy strategies clinically. The four cell lines varied in invasion and migration abilities in vitro, but there was no statistical difference in the metastatic ability in vivo, and the reason need to be explored further in future.

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体外生长曲线检测四种细胞之间群体细胞增殖能力。^*P < 0.05，差异具有统计学意义。 In vitro cell growth curve assay of four cell lines. ^*P < 0.05, there was statistical significance among the proliferation ability of the four cell lines.

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体外侵袭、迁移实验检测四种肺腺癌细胞系之间侵袭、迁移能力的差异。A：四种肺腺癌细胞之间迁移实验（×100）；B：四种肺腺癌细胞之间侵袭实验（×100）；^*P < 0.05，四种细胞之间侵袭、迁移能力差异均有统计学意义。 In vitro invasion and migration assays of 4 lung adenocarcinoma lines. A: The migration ability of 4 lung adenocarcinoma lines using the migration assay (×100); B: The invasion ability of the 4 lung adenocarcinoma lines using the matrigel invasion assay (×100); ^*P < 0.05, there was statistical significance.

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对照组及各实验组小鼠MicroCT。A1-D1：对照组小鼠正常脊柱、肋骨、胫骨、肩胛骨MicroCT结果；A2：A549实验组内出现的脊柱转移；B2：H1299实验组内出现的肋骨转移；C2：SPC-A-1实验组内出现的胫骨破坏；D2：XL-2实验组内出现的肩胛骨变化；箭头所示处为各部位的骨质破坏。 MicroCT of the mice in the assay. A1-D1: MicroCT of the mice in the control group show the normal spinal column, rib, tibia and scapula; A2: MicroCT of the mice in the A549 experimental group shows the spinal column metastasis; B2: MicroCT of the mice in the H1299 experimental group shows rib metastasis; C2: MicroCT of the mice in the SPC-A-1 experimental group indicates tibia metastasis; D2: MicroCT of the mice in the XL-2 experimental group indicates scapula metastasis; Arrows show different bone metastatic sites of lung cancer.

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实验组及对照组小鼠骨切片HE染色。A1：对照组小鼠正常脊柱；A2：A549实验组小鼠脊柱发生肿瘤转移的情况；B1：对照组小鼠正常肋骨；B2：H1299实验组小鼠肋骨处肿瘤转移情况；C1：对照组小鼠正常胫骨；C2：SPC-A-1实验组肿瘤胫骨转移；D1：对照组小鼠正常肩胛骨；D2：XL-2实验组小鼠发生肿瘤肩胛骨转移。 HE staining was performed to confirm the bone lesion in the control group and experimental groups. A1: The normal spinal column in the control group; A2: Spinal column with tumor metastasis in the A549 experimental group; B1: Normal rib in the control group; B2: Rib with tumor metastasis in the H1299 experimental group; C1: Normal tibia in the control group; C2: Tibia with tumor metastasis in the SPC-A-1 experimental group; D1: Normal scapula in the control group; D2: Scapula with tumor metastasis in the XL-2 experimental group; T: Tumor, B: Bone.