In vivo metastasis assay using a nude mouse model

BALB/c nude mice aged 4−5 weeks old (18 g, female, Dalian Medical University Laboratory Animal Center) were used to assess the in vivo metastatic capability of tumor cells. Mice were divided into three groups according to type of transplanted tumor cells: the MCF7 group, the MDA-MB-231 group, and the ACC-M group, with five mice in each group. Cells were harvested and resuspended at a concentration of 2 × 10⁶ cells/mL in PBS, then injected into the tail vein. The nude mice were raised in sterile conditions for 30 days, then euthanized and the lung tissues removed. Tissues were fixed in 10% formalin for 24 hours and embedded in paraffin. Sections of 4 μm thickness were prepared and stained with hematoxylin and eosin (H.E.). Immunohistochamical staining with anti-humna Pan CK (Merckmillipore, USA) was performed to detect the metastatic colonies. The images were recorded by a microscope (Olympus BX43, Olympus Corp., Tokyo, Japan). The images were recorded by a microscope (Olympus BX43, Olympus Corp., Tokyo, Japan). Metastatic rate and metastatic colony areas per lung section were used to evaluate the metastatic capability of each tumor cell line. Metastasis was regarded as positive even if only one metastatic colony was found in the lung. Metastatic rate was represented as the ratio of animals with positive metastasis in all experimental animals. High power view images (400×) were used to calculate metastatic colony areas. Firstly, the colonies were confirmed by H.E. and anti-human Pan cytokeratin immunostaining and circumscribed using Image-Pro^® Plus version 6.0 software. The metastatic areas were determined as the total areas of all metastatic colonies in the lung.