Establishment and validation of lung-specific metastatic cell subline of mouse breast cancer

Abstract

Objective To screen and establish a spontaneously high organotrophic cell subline of metastatic breast cancer and to validate characteristic and the organotropism of the lung metastasis cell subline obtained from the screening. Methods By using the experimental lung metastasis, mouse breast cancer cells line 4T07 were injected into tail vein of mouse. Retrieving metastatic tumor cells in the lung and enriching them in vitro; By using the spontaneous metastasis model, the mammary fat pad was inoculated with tumor cells. Combined with the in vitro expansion culture of lung metastasis and repeated three times, a lung organotrophic cell sub-line and model of spontaneous lung metastasis were obtained; A highly metastatic cell subline stably expressing luciferase was obtained by transfection and screening. Cell morphology was microscopied with HE staining and an in vivo imaging system was performed to detect the migration and lung-specific metastasis. Flow cytometry was used to analyze the changes of immune cells in primary site, metastasis and spleen. Wound healing and Transwell assays were performed to detect migration of cell lines in vitro. Results A mouse breast cancer lung-specific metastatic cell subline (4T07LuM) was successfully established. in vivo imaging results validated that the fluorescent signal was mainly concentrated in the lung after injection of 4T07LuM cells labeled with luciferase in situ. HE staining microscopy results verified tumor metastasis in the lung. In addition, according to the results of Transwell and wound healing assays, 4T07LuM, compared to the parental cells 4T07, had shown a significantly more invasive migration(P＜0.001). Finally, according to the results of flow cytometry detection, 4T07LuM cell had showed significantly altered the immune cell ratios as compared to that of 4T07 cell. The immune microenvironment in vivo was more inclined to an immuno-suppressed state. Conclusions Cell sublines with high organ-specific metastasis ability are obtained through combination of serial screening of experimental and spontaneous metastasis and in vitro culture expansion.

Keywords

• breast cancer|lung metastasis|organ-specificity|spontaneous metastasis model