Simulated microgravity with floating environment promotes migration of non-small cell lung cancers

Abstract

A migration of cancer is one of the most important factors affecting cancer therapy. Particularly, a cancer migration study in a microgravity environment has gained attention as a tool for developing cancer therapy. In this study, we evaluated the proliferation and migration of two types (adenocarcinoma A549, squamous cell carcinoma H1703) of non-small cell lung cancers (NSCLC) in a floating environment with microgravity. When we measured proliferation of two NSCLCs in the microgravity (MG) and ground-gravity (CONT), although initial cell adhesion in MG was low, a normalized proliferation rate of A549 in MG was higher than that in CONT. Wound healing results of A549 and H1703 showed rapid recovery in MG; particularly, the migration rate of A549 was faster than that of H1703 both the normal and low proliferating conditions. Gene expression results showed that the microgravity accelerated the migration of NSCLC. Both A549 and H1703 in MG highly expressed the migration-related genes MMP-2, MMP-9, TIMP-1, and TIMP-2 compared to CONT at 24 h. Furthermore, analysis of MMP-2 protein synthesis revealed weaker metastatic performance of H1703 than that of A549. Therefore, the simulated microgravity based cancer culture environment will be a potential for migration and metastasis studies of lung cancers.

Figure 1

System configuration of simulated microgravity with a floating environment. Schematic diagram of non-small cell lung cancer (NSCLC) culture system (A). Installation of microgravity system in the CO₂ incubator (B).

Figure 2

Proliferation analysis of A549 and H1703 cells. Comparison of A549 proliferation between ground (CONT) and microgravity (MG) (A). Comparison of H1703 proliferation between CONT and MG (B) (*p < 0.05, **p < 0.01).

Figure 3

Wound healing analysis of A549 and H1703 cells on CONT and MG. Migration potential of A549 cells in wound healing assay (A). Calculated wound healing areas in A549 cells (B). Migration potential of H1703 cells in wound healing assay (C). Calculated wound healing areas in H1703 (D) (*p < 0.05, **p < 0.01).

Figure 4

Wound healing analysis of A549 and H1703 at a low cell proliferation condition (1% FBS condition) on CONT and MG. Migration potential of A549 cells in wound healing assay (A). Calculated wound healing areas in A549 cells (B). Migration potential of H1703 cells in wound healing assay (C). Calculated wound healing areas in H1703 (D) (**p < 0.01).

Figure 5

Comparison of expression levels of cell migration related genes between CONT and MG. Relative gene expression levels of MMP-2 (A), MMP-9 (B), TIMP-1 (C), and TIMP-2 (D) in A549 cells. Relative gene expression levels of MMP-2 (E), MMP-9 (F), TIMP-1 (G), and TIMP-2 (H) in H1703 cells (*p < 0.05, **p < 0.01).

Figure 6

Protein expression evaluation by western blotting of A549 and H1703 on CONT and MG. Comparison of MMP-2 and MMP-3 protein expression between CONT and MG in A549 cells (A). Relative MMP-2 protein expression levels of CONT and MG over time in A549 cells (B). Relative MMP-3 protein expression levels of CONT and MG over time in A549 cells (C). Comparison of MMP-2 and MMP-3 protein expression between CONT and MG in H1703 cells (D). Relative protein expression levels of CONT and MG over time in H1703 cells (E). Relative protein expression levels of CONT and MG over time in H1703 cells (F) (*p < 0.05, **p < 0.01).