Cytoskeletal Actin Structure in Osteosarcoma Cells Determines Metastatic Phenotype via Regulating Cell Stiffness, Migration, and Transmigration

Abstract

Osteosarcoma is the most common primary malignant bone tumor. The cause of death due to osteosarcoma is typically a consequence of metastasis to the lung. Controlling metastasis leads to improved prognosis for osteosarcoma patients. The cell stiffness of several tumor types is involved in metastatic potential; however, it is unclear whether the metastatic potential of osteosarcoma depends on cell stiffness. In this study, we analyzed the cell stiffness of the low metastatic Dunn cell line and its highly metastatic LM8 subline, and compared actin organization, cell proliferation, and metastasis. Actin cytoskeleton, polymerization, stiffness, and other cellular properties were analyzed. The organization of the actin cytoskeleton was evaluated by staining F-actin with Alexa Fluor 488 phalloidin. Cell stiffness was measured using Atomic Force Microscopy (AFM). Cell proliferation, migration, invasion, and adhesion were also evaluated. All experiments were performed using mouse osteosarcoma cell lines cultured in the absence and presence of cytochalasin. In LM8 cells, actin polymerization was strongly suppressed and actin levels were significantly lower than in Dunn cells. Stiffness evaluation revealed that LM8 cells were significantly softer than Dunn. Young's modulus images showed more rigid fibrillar structures were present in Dunn cells than in LM8 cells. LM8 cells also exhibited a significantly higher proliferation. The migration and invasion potential were also higher in LM8 cells, whereas the adhesion potential was higher in Dunn cells. The administration of cytochalasin resulted in actin filament fragmentation and decreased actin staining intensity and cell stiffness in both LM8 and Dunn cells. Cells with high metastatic potential exhibited lower actin levels and cell stiffness than cells with low metastatic potential. The metastatic phenotype is highly correlated to actin status and cell stiffness in osteosarcoma cells. These results suggest that evaluation of actin dynamics and cell stiffness is an important quantitative diagnostic parameter for predicting metastatic potential. We believe that these parameters represent new reliable quantitative indicators that can facilitate the development of new drugs against metastasis.

Keywords: actin cytoskeleton; atomic force microscopy; cell stiffness; metastasis; osteosarcoma cell line.

Figure 1

Evaluation of lung metastases between Dunn and LM8. (a) Hematoxylin and eosin staining was performed (Left: Dunn, Right: LM8). The metastatic foci was counted. (b) The number of metastatic foci of LM8 in the lung was significantly higher than Dunn (* p < 0.01).

Figure 2

Actin polymerization, intensity, and stiffness between Dunn and LM8. (a) Actin cytoskeleton was stained with Alexa 488-conjugated phalloidin. Left: Dunn, Right: LM8. (b) Actin staining intensity was evaluated by arbitrary units (AU) using Image J software. * p < 0.005. (c) Young’s modulus was measured by AFM. Mean ± SD, Dunn: 9.20 ± 0.27 kPa. LM8: 8.11 ± 0.51 kPa. * p < 0.005.

Figure 3

Change of Young’s modulus by cytochalasin. (a) Measured area by AFM was indicated in the scheme. (b) Young’s modulus images in Dunn (left) and LM8 (right) with or without cytochalasin were shown. (c) Young’s modulus was compared between Dunn and LM8 with or without cytochalasin. Dunn (−: 9.20 ± 0.27 kPa, +: 7.69 ± 0.35 kPa, p < 0.005), LM8 (−: 8.11 ± 0.51 kPa, +: 7.77 ± 0.26 kPa, * p < 0.005, n.s.: not significant).

Figure 4

Actin polymerization and intensity with or without cytochalasin. (a) Actin filaments were visualized and compared between Dunn (left) and LM8 (right) with or without cytochalasin. (b) Actin intensity was evaluated between Dunn and LM8 with or without cytochalasin.

Figure 5

Functional analysis of LM8 and Dunn cells. (a) Cell proliferation and (b) migration were measured after 3, 24, 48, and 72 h in culture. (c) Invasion activity were calculated after 48 h treatment. (d) Adhesion activity was evaluated after 1 h treatment.