Basic Research for Osteosarcoma Lung Metastasis (LM8)

Fig. 2.1

Establishments of highly metastatic OS cell line, LM8

2.3 Candidate Target Molecules

When we compared the expression of some of the proteins related to the cancer metastasis, most of the metastasis-related genes (proteins) MMP, VEGF, etc. are upregulated in LM8 compared to the parental Dunn cells, as expected (Table 2.1).

Table 2.1

Comparison of several biological characters between LM8 and Dunn

			LM8		Dunn
Metastasis	Subcutaneous transplantation		Massive pulmonary metastasis in 4 weeks		No pulmonary metastasis
Morphology	Filopodia formation		2.2 filopodia/cell		1.4 filopodia/cell
	Stress fiber formation		Few	<	Numerous
	Focal adhesion	Integrin β1, paxillin, vinculin	Localized in cell edge		Cell edge and cytoplasm
	Focal adhesion	FAK Y397 phosphorylation	High	>	Low
Motility	Vertical migration		20 % of applied cells	>	12 % of applied cells
	Horizontal migration		Fast	>	Slow
	Horizontal migration		Fibroblastic shape	>	Rounded shape
VEGF	RNA and protein expression		High	>	Low
MMP	MMP-2 secretion		High	>	None
Cdc42	GTP-Cdc42		High	>	Low
Cdc42	Cdc42 siRNA		20 % → 12 %		No change
Rho-ROCK-myosin	Myosin II phosphorylation		Low	<	High
	Y-27632 (10 μM)		20 % → 32 % (haptotaxis)		Slightly decrease (haptotaxis)
	Y-27632 (10 μM)		MLC-P↓		Slightly decrease (haptotaxis)
	Blebbistatin (2.5 μM)		20 % → 28 % (haptotaxis)		No change

2.3.1 MMP

Since MMP played several important biological roles in metastatic spread, a number of pharmaceutical companies tried to develop specific inhibitors for clinical trials. Indeed, several compounds were undertaken for the phase III clinical trials in the late 1990s; however, no drug has been approved so far. A number of reasons were pointed out, including inappropriate clinical protocols, no surrogate markers, heterogeneity of cancer cells, etc. [5]. We also tried several MMP inhibitors for the LM8 animal experiments, but no positive effect was found for the lung metastasis.

2.3.2 Motility

When we established LM8 cells from parental Dunn cells, we already recognized the clear morphological difference between the two. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin beta 1, and biochemical analyses present the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single-cell migration mode. LM8 migration was increased by a Rho-associated protein kinase (ROCK) inhibitor, Y-27632, while it was decreased by Cdc42 silencing using RNA interference system (Table 2.1) [6].

We also found that a clinically approved camptothecin analog irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK and attenuated integrin beta 1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice (Fig. 2.2). The concentrations of irinotecan-active metabolites SN38 were equivalent to in vitro experiments to inhibit motility of LM8. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma [6].