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

FAK Y397 phosphorylation

High

>

Low

Motility

Vertical migration

20 % of applied cells

>

12 % of applied cells

Horizontal migration

Fast

>

Slow

Fibroblastic shape

Rounded shape

VEGF

RNA and protein expression

High

>

Low

MMP

MMP-2 secretion

High

>

None

Cdc42

GTP-Cdc42

High

>

Low

Cdc42 siRNA

20 % → 12 %
 
No change

Rho-ROCK-myosin

Myosin II phosphorylation

Low

<

High

Y-27632 (10 μM)

20 % → 32 % (haptotaxis)
 
Slightly decrease (haptotaxis)

MLC-P↓

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].
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Jun 4, 2017 | Posted by in ORTHOPEDIC | Comments Off on Basic Research for Osteosarcoma Lung Metastasis (LM8)

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