The findings of the three-dimensional culture of SCLs.

<p>Cells were incubated for 12(A and B) or 24 hours (C and D). SCLs proliferated linearly and formed tube-like structures and network-like structures within Matrigel (A), but the A549 (lung adenocarcinoma) cells formed no network structures (B). SCLs incubated with EBM-2 also formed the tube-like structures in the Matrigel (C). Exposure to 10 µM batimastat inhibited the formation of the three-dimensional structures (D). (Scale bars show 300 µm.)</p

Batimastat suppressed the growth of SCLs in xenograft models.

<p>Subcutaneous tumors were formed on the backs of the mice in the control group (A) and batimastat group (B). It was revealed that there were significant differences in the tumor weights between the control group (n = 6) and batimastat group (n = 4) (C). (* p-value <0.05, versus control group, by Student's t-test)</p

Batimastat suppressed the biological activities of SCLs in <i>in vivo</i> studies.

<p>(A) Batimastat, a synthetic matrix metalloproteinase inhibitor, suppressed the proliferation of SCLs 72 hours after incubation with batimastat. The number of SCLs that survived following the culture with batimastat was lower than that in the control group. (* p-value <0.05, **p-value<0.01 versus control group, by Student's t-test, n = 6). (B) The invasion assays revealed that the SCLs incubated with batimastat were less invasive than those in the control group. (* p-value <0.05 versus control group, **p-value<0.01 versus control group, by Student's t-test, n = 8)</p

Tumors which grew along the intimal surface of the pulmonary arteries.

<p>(A) A resected lung from a mouse. SCID mice were sacrificed 28 days after the injection of SCLs. Many nodules were located on the surface of the lungs. Arrow heads showed some of these nodules. (B) HE staining showed that the tumors were composed of a central area with necrosis and a peripheral zone filled with SCLs. (C) Immunohistochemical staining for MMP-14 revealed that the peripheral area of tumors was positive for MMP-14, and the central necrotic area was negative for MMP-14. (D) Immunohistochemical staining for MMP-2 showed that MMP-2 was weakly positive in the peripheral zone. (Scale bars show 100 µm.)</p

The results of the immunohistochemical analysis of the SCLs.

<p>The SCLs were positive for vimentin (A), MMP-14 (F), MMP-2 (G) and CD44 (H). In contrast, the staining for α-SMA (B), VWF (C), CD31 (D) and desmin (E) was negative. (Scale bars show 50 µm.)</p

The results of the Western blot analysis.

<p>(A) The Western blot analysis revealed that the MMP-14 protein expression in SCLs was higher than that in A549 cells. There was no significant difference in the protein expression of MMP-14 in SCLs incubated in the media with or without growth factors. (B) Batimastat did not suppress the SCLs' expression of the MMP-14 and MMP-2 proteins. (C)(D) The band signal strength of MMP-14 (c) and MMP-2 (d) expressed as a ratio to beta-actin. No significant differences in the signal strength were recognized 24 hours and 48 hours after batimastat exposure between the batimastat and control groups. (“N.S.” showed no significant difference between two groups analyzed by Student's t-test.)</p