Dual roles of tumour cells-derived matrix metalloproteinase 2 on brain tumour growth and invasion

Abstract

Background: A previous study on a murine astrocytoma cell-line ALTS1C1 showed a highly invasive pattern similar to clinical anaplastic astrocytoma in vivo. This cell-line also expressed a high level of matrix metalloproteinase 2 (MMP2). This study aimed to verify the role of MMP2 in brain tumour progression.

Methods: ALTS1C1 and MMP2 knockdown (MMP2kd) cells were inoculated intracranially, and tumour microenvironment was assessed by immunohistochemistry staining.

Results: MMP2 expression was co-localised with CD31-positive cells at invading the tumour front and correlated with an invasive marker GLUT-1. The suppression of MMP2 expression prolonged the survival of tumour-bearing mice associated with tumours having smoother tumour margins, decreased Ki67-proliferating index, and down-regulated GLUT-1 antigen. Although the reduction of MMP2 expression did not alter the vessel density in comparison to parental ALTS1C1 tumours, vessels in MMP2kd tumours were less functional, as evidenced by the low ratio of pericyte coverage and reduction in Hoechst33342 dye perfusion.

Conclusions: This study illustrated that tumour-derived MMP2 has at least two roles in tumour malignancy; to enhance tumour invasiveness by degrading the extracellular matrix and to enhance tumour growth by promoting vessel maturation and function.

Figure 1

The association of MMP2 and GLUT-1 expression with tumour invasion in an orthotopic ALTS1C1 tumour model. The co-localisation of MMP2/CD31 and GLUT1/CD31 in ALTS1C1 tumours was stained by fluorescent IHC staining. Representative images of MMP2/CD31 and GLUT1/CD31 staining at (A) invasion tumour fronts, (B) infiltrating islands, and (C) the tumour core. The star indicated the co-localisation of MMP2 or GLUT1 on CD31-positive cells. Scale bar: 200 μm in A and C, and 50 μm in B.

Figure 2

Effects of MMP2 inhibition on cell proliferation, cell invasion, and the survival of brain tumour-bearing mice. (A) In vitro cell proliferation rate had no difference between ALTS1C1 and MMP2kd cells. (B) MMP2 knockdown altered the invasive ability of ALTS1C1 cells, but not the migration ability. (C) Kaplan–Meier survival curves of ALTS1C1 (n=18) and MMP2kd (n=11) tumour-bearing mice. (D) Representative fluorescent images of MMP2 and DAPI staining in ALTS1C1 and MMP2kd tumours. Scale bar: 50 μm. *P<0.05, ****P<0.0001.

Figure 3

MMP2 knockdown reduced the proliferation rate in vivo. (A) The change of tumour size was followed at the indicated day after tumour inoculation. The mean sectional areas of tumours were measured at the maximum cross section with H&E stain. (B) Representative images of Ki67 and DAPI staining in ALTS1C1 and MMP2kd tumours. Scale bar: 50 μm. (C) Quantification of Ki67⁺ cells was performed by counting the average of × 400 magnification field for sections in each tissue. *P<0.05, **P<0.01.

Figure 4

MMP2 expression promoted tumour invasion. (A) Representative images of tumour borders stained with DAPI in ALTS1C1 and MMP2kd tumours from day 17 tissues. ALTS1C1 had an invasive front and small tumour-infiltrating islands, but MMP2kd tumours had smooth boundaries and few islands. T indicated the local tumour site and N indicated the normal brain tissue. (B) Number of infiltrating islands in the brain was counted at the indicated day. The plot represents the average island numbers of three brains on each group observed under × 100 magnification and repeated in three independent experiments. (C) Representative images of GLUT-1 (upper panel) and Ki67 (lower panel) expression in the tumour margin of ALTS1C1 and MMP2kd tumours from day 17 tissues. The nucleus was visualised with haematoxylin staining (blue). (D) The quantification data for the comparison of GLUT-1 and Ki67 in the tumour margin of ALTS1C1 and MMP2kd tumours. *P<0.05, **P<0.01.

Figure 5

Suppression of MMP2 expression in tumour cells did not affect MVD, but increased VEGF expression. (A) Representative images of vessel density in tumours. Scale bar: 200 μm. (B) Quantification of MVD at the indicated day after tumour implantation. (C) Higher level of VEGF in MMP2kd tumours was detected by IHC staining. Scale bar: 200 μm. (D) The level of VEGF in normal brain, ALTS1C1, and MMP2kd tumour lysates was measured by western blotting.

Figure 6

MMP2 expression was associated with vascular function. (A) Representative images of ALTS1C1 and MMP2kd tumours stained with CD31 and NG2. Scale bar: 50 μm. (B) Quantification of the ratio of NG2 covered on the blood vessels at the indicated day after tumour implantation. (C) Representative images of blood perfusion and hypoxia in ALTS1C1 and MMP2kd tumours. CD31 and PIMO were stained to identify vessels and tumour hypoxia, respectively, and the blood perfusion was visualised using Hoechst33342. Scale bar: 200 μm. (D) Quantification of the blood perfusion (left panel) and hypoxia area (right panel) in tumours. *P<0.05, **P<0.01.