Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion In vivo

Abstract

Malignant gliomas (primary brain tumors) aggressively invade the surrounding normal brain. This invasive ability is not demonstrated by brain metastases of nonglial cancers. The brain-specific, brain-enriched hyaluronan binding (BEHAB)/brevican gene, which encodes an extracellular hyaluronan-binding protein, is consistently expressed by human glioma and is not expressed by tumors of nonglial origin (Jaworski et al., 1996). BEHAB/brevican can be cleaved into an N-terminal fragment that contains a hyaluronan-binding domain (HABD) and a C-terminal fragment (Yamada et al., 1995). Here, using antisera to peptides in the predicted N-terminal and C-terminal proteolytic fragments, we demonstrate that the BEHAB/brevican protein is cleaved in invasive human and rodent gliomas. A role for this protein in glioma cell invasion was tested by transfecting a noninvasive cell line with the BEHAB/brevican gene. The noninvasive 9L glioma cell was transfected with either full-length BEHAB/brevican or the HABD and tested for invasion in in vitro and in vivo invasion assays. Although both constructs increased invasion in vitro, only the HABD increased invasion by tumors growing in vivo. Experimental intracranial tumors from full-length transfectants showed no increase in invasion over control tumors, whereas tumors from HABD transfectants showed a marked potentiation of tumor invasion, producing new tumor foci at sites distant from the main tumor mass. This work demonstrates a role for a brain-specific extracellular matrix protein in glioma invasion, opening new therapeutic avenues for a uniformly fatal disease.

Fig. 1.

Expression of BEHAB/brevican in rodent and human brain tumors. A, Western blots of invasive rodent brain tumors established from CNS-1 cells show full-length 140 kDa BEHAB/brevican, as well as 90 and 50 kDa proteolytic fragments. Full-length and cleaved 90 kDa bands are visualized with an antibody to a peptide in the C-terminal portion of BEHAB/brevican; the 50 kDa band is visualized with an antibody to a peptide in the N-terminal portion of BEHAB/brevican (see Materials and Methods). B, Western blots of surgical samples from neuropathologically diagnosed glioblastoma multiforme show 150 kDa full-length BEHAB/brevican and a 97 kDa proteolytic fragment. These bands are immunoreactive with the antibody to the C-terminal peptide; however, the antibody to the N-terminal peptide does not recognize the human protein.

Fig. 2.

Transfected 9L cells express full-length BEHAB/brevican or the HABD. Western blots of cell homogenates (lanes 1–3) and conditioned media (lanes 4–6) are from transfected cells. 9L-GFP cell homogenates (lane 1) and conditioned media (lane 4) show no immunoreactivity for BEHAB/brevican or its cleavage products. 9L-BEHAB/brevican cell homogenates (lane 2) and conditioned media (lane 5) contain a 140 kDa-immunoreactive species. 9L-HABD cell homogenates (lane 3) and conditioned media (lane 6) contain a 50 kDa species. The immunoreactive species in conditioned media from both 9L-BEHAB/brevican and 9L-HABD transfectants are polydisperse (lanes 5, 6), possibly reflecting glycosylation.

Fig. 3.

Full-length BEHAB/brevican and the HABD increase invasion and motility of 9L cells in vitro.A, Invasion in the Matrigel invasion assay is potentiated by the expression of either full-length BEHAB/brevican or the HABD. Two independent cell lines for each of the constructs (B/b-1 and B/b-2for 9L-BEHAB/brevican; HABD-1 and HABD-2for 9L-HABD) were assayed for invasion in the Matrigel invasion assay (see Materials and Methods). Invasion is expressed relative to that observed for the 9L-GFP transfectants. Both constructs markedly increased invasion. Invasion was equivalent when either fibronectin or hyaluronan was the attractant in the lower chamber. B, Motility in the absence of Matrigel is also increased by the expression of either full-length BEHAB/brevican or the HABD. As seen in the Matrigel assay, both constructs similarly increased motility in a Matrigel-independent assay. The degree of invasion or motility seen for all four transfected cell lines was statistically different from 9L-GFP cells at the p < 0.01 level. There was not a statistically significant difference between the behavior of 9LBEHAB/brevican versus 9L-HABD transfectants. Error bar indicates SEM.

Fig. 4.

Intracranial tumors established from 9L-HABD, but not from 9L-BEHAB/brevican, cells show increased invasion into the surrounding brain. A,A′, Intracranial tumors (asterisks) derived from 9L-GFP cells grow as compact cell masses, with little infiltration into the surrounding brain. The border with the underlying thalamus (A′) is smooth, with very few clusters of cells seen beyond the border between the tumor and the normal brain.B, B′, Intracranial tumors (asterisks) derived from 9L-BEHAB/brevican cells showed identical behaviors to those of the control transfectants. These tumors also grew as compact cell masses, with little infiltration of the surrounding brain. Here again, very few cell clusters were observed in the normal brain adjacent to the tumor (B′).C, C′, Intracranial tumors (asterisks) derived from 9L-HABD cells showed a marked increase in invasive ability compared with the other two cell lines. Although the main tumor mass was a compactgroup of cells, many cell clusters were seen in the surrounding normal brain. The border of the 9L-HABD tumors with the underlying thalamus (C′) was often interrupted by peninsulas of cells extending out from the main tumor mass.

Fig. 5.

Intracranial tumors established from 9L-BEHAB/brevican and 9L-HABD cells express full-length or HABD proteins. Western blots of tumors produced after intracranial injection of 9L-BEHAB/brevican (lane 1) or HABD (lane 2) transfectants are shown. In 9L-BEHAB/brevican tumors (lane 1), immunoreactivity for BEHAB/brevican is at 140 kDa. No evidence of a cleavage product is seen. In 9L-HABD tumors (lane 2), only a 50 kDa product is observed.