Treatment strategies for medulloblastoma include surgery, irradiation and/or chemotherapy (Raffel, 2004
). These approaches do not always control invading tumor cells and local recurrence, cerebral spinal fluid and systemic metastasis result in a 60–70% 5 year survival rate (Louis et al., 2002
We provide evidence for Slit2 inhibition of invading medulloblastoma cells. This was shown using medulloblastoma spheroid-Slit2 aggregate cocultures, sodium alginate bead bioreactors in collagen and also in confrontation cocultures with astrocyte aggregates. Slit proteins have been associated with chemorepellent roles in axon guidance; however, inhibitory effects have been described. Cells migrating from neurospheres generated from SVZ of P0-P3 Slit1 −/− mice were found to migrate further and in a more dispersed pattern than cells from Slit1 +/− neurospheres suggesting that Slit1 plays an inhibitory role in addition to its function as a repellent (Nguyen-Ba-Charvet et al., 2004
). Other studies have suggested that Slit inhibits migration from subventricular zone explants; however, when combined with an astrocyte-derived migration inducing activity, Slit acts as a repellent (Mason et al., 2001
). Slit2 has also been shown to inhibit leukocyte chemotaxis induced by chemokines (Wu et al., 2001
Microencapsulation bioreactor technology has been used to assess the efficacy of antiangiogenic compounds on glioma growth (Joki et al., 2001
; Read et al., 2001
). Sodium alginate bioreactors provide an isolated environment for continuous protein delivery. We have shown sustained bioreactor Slit2 secretion for several weeks in our models (unpublished results). This model also demonstrates that the inhibition of medulloblastoma cell invasion by continuous Slit2 delivery can be regulated and controlled by mixed beads encapsulating both Slit2 and RoboN cells. Bioreactor technology can potentially be used for combination treatment with encapsulated cells overexpressing multiple anti-invasive, antiproliferative and proapoptotic molecules.
Marillat et al. (2004)
have demonstrated a role for Rig1/Robo3 in controlling midline crossing of hindbrain precerebellar neurons and axons. Human patients with horizontal gaze palsy with progressive scoliosis (HGPPS) were reported to have mutations in Rig1/Robo3, and functional studies have shown defects in commissural hindbrain projections and pontine nuclei (Jen et al., 2004
). Gilthorpe et al. (2002)
have shown that early and late stage chick cerebellar rhombic lip fragments are repelled by Slit2. Medulloblastoma cells are thought to arise from external granular layer cerebellar precursors derived from the rhombic lip, and these results provide an obvious developmental parallel with our studies of Slit2 inhibition of medulloblastoma cell invasion. Slit also inhibits CXCR4-induced motility in breast cancer cells and CXCR4 antagonists have been shown to inhibit medulloblastoma tumor growth in vivo
(Rubin et al., 2003
; Prasad et al., 2004
). However, in contrast to our studies, Slit treatment alone did not influence the motility of the breast cancer cells (Prasad et al., 2004
The lack of responsiveness of highly invasive glioma cells to Slit2 may be attributed to a variety of mechanisms. Dallol et al. (2003a)
have found by quantitative real-time RT–PCR that Slit2 expression was downregulated in some gliomas with methylated SLIT2 promoter compared to gliomas and normal brain samples showing no methylation of this promoter. Our results demonstrate that Slit2 and Robo1 are expressed by a variety of glioma and medulloblastoma cell lines and primary tumors. Marillat et al. (2002)
have shown that most CNS neurons in the rat brain express at least one Robo and one Slit during their development, and that levels are maintained from the embryonic to the adult stage. These authors suggested that neurons expressing Robo mRNA could be unresponsive to Slit if molecules or mechanisms regulating Robo expression and function were present (Marillat et al., 2002
). We utilized the demethylating agent 5′ aza-2′-deoxycytidine to test for methylation silencing as a potential mechanism for loss of Robo1 and Slit2 protein expression in our cell lines. This treatment had no effect on expression levels of both Slit2 and Robo1 in the cell lines tested.
Wong et al. (2001)
have demonstrated a role for the srGAPs (Slit-Robo GAPs) in Slit-Robo signaling. Interaction of srGAP’s with the Rho-GTPase, Cdc42 is essential for the repulsive activity of this guidance cue (Wong et al., 2001
). Other studies support a role for Phosphatidyl inositol 3 kinase, Dock and Pak, and the Abelson (Abl) kinase and Enabled (Ena) proteins further implicating actin and microtubule reorganization in Slit-Robo signaling (Bashaw et al., 2000
; Fan et al., 2003
; Wang et al., 2003
). Our time-lapse studies showed a change in cell morphology, as the presence of Slit2 aggregates increased the number of noninvading ruffling cells. We have shown that Slit2 does not influence cell proliferation suggesting that these stationary cells are not undergoing cell death. The presence of noninvading ruffling cells has also been demonstrated in glioblastoma spheroid cocultures in response to a secreted chemorepellent (Werbowetski et al., 2004
). This prolonged cell ruffling may be associated with a failure of the cell to organize its actin-myosin cytoskeleton (Del Maestro et al., 2001
We have assessed Robo1 surface expression levels by biotinylation of cell surface proteins and our results confirm that differential responses to Slit2 are attributed to mechanisms downstream of the cell membrane. We have demonstrated decreased Cdc42 activation for medulloblastoma cells in response to Slit2. This agrees with previous results obtained by Wong et al. (2001)
. Therefore, there may be a conserved mechanism regulating both the repellent and inhibitory response to Slit2. Cdc42 is known to regulate actin-rich filopodia formation and nuclear repositioning during the initiation of cell migration (reviewed by Luo, 2000
; Gomes et al., 2005
). The inhibitory effect of Slit2 on medulloblastoma cell invasion may be mediated by Cdc42-dependent effects on the actin cytoskeleton and/or cell polarity (reviewed by Luo, 2000
; Etienne-Manneville and Hall, 2001
; Palazzo et al., 2001
; Gomes et al., 2005
). Slit2-Robo1 interaction may be insufficient to inhibit the extensive invasion seen in glioblastoma cells lines such as U251 and C6 but effective in modulating the cell movement in the less invasive medulloblastoma cell lines.
Slit2 and Robo1 expression were not correlated with the degree of malignancy of the tumor samples or cell lines tested in this study. Rossi et al. (2005)
obtained similar results using RT–PCR analysis of Slit2 expression in the cerebral tumors and glioma cell lines. Although Slit2-Robo1 mRNA can be detected in many human tumors, this does not necessarily correlate with Slit2 protein content or activity. Our results would suggest that only cells from specific tumors may respond to this signaling by modulating their invasive paradigm. Analysis of the regional expression of Slit2 and Robo1 in highly invasive tumor samples may shed light on this issue. In the absence of specific Slit2 and Robo1 antibodies, a comparison of Slit2 and Robo1 expression at the leading edge versus the tumor core by in situ
hybridization would help to reconcile the RT–PCR and methylation data, and further delineate the role of Slit2 signaling in invading medulloblastoma cells.
We have demonstrated a slight decrease in Rac1 activity in response to Slit2. Slit stimulation leads to recruitment of a Dock and Pak complex to the Robo receptor and increases Rac activation (Fan et al., 2003
). In contrast, Vilse, a conserved family of RhoGAPs, regulate the repellent response to Slit by binding to the intracellular domain of Robo receptors and promoting Rac inactivation (Lundstrom et al., 2004
). Our results are indicative of an inhibitory, and not a directional, effect of Slit2 on medulloblastoma cells. A number of different signaling pathways may be involved in Slit2 inhibition of medulloblastoma invasion.
A new role for Slit2 as an inhibitor of medulloblastoma invasion has been identified. Our results reinforce the concept that selective neurodevelopmental cues may provide significant insights into tumor invasion and outline the need for detailed assessments of how to implement this strategy for other tumor types.