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Dispersal of Glioblastoma (GBM) renders localized therapy ineffective and is a major cause of recurrence. Previous studies have demonstrated that Dexamethasone (Dex), a drug currently used to treat brain tumor-related edema, can also significantly reduce dispersal of human primary GBM cells from neurospheres. It does so by triggering α5 integrin activity, leading to restoration of fibronectin matrix assembly (FNMA), increased neurosphere cohesion, and reduction of neurosphere dispersal velocity (DV). How Dex specifically activates α5 integrin in these GBM lines is unknown. Several chaperone proteins are known to activate integrins, including calreticulin (CALR). We explore the role of CALR as a potential mediator of Dex-dependent induction of α5 integrin activity in primary human GBM cells. We use CALR knock-down and knock-in strategies to explore the effects on FNMA, aggregate compaction, and dispersal velocity in vitro, as well as dispersal ex vivo on extirpated mouse retina and brain slices. We show that Dex increases CALR expression and that siRNA knockdown suppresses Dex-mediated FNMA. Overexpression of CALR in GBM cells activates FNMA, increases compaction, and decreases DV in vitro and on explants of mouse retina and brain slices. Our results define a novel interaction between Dex, CALR, and FNMA as inhibitors of GBM dispersal., Competing Interests: The authors declare that they have no conflict of interest.

Glioblastoma is highly aggressive. Early dispersal of the primary tumor renders localized therapy ineffective. Recurrence always occurs and leads to patient death. Prior studies have shown that dispersal of Glioblastoma can be significantly reduced by Dexamethasone (Dex), a drug currently used to control brain tumor related edema. However, due to high doses and significant side effects, treatment is tapered and discontinued as soon as edema has resolved. Prior analyses of the dispersal inhibitory effects of Dex were performed on tissue culture plastic, or polystyrene filters seeded with normal human astrocytes, conditions which inherently differ from the parenchymal architecture of neuronal tissue. The aim of this study was to utilize an ex-vivo model to examine Dex-mediated inhibition of tumor cell migration from low-passage, human Glioblastoma neurospheres on multiple substrates including mouse retina, and slices of mouse, pig, and human brain. We also determined the lowest possible Dex dose that can inhibit dispersal. Analysis by Two-Factor ANOVA shows that for GBM-2 and GBM-3, Dex treatment significantly reduces dispersal on all tissue types. However, the magnitude of the effect appears to be tissue-type specific. Moreover, there does not appear to be a difference in Dex-mediated inhibition of dispersal between mouse retina, mouse brain and human brain. To estimate the lowest possible dose at which Dex can inhibit dispersal, LogEC50 values were compared by Extra Sum-of-Squares F-test. We show that it is possible to achieve 50% reduction in dispersal with Dex doses ranging from 3.8 x10-8M to 8.0x10-9M for GBM-2, and 4.3x10-8M to 1.8x10-9M for GBM-3, on mouse retina and brain slices, respectively. These doses are 3-30-fold lower than those used to control edema. This study extends our previous in vitro data and identifies the mouse retina as a potential substrate for in vivo studies of GBM dispersal.