Adhesion Plasticity and Paracrine “Education” Shape the Invasive Trajectory of Glioblastoma (GBM)

Introduction: : Glioblastoma (GBM) is a primary brain tumor that has a short median survival time because its highly invasive cells evade surgical resection. Invasive GBM cells often express a constitutively active epidermal growth factor receptor variant (EGFRvIII), and while EGFRvIII's kinase activity has been extensively studied, its cell intrinsic role in migration through adhesion receptors and cell extrinsic role in cooperative invasion with wild-type EGFR (wtEGFR) cells remains unexplored. Highly invasive tumors require both wtEGFR and EGFRvIII, hence here we assessed adhesion alone and in co-culture to investigate co-operative mechanisms contributing to a highly invasive phenotype.

Materials and

Methods: : Mouse astrocytes expressing either wtEGFR or EGFRvIII were cultured as previously described (Bachoo et al.,Cancer Cell, 2002; Inda et al., Genes and Development, 2010). To measure cell-ECM adhesion strength, 104 cells/cm2 cells were seeded onto 25 mm fibronectin coated glass coverslips and exposed to a range of fluid sheer in a custom-built spinning-disk device for 5min. Once spun, cells were fixed, DAPI stained and imaged using the Keyence microscope. A custom-written Python program was used to quantify average cell-adhesion. For the 3D system, spheroids made in an AggreWell-400 plate were embedded in rat tail type-I collagen and starting day 0, cellular invasion into the matrix was tracked every 24hrs on a ZEISS Confocal Laser-Scanning microscope. Cytokines in the media were detected using the Proteome Profiler Mouse-XL Cytokine-Array kit. Bulk-RNA sequencing was performed on ILLUMINA NOVASEQ-6000 platform. For intracranial injection, a total of 5 x 105 cells in a 5-uL volume were injected into 4- to 5-wk-old NSG mice using a guide-screw system. Whole brains with tumor-cells were cleared using the Cubic-X2 protocol(Murakami et al., Nature Neuroscience, 2018) and imaged on Zeiss Z.1 Light-Sheet microscope.

Results, Conclusions, and Discussions:: EGFRvIII cells are more weakly adherent, enabling their ability to migrate faster than their wtEGFR counterparts. EGFRvIII conditioned media reduced adhesion strength of wtEGFR cells, but direct co-culture showed that extrinsic signaling was bidirectional with both populations experiencing a two-fold decrease in adhesion strength (Fig. 1A). Cooperative adhesion reduction was induced by a secretome unique to co-cultured cells, further suggesting bidirectional communication (Fig. 1B). Adhesion changes also cause post-“education” differences in invasion mode; EGFRvIII cells alone disseminate in a follow-the-leader mode but switch to single cell scattering when in co-culture with wtEGFR (and similar to the wt cells only condition) (Fig. 1C). In addition to mode, the extent of migration is impacted by co-culture; mixed co-culture spheroids invade the matrix more extensively than spheroids of either population alone, mirroring how patient tumors heterogeneity scales with invasiveness and prognosis (Fig 1D). Moreover, RNA-seq, cytokine arrays, and intracranially injected in vivo mouse models have identified transcriptional and signaling alterations that could drive GBM adhesion.

Together, these data indicate that intrinsic and extrinsic signaling both contribute to glioma cell migration and suggest novel GBM targets for its treatment. Our findings demonstrate that the bidirectional communication between wtEGFR and EGFRvIII cells creates a unique microenvironment that enhances invasion through cooperative modulation of cell adhesion strength and migration modes. These insights into the molecular mechanisms driving heterogeneous GBM invasion provide potential therapeutic targets for addressing both cell-intrinsic and extrinsic signaling pathways that could inhibit tumor cell dissemination and improve patient outcomes.

Acknowledgements and/or References (Optional)::