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Therapeutic Targets to Limit Glioblastoma Recurrence Uncovered

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“Cellular Plasticity Confers Migratory and Invasive Advantages to a Population of Glioblastoma-Initiating Cells that Infiltrate Peritumoral Tissue”

From Stem Cells

Treatment of glioblastoma multiforme (GBM) is often problematic due to infiltration of the peritumoral (PT) parenchyma by tumour cells (Kelly et al, Martiroysan et al and Stevenson et al) leading to tumour regrowth.   Additionally, studies into GBM also support the existence of cancer stem cells (CSCs) which survive traditional treatments and initiate tumour recurrence (Eyler and Rich).   As CSCs from GBM (or Glioblastoma Initiating Cells – GICs) have been found in peritumoral regions (Glas et al), studies into the mechanisms behind the ability of GICs to migrate and infiltrate may allow the development of new therapeutic interventions.   In their current study, researchers from the laboratory of Jose L. Fernandez-Luna at the Hospital Universitario Marqués de Valdecilla and Instituto de Formación e Investigación Marques de Valdecilla (IFIMAV), Santander, Spain have shown that GICs isolated from the PT have an invasive advantage over GICs from the tumour mass (TM), and that this is governed by an upregulation in αVβ3 integrin, which promotes Rac1 activation (Dey et al) and is involved in sensing matrix rigidity during cell spreading (Jiang et al) and the downregulation of p27, which can inhibit RhoA activation (Besson et al) (Ruiz-Ontañon et al).

Paired cultures of TM and PT GICs from three GBM patients were established and analysed for stem-like properties.   GICs from both TM and PT formed neurospheres, with neurosphere cells expressing Nestin and Sox2 and being able to differentiate towards GFAP+ and Tuj1+ neural cells.   Both cells types also contained copy number variations described in GBM neurosphere lines (Chen et al) and retained tumour formation capacity following injection into mouse brain.   However TM cells formed bigger tumours compared to PT cells and demonstrated a reduced doubling time and a higher proportion of cells in S and G2/M phases of the cell cycle.   Interestingly, lower proliferation rate, as seen in the PT cells, has been linked to an increased migration and infiltration capacity (Hoek et al).   Comparative microarray analysis of TM and PT cells found 64 differences including genes associated with cell adhesion, migration, cell cycle, and immune or inflammatory responses.  

Migrational analysis of PT cells found no effect from differing matrix stiffness, whereas TM cell migration speed fell with increasing matrix stiffness, while PT cells displayed high plasticity in cellular protrusion formation and migrated through cell thinning and extension.   Additionally, PT cells were highly invasive compared to TM cells (measured during culture in collagen matrices), again associated with long protrusions, and PT cells were also more adhesive to collagen matrices.   Analysis after GIC injection into immunodeficient mice found that while TM cells formed small masses, PT cells formed a dispersed distribution pattern with cells infiltrating different regions of the brain and tissue proximal to blood vessels, unlike TM cells, and exhibited an irregular cell shape and nuclei.   Furthermore, PT cells cultured on human umbilical vein endothelial cells were more adherent than TM cells, while analysis using a chicken embryo xenograft model found that only PT cells invaded the embryo tissue, including the vascular endothelium, from the injection point at the limb bud.

Investigations into the molecular mechanisms behind the enhanced migration and invasion of PT cells first addressed the activity of Rho GTPases; key regulators of cell migration.   Both Rac1 and RhoA were active in PT cells, consistent with their high plasticity, and silencing of Rac1 in PT cells reduced their invasiveness, which was augmented by inhibiting ROCK signalling.   Analysis of the RhoA inhibitor p27 found it to be downregulated in PT cells, and artificial downregulation by siRNA in TM cells increased cell protrusion length and increased the capacity of TM cells to infiltrate surrounding tissue in the chicken embryo xenograft model.   Additional analysis found that β3 integrin was upregulated in PT cells which is known to be involved in mechanotransduction (as an Integrin αVβ3 heterodimer) and Rac1 activation.   Blocking αVβ3 reduced cell invasion, and was again augmented with addition of the ROCK inhibitor.  

In conclusion, glioblastoma initiating cells found in peritumoral areas are more mobile and invasive than those found within the tumour mass. The molecular changes detected in more highly invasive cells in this study, including increased αVβ3 integrin and low levels of cytoplasmic p27 and Rac and RhoA GTPases, may facilitate this invasive nature and highlights these as possible therapeutic targets for the treatment of glioblastoma to stop tumour spread and recurrence.   The authors also note that their microarray findings identified genes which distinguish PT and TM cells, and so further research in this direction could allow the identification of additional therapeutic targets.   Of particular interest is the HEG1 gene, which is unique to endothelial cells and required for blood vessel formation (Kleaveland et al) and may promote cell migration.

 

References

  • Besson A et al.p27Kip1 modulates cell migration through the regulation of RhoA activation.Genes Dev2004;18:862876
  • Chen R et al. A hierarchy of self-renewing tumor-initiating cell types in glioblastoma. Cancer Cell 2010; 17: 362–375
  • Dey N et al.The protein phosphatase activity of PTEN regulates SRC family kinases and controls glioma migration.Cancer Res2008;68:18621871.
  • Eyler CE,Rich JN.Survival of the fittest: Cancer stem cells in therapeutic resistance and angiogenesis.J Clin Oncol2008;26:28392845
  • Glas M et al.Residual tumor cells are unique cellular targets in glioblastoma.Ann Neurol2010;68:264269
  • Hoek KS et al. In vivo switching of human melanoma cells between proliferative and invasive states. Cancer Res 2008; 68: 650–656
  • Jiang G et al.Rigidity sensing at the leading edge through alphavbeta3 integrins and RPTPalpha.Biophys J2006;90:18041809
  • Kelly PJ et al.Imaging-based stereotaxic serial biopsies in untreated intracranial glial neoplasms. J Neurosurg1987;66:865874
  • Kleaveland B et al. Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway. Nat Med 2009; 15: 169–176
  • Martirosyan NL et al.Use of in vivo near-infrared laser confocal endomicroscopy with indocyanine green to detect the boundary of infiltrative tumor.J Neurosurg2011;115:11311138
  • Stevenson CB et al.CXCR4 expression is elevated in glioblastoma multiforme and correlates with an increase in intensity and extent of peritumoral T2-weighted magnetic resonance imaging signal abnormalities.Neurosurgery2008;63:560569

From Stem Cells.

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.