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Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (Ͼ10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients.

Both the perivascular niche (PVN) and the integration into multicellular networks by tumor microtubes (TMs) have been associated with progression and resistance to therapies in glioblastoma, but their specific contribution remained unknown. By long-term tracking of tumor cell fate and dynamics in the live mouse brain, differential therapeutic responses in both niches are determined. Both the PVN, a preferential location of long-term quiescent glioma cells, and network integration facilitate resistance against cytotoxic effects of radiotherapy and chemotherapy—independently of each other, but with additive effects. Perivascular glioblastoma cells are particularly able to actively repair damage to tumor regions. Population of the PVN and resistance in it depend on proficient NOTCH1 expression. In turn, NOTCH1 downregulation induces resistant multicellular networks by TM extension. Our findings identify NOTCH1 as a central switch between the PVN and network niche in glioma, and demonstrate robust cross-compensation when only one niche is targeted.

The newly proposed glioma stem cell (GSC) hypothesis may re-model the way we diagnose and treat the tumor, which highlights the need for a complete knowledge on the genetic and epigenetic "blueprints" of GSCs. To identify the true "stemness" signatures, pure GSC populations are primarily needed. Reliable in vitro methods enriching for GSCs and thereby identifying the key stem-like characteristics constitute the preliminary step forward. We discuss in this review the current widely used methods for enriching and isolating GSCs, namely neurosphere assay, CD133 Immunophenotyping and side population assay, and detail their limitations and potential pitfalls that could complicate interpretation of corresponding results.

Background Malignant gliomas including glioblastomas are characterized by a striking cellular heterogeneity, which includes a subpopulation of glioma cells that becomes highly resistant by integration into tumor microtube (TM)-connected multicellular networks. Methods A novel functional approach to detect, isolate, and characterize glioma cell subpopulations with respect to in vivo network integration is established, combining a dye staining method with intravital two-photon microscopy, Fluorescence-Activated Cell Sorting (FACS), molecular profiling, and gene reporter studies. Results Glioblastoma cells that are part of the TM-connected tumor network show activated neurodevelopmental and glioma progression gene expression pathways. Importantly, many of them revealed profiles indicative of increased cellular stemness, including high expression of nestin. TM-connected glioblastoma cells also had a higher potential for reinitiation of brain tumor growth. Long-term tracking of tumor cell nestin expression in vivo revealed a stronger TM network integration and higher radioresistance of the nestin-high subpopulation. Glioblastoma cells that were both nestin-high and network-integrated were particularly able to adapt to radiotherapy with increased TM formation. Conclusion Multiple stem-like features are strongly enriched in a fraction of network-integrated glioma cells, explaining their particular resilience.

Clinically relevant concentrations and incubation times of isoflurane could promote the viability and mobility of U251-GSCs, suggesting this general anaesthetic may have detrimental effects in glioblastoma by facilitating its growth and migration.

The human leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family contains LRIG1, 2 and 3, encoding integral membrane proteins with an ectodomain, a transmembrane domain and a cytoplasmic tail. LRIG1 negatively regulates multiple receptor tyrosine kinases signaling including the epidermal growth factor receptor (EGFR) and is a proposed tumor suppressor. The soluble LRIG1 ectodomain is demonstrated to be shed naturally and inhibit the progression of glioma. However, little is known regarding the functions of LRIG2. In oligodendroglioma, LRIG2 expression is associated with poor survival, suggesting that LRIG2 might have different functions compared with LRIG1. Since soluble LRIG1 ectodomain has a similar function to the full-length LRIG1, we hypothesize that the different roles exerted by LRIG2 and LRIG1 result from the difference of their ectodomains. Here, we addressed the functions of LRIG2 and LRIG2 ectodomain in the proliferation and apoptosis of glioma and the possible underlying mechanisms. Firstly, we found that LRIG2 expression levels positively correlated with the grade of glioma. Further, we demonstrated for the first time that soluble LRIG2 ectodomain was capable of being released from glioblastoma cells and exerted a pro-proliferative effect. Overexpression of LRIG2 ectodomain promoted the proliferation and inhibited the apoptosis of glioblastoma cells in vitro and in vivo in a similar manner to the full-length LRIG2. Both full-length LRIG2 and LRIG2 ectodomain were found to physically interact with EGFR, enhance the activation of EGFR and its downstream PI3 K/Akt pathway. To our knowledge, this is the first report demonstrating that soluble LRIG2 ectodomain is capable of being released from glioblastoma cells and exerts a similar role to the full-length LRIG2 in the regulation of EGFR signaling in the progression of glioblastoma. LRIG2 ectodomain, with potent pro-tumor effects, holds promise for providing a new therapeutic target for the treatment of glioblastoma.