LGR5 promotes tumorigenicity and invasion of glioblastoma stem‐like cells and is a potential therapeutic target for a subset of glioblastoma patients

Abstract

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine‐rich repeat‐containing G‐protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient‐derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5‐responding cultures could be identified by their significantly higher self‐renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high‐ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high‐ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5‐responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient‐derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A molecularly distinct subset of glioblastoma requires serum‐containing media to establish sustainable bona fide glioblastoma stem cell cultures

“…We analyzed the invasion capacity of ESCs in vitro using the collagen invasion assay. Interestingly, we found that all three ESCs were equally or more invasive than U3047MG_SFC (Figure j), that displayed invasive growth in vivo (Figure S1) and previously has been shown to be highly invasive in vitro compared to other SFCs (Xie et al, ). This suggested that the invasive properties of ESCs in vivo might have been hampered by the lack of exogenous factors in the immune‐deficient mouse brain microenvironment.…”

“…The expression of stem cell markers and extended proliferative capacities of ESCs prompted us to analyze their self‐renewal properties. We used extreme limiting dilution assay (ELDA) and found that the ESCs formed tumorspheres (Figure a–c) in a range previously described for several SFCs (Xie et al, ). In line with their more stem‐like phenotype upon switch to SF media (Figure ), this was also accompanied by a significant increase in self‐renewal (Figure a–c, ESC_to_SFC), where the pGBM cultures (Figure a,b) had similar sphere‐forming capacities as the reference culture U3047MG_SFC (Figure d).…”

“…Cell spheres were obtained by seeding 100 cells/well in 96‐well plates that were left undisturbed in the incubator for 7 days. Collagen gel mix was prepared and 400 μl per well was added in 24‐well plates as described (Xie et al, ). Five to six spheres were transferred to each collagen‐covered well and these were covered with an additional 400 μl of collagen mix.…”

“…Before NSC media became the standard culture method, GBM cells were for decades established and maintained in serum‐containing media (Ponten & Macintyre, ), and many such GBM cell lines have been extensively used in research (Allen, Bjerke, Edlund, Nelander, & Westermark, ). In an effort to create an up‐to‐date library of GBM cell cultures we recently presented a panel of authenticated in vitro cell models established under defined SF conditions representing a large part of GBM inter‐tumor heterogeneity (Jiang et al, ; Xie et al, , ). In doing so we observed that a significant part of GBM samples did not form long‐term SFCs excluding almost half of the GBM patients from being represented by cell models, a notion also supported by others (Balvers et al, ; Behnan et al, ; Galli et al, ; Ledur, Onzi, Zong, & Lenz, ).…”

LGR6 is a potential diagnostic and prognostic marker for esophageal squamous cell carcinoma

“…Leucine‐rich repeat‐containing G protein–coupled receptors (LGRs) are a subgroup of the seven‐transmembrane G protein‐coupled superfamily that regulates various physiological processes associated with various diseases, and its member LGR4‐6 has high homology . Many studies have recently explored the biological functions of LGR4‐6 in various human cancer types . LGR4‐6 plays an important role in activating the Wnt/β‐catenin pathway by binding with R‐spondin (RSPO) ligands, which are closely related to tumor progression and invasion …”