Patient-derived glioblastoma stem cells respond differentially to targeted therapies

Abstract

The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development.

Novel approach to temozolomide resistance in malignant glioma: connexin43-directed therapeutics

“…Lathia and colleagues reported that the ratio of Cx46 to Cx43 was elevated in GSCs, whereas following differentiation into non-CSC GBM cells, Cx46 is reduced and Cx43 is increased. Furthermore, characterization of 10 GSCs lines derived from established cell lines, xenografts, and freshly dissected patient tumor tissues indicated variation in self-renewal capabilities, gene expression profiles, and chemotherapeutic susceptibility [16 •• ]. Recent reports also emphasize the importance of Cx43 subcellular localization and how these findings may translate to recently reported in vitro findings [21 • ].…”

“…Given that Cx43 hemichannels facilitate secretion of certain small molecules such as ATP or glutamate [56], it is possible that inhibition of Cx43 hemichannels by Cx43 peptidomimetics helps retain TMZ in tumor cells, thereby increasing TMZ cytotoxicity. When used in combination with TMZ, targeting Cx43 with this peptide also inhibited GSC cell self-renewal and viability [16 •• ]. Ongoing efforts are geared towards reformulating aCT1 for internal use in brain tumors.…”

“…An added layer of complexity is afforded through channel-independent mechanisms via the stabilization or destabilization of multiprotein complexes mediated through protein binding sites on the connexin protein. Connexin43 (Cx43) is the most ubiquitous connexin and in addition to having critical regulatory and developmental roles in many tissues, has been implicated as a driver of tumor invasion [11 •• ], a marker of tumor progression [12], and an inducer of TMZ resistance in GBM cells [6 •• ,13–15,16 •• ]. Furthermore, the expression and function of connexins in the microenvironment of malignant glioma is an evolving field, where recent studies investigating the function of Cx43 in the tumor microenvironment reveal that Cx43 in peritumoral cells regulates gliomagenesis, tumor progression, and treatment resistance.…”

Importance of GFAP isoform‐specific analyses in astrocytoma

“…Since then, GFAP is often used to mark cells with stem cell characteristics in glioma and to target neural stem cells to induce gliomagenesis in animal models (Kwon et al, 2008;J. Chen et al, 2012;Bradshaw et al, 2016;Guichet et al, 2016;Kanabur et al, 2016;Jiang et al, 2017;Welker, Jaros, An, & Beattie, 2017). In addition, GFAP is up-regulated in non-neoplastic astrocytes that become reactive in response to the growth of the tumor and do not reflect the differentiation state of neoplastic cells (Gullotta, Schindler, Schmutzler, & Weeks-Seifert, 1985;Yoshii et al, 1992; H. Y.…”

The feasibility of using dielectrophoresis for isolation of glioblastoma subpopulations with increased stemness

“…Recent attempts to characterize GBM cellular diversity have revealed the presence of malignant cells with increased stem‐like properties known as glioblastoma stem cells (GSCs) . GSCs reside in the perivascular and hypoxic niches of glioblastoma tumors and exhibit plasticity, self‐renewal properties, resistance to therapies, and ability to cause relapse . Hence, it is important to characterize properties of GSCs with the goal of finding therapies that target this subpopulation .…”