High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma

Abstract

BackgroundGlioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation.MethodsSeven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis.ResultsCell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines.ConclusionThe study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells.

Large-Scale Drug Screening in Patient-Derived IDHmut Glioma Stem Cells Identifies Several Efficient Drugs among FDA-Approved Antineoplastic Agents

“…However, they mostly rely on widely-used long-term cultured adherent cell lines and include only IDH wt glioma cell lines. This limits the usability of their results for the treatment of IDH mut glioma, since glioma stem cells grown as neurospheres seem to have a distinct drug sensitivity compared to cells grown adherently [ 55 ]. Furthermore, cells grown as neurospheres mimic the parental tumor more closely.…”

Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma

“…DNA for patient samples for lines TD1–3 and BK1 and BK3 was analyzed by the free online brain tumor classifier 17 at ( www.molecularneuropathology.org ) for methylation subgrouping. Patient lines GS1-TS20 are described further by Yu et al 16 …”

“…Patient cell line derivation and expansion were performed as described 16 using serum-free methods. All cells were cultured in neurobasal medium (NBM) supplemented with 1x N-2 and 1x B-27 and growth factors epidermal growth factor and human fibroblast growth factor (20 ng/mL).…”

“…Whole spheroids were processed and imaged as described. 16 Briefly, spheroids were incubated with primary antibodies at the following dilutions: Nestin 1:1000 (Abcam) and Sox2 1:250 (Abcam). Secondary staining was performed using Alexa Fluor 488 and 594 secondary antibodies (Invitrogen), followed by nuclear counterstaining with 4′,6′-diamidino-2-phenylindole (DAPI) (300 mM).…”

“…Therapeutics that could potentially abrogate the oncogenic effects of Sox2 in GBM stemlike cells are therefore an interesting prospect, especially those that are already approved for alternative clinical applications such as ACTD. Work done previously in our group 16 has demonstrated that automated screening of repurposed drug libraries is feasible in clinically relevant time-frames, while revealing ACTD to be a potent cytotoxic agent in seven GSC lines. Here we use a refined screening process to confirm our findings in an additional 5 GSC lines and validate ACTD as a potential therapeutic for recurrent GBM in multiple patient-derived in vivo models.…”