Feasibility study of using high-throughput drug sensitivity testing to target recurrent glioblastoma stem cells for individualized treatment

Abstract

Background: Despite the well described heterogeneity in glioblastoma (GBM), treatment is standardized, and clinical trials investigate treatment effects at population level. Genomics-driven oncology for stratified treatments allow clinical decision making in only a small minority of screened patients. Addressing tumor heterogeneity, we aimed to establish a clinical translational protocol in recurrent GBM (recGBM) utilizing autologous glioblastoma stem cell (GSC) cultures and automated high-throughput drug sensitivity and resistance testing (DSRT) for individualized treatment within the time available for clinical application.

Results: From ten patients undergoing surgery for recGBM, we established individual cell cultures and characterized the GSCs by functional assays. 7/10 GSC cultures could be serially expanded. The individual GSCs displayed intertumoral differences in their proliferative capacity, expression of stem cell markers and variation in their in vitro and in vivo morphology. We defined a time frame of 10 weeks from surgery to complete the entire pre-clinical work-up; establish individualized GSC cultures, evaluate drug sensitivity patterns of 525 anticancer drugs, and identify options for individualized treatment. Within the time frame for clinical translation 5/7 cultures reached sufficient cell yield for complete drug screening. The DSRT revealed significant intertumoral heterogeneity to anticancer drugs (p < 0.0001). Using curated reference databases of drug sensitivity in GBM and healthy bone marrow cells, we identified individualized treatment options in all patients. Individualized treatment options could be selected from FDA-approved drugs from a variety of different drug classes in all cases.

Conclusions: In recGBM, GSC cultures could successfully be established in the majority of patients. The individual cultures displayed intertumoral heterogeneity in their in vitro and in vivo behavior. Within a time frame for clinical application, we could perform DSRT in 50% of recGBM patients. The DSRT revealed a remarkable intertumoral heterogeneity in sensitivity to anticancer drugs in recGBM that could allow tailored therapeutic options for functional precision medicine.

Keywords: Drug sensitivity; Drug sensitivity and resistance testing; Glioblastoma; Glioblastoma stem cells; High-throughput drug screening; Individualized medicine; Recurrent glioblastoma.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Course of the disease and time frame for clinical protocol. Glioblastoma patients typically undergo surgery followed by combined radio- and chemotherapy for 6 weeks and thereafter monthly adjuvant chemotherapy. Despite this multimodal treatment the disease almost invariably recurs within 9 months. The time frame for this clinical protocol was defined as 10 weeks following surgery for recurrent GBM, which included expansion of individualized GSC cultures for 6 weeks, automated high-throughput drug screening and data analysis for 1 weeks and scheduling a treatment plan and initiation within 3 weeks

Fig. 2

Characterization of glioblastoma stem cells from recurrent GBM. a–c Pre- and post-operative T1-weighted, contrast-enhanced MRI of three recurrent GBM with the corresponding sphere-, cellular- and xenograft morphology. The individual cultures displayed extensive tumor-to-tumor heterogeneity in their in vitro morphology (e.g. adherent growth in T1608, various differentiation morphology) and in their induced tumor phenotype (e.g. mainly bulk formation In T1534, mainly invasive in T1608). Arrow points to compressed lateral ventricle. Xenografts stained with hematoxylin & eosin. In the recGSC cultures the tumors were harvested after 15 weeks following xenografting. d Total cell yield following serial passages revealed intertumoral variability in their capacity for cell proliferation. Dashed lines represent tumors that could not be serially expanded. e Intertumoral heterogeneity in the expression of stem cell related markers evaluated by flow cytometry. f Upon differentiation all cultures evaluated increased their expression of glial lineage marker GFAP, and all but one (T1513) increased the expression of the neuronal lineage marker β3-tubulin. Scale bar in the light microscopy images: 100 µm. Scale bar in fluorescent images 20 µm. Scale bar in brain sections 1 mm. T tumor, CC corpus callosum

Fig. 3

Heterogeneity in drug sensitivity in recGSCs. a Dose–response curves of the pan-HER inhibitor canertinib display the variation in drug efficacy in the recGSC cultures. Three responses are classified below the threshold for moderate activity (DSS ≥ 10). b Distribution of the number of drugs displaying a DSS ≥ 10 across the recGSC cultures. c Using a non-parametric one-way ANOVA of ranks, a significant difference was observed in the overall drug sensitivity across the cultures (p < 0.0001). According to the individual culture’s sensitivity to the entire drug collection (n = 525 drugs), they separated into two major clusters as the most and least sensitive. d Clustering of recGSC cultures by correspondence analysis based on all drug responses (n = 525) in all tumors (n = 6). The dots in the scatter plot represents the drugs in the DSRT and the color shading represent a heat map of where the average of the data is located. The scattering of tumors in the plot display both how they differ from the average and how tumors cluster together based on similarities in drug sensitivity patterns. e In the DSRT there were four pan-HER inhibitors that displayed a DSS ≥ 10 in recGSC cultures. f The consistency of T1532 being the most sensitive and T1544 the most resistant displayed an excellent correlation in correlation matrices (Spearman, ρ). g p-values in the correlation matrix of the pan-HER inhibitors. h Selecting for drugs with at least moderate efficacy (DSS ≥ 10) and increased patient-selectivity (sDSSGBM ≥ 5) the distribution of individual classes of drugs with selective efficacy revealed a considerable tumor heterogeneity in drug sensitivity in recGSC cultures

Fig. 4

Unsupervised hierarchical clustering of drug sensitivity patterns in recGBM. Heat map and unsupervised hierarchical clustering of patient-specific drug responses (sDSSGBM) with Euclidian distance (cultures and drugs). The heat map is filtered by DSS ≥ 10 and sDSS ≥ or ≤ 7 (n = 76 drugs). PN proneural, M mesenchymal, UN unmethylated MGMT promoter, ME methylated MGMT promoter

Fig. 5

Individual therapeutic options in recGSC cultures. a Waterfall plot of the 15 most (red) and 15 least selective (blue) drug responses in T1534 by sDSSGBM. The plot displays the sensitivity to e.g. statins and estrogen receptor inhibitors, and the resistance to MDM2 inhibitors (SAR405838, AMG-232, Idasanutlin). b Dot plot of sDSS in T1534 using both the GBM (x-axis) and healthy bone marrow (y-axis) reference databases. Classes (color coded) and single drugs with patient-specific activity in T1534 are highlighted. c The corresponding dose–response curves of selected drug responses in T1534. d, e Similar dot plot and selected dose–response curves in T1516. T1516 displayed a remarkable sensitivity to EGFR- and HER-inhibitors, of which several with approval status available for fast translation. f, g Dot plot and selected dose-response curves in T1544. T1544 was among the least sensitive tumors, and displayed an increased sensitivity to MDM2-inhibitors, that currently are evaluated in clinical trials of GBM (NCT03158389)