Bland-Altman method comparison of assay models demonstrated no overall bias for potency, whilst highlighting specific drugs to take forward for validation.

<p>All pEC50s were stacked by patient-derived line. Mean difference and lower and upper limits of agreements (95% CI) are indicated by dotted lines. Values outside these limits can be considered real biological differences.</p

Patient demographics, clinical features and cell line designations.

<p>Patient demographics, clinical features and cell line designations.</p

pEC50 values highlighted general resistance of GBM cell lines and cell line specific responses to chemotherapeutics and irradiation.

<p>Heat Map was populated using best-fit pEC50 values generated from dose-response curves, then color mapped categorically according to the legend. Each heat map color and corresponding number (1,2,3 etc.), corresponds to a log change in concentration of drug (100,000 μM, 10,000 μM, 1000 μM etc.). Red values of a pEC50 of 9 or above would indicate an EC50 of 1nm or below, indicating high efficacy, whereas any values categorized green-blue or below would likely be poor drug candidates as they correspond to EC50 values above 10μM. Any data Prism was unable to fit was colored black—this was typically due to an inability to generate an EC50 with that drug candidate because of inefficiency at high concentrations.</p

<i>In vivo</i> assessment of drug activity demonstrated strong correlation with neurosphere <i>in vitro</i> results.

<p>(A) NSG mice were dosed at sub-maximal tolerated doses with reference to accepted published data^18-24. Tumors were excised seven days post-treatment then measured ex-vivo for accuracy. Results are expressed as the percentage volume compared to the control group (*p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001). (B) Rank order of drugs based on pEC50 values for the U87 cell line are shown here to facilitate comparison. pEC50s were generated for 66 drugs in one, or both, of the U87 screening models, thus rankings are from 1 to 67, with 1 having the highest pEC50 and therefore the highest cytotoxicity. Mitoxantrone, bortezomib and actinomycin D all ranked in the top 10 in the neurosphere assay, which appears to correlate with the <i>in vivo</i> efficacy seen in these three drugs. Paclitaxel and doxorubicin demonstrated low efficacy <i>in vivo</i>, yet ranked highly in the monolayer assay.</p

Response space analysis identifies highly effective drugs and patient cell–line resistance.

<p>(A) Responses for TS1, TS18, TS9 and GS1 (0Gy) are depicted. Response is plotted onto a response space for each drug by the change in cell death (as measured by DRAQ7 intensity/spheroid cross sectional area), and fold change in neurosphere count. Highly responsive drugs are highlighted in dashed circles, with negative controls sitting at the intersection of the dotted lines (0, 1). (B) Sub -classification of drug response in neurosphere culture. Blue shaded areas represent responsive drugs, and yellow/orange/red areas represent resistance. (C) Summarized neurosphere response sub-classifications. The combined height of the yellow/orange/red columns provides an indication of the overall chemotherapeutic resistance of the cell line.</p

Cell line characterization demonstrated stem-cell characteristics and tumorigenicity <i>in vivo</i>.

<p>(A-B) U87 neurosphere culture demonstrating Nestin (green) and Sox2 (red) staining in serum-free culture (s.b. = 100μm). (C) U87 orthotopic implantation model showing large well-defined masses within the brain parenchyma, showing staining for anti-human nuclear antigen (green) (s.b. = 1000μm). (D) High power field (x40) of well-demarcated tumour edge showing anti-human nuclear antigen staining of tumour cells (s.b. = 50μm). (E-F) TS18 in serum free neurosphere culture showing uniform Nestin and Sox2 staining (s.b. = 100μm). (G) Orthotopic implantation of TS18-GFP cells with combined GFP (green) and anti-human nuclear antigen (red) staining to show diffuse infiltration of cells both within the parenchyma and across the corpus callosum into the opposite hemisphere (s.b. = 1000μm). (H) High power field (x20) showing area of diffuse infiltration and co-expression of GFP and anti-human nuclear antigen in implanted cells (s.b. = 100μm). (I-J) GS1 neurosphere culture showing uniform Nestin and Sox2 staining in serum free culture. (K) Orthotopic implantation of GS1 cells into the striatum with anti-vimentin staining. Vimentin staining is characteristic of gliosarcoma and is not found in the parenchyma, and was used to trace GS1 cells within the parenchyma (s.b. = 1000μm). (L) High power field (x20) showing diffusely infiltrating vimentin positive cells (green) within the corpus callosum (s.b. = 100μm).</p