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, 9 (1), 1407

A Human iPSC-derived 3D Platform Using Primary Brain Cancer Cells to Study Drug Development and Personalized Medicine

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A Human iPSC-derived 3D Platform Using Primary Brain Cancer Cells to Study Drug Development and Personalized Medicine

Simon Plummer et al. Sci Rep.

Abstract

A high throughput histology (microTMA) platform was applied for testing drugs against tumors in a novel 3D heterotypic glioblastoma brain sphere (gBS) model consisting of glioblastoma tumor cells, iPSC-derived neurons, glial cells and astrocytes grown in a spheroid. The differential responses of gBS tumors and normal neuronal cells to sustained treatments with anti-cancer drugs temozolomide (TMZ) and doxorubicin (DOX) were investigated. gBS were exposed to TMZ or DOX over a 7-day period. Untreated gBS tumors increased in size over a 4-week culture period, however, there was no increase in the number of normal neuronal cells. TMZ (100 uM) and DOX (0.3 uM) treatments caused ~30% (P~0.07) and ~80% (P < 0.001) decreases in the size of the tumors, respectively. Neither treatment altered the number of normal neuronal cells in the model. The anti-tumor effects of TMZ and DOX were mediated in part by selective induction of apoptosis. This platform provides a novel approach for screening new anti-glioblastoma agents and evaluating different treatment options for a given patient.

Conflict of interest statement

S.P. and S.W, are employees of MicroMatrices Associates ltd. D.P. and T.H. are named inventors of a Johns Hopkins patent for the mini-brain technology. T.H. is founder of Organome LLC aiming to commercialize this technology.

Figures

Figure 1
Figure 1
Growth of glioblastoma tumors in the gBS model. Tumour cell growth was monitored by segmenting tumour cells in the GFP channel so that we could measure tumour and normal neuronal cell numbers inside the spheroids independently. Panel (A) shows the growth of glioblastoma cells and non-tumor (normal) cells in the gBS spheroids over time (4–7 weeks). a–c H&Es (x10) show the glioblastoma cells (‘pink’ eosinophylic cells) and normal neuronal cells (lighter pink, less eosinophylic) in the gBS spheroids at different times after seeding the cultures: a - 4 weeks; b - 5 weeks; c - 7 weeks; d–f are higher powered (x25) images of the same spheroid sections shown in the top panels. Panel (B) box plots of image analysis data showing total number of glioblastoma cells and total number of normal neuronal cells in the gBS over the time course (3–7 weeks) expressed as the total numbers of cells in the spheroid section. Box plots show the median (black horizontal line) and the upper and lower quartiles (ends of the boxes). Open circles show outliers >1.5 times the interquartile range away from the upper and lower quartiles. **Significantly different from 4 wk P < 0.001, n = 20; ***significantly different from 4 wk P < 0.00001, n = 20. Panel (C) Shows representative H&E and GFP/Tuj1, GFAP/O1 and Vimentin/NF200 dual IF staining photomicrographs of untreated gBS (parallel sections): a, H&E; b GFP(red)/Tuj1(green) IF staining at low power (x20); c GFP/Tuj1 IF staining at high power (x63); d, H&E; e, GFAP (red)/O1 (green) IF staining at low power (x20); f, GFAP/O1 IF staining at high power (x63); g, H&E; h, NF200 (red)/vimentin (green) IF staining at low power (x20); i, NF200/vimentin IF staining at high power (x63). Blue staining is DAPI (nuclear counterstain).
Figure 2
Figure 2
Effect of temozolomide (TMZ) treatment (10 and 100 uM) on the size of glioblastomas in vitro (gBS) and in vivo (nude mouse xenografts). Image analysis in the spheroid sections was performed by segmentation of tumour cells in the GFP channel in order to quantify their number relative to that of normal neuronal cells which were GFP negative (see materials and methods section 4.8 for details). Panel (A) a - H&E control; b - H&E 10 uM TZ; c - H&E 100 uM TMZ (glioblastomas are eosinophylic ‘pink’ cells); d–f show dual immunofluorescence (IF) stain of GFP (red) and cleaved caspase (green) with hoechst (blue) counterstain for control, 10 uM TMZ and 100 uM TZ, respectively. Panel (B) shows the total number of normal neuronal cells (N) and the total number of glioblastoma cells (T) in the spheroid sections.Box plots show the median (black horizontal line) and the upper and lower quartiles (ends of the boxes); whiskers show maximum and minimum values; open circles show outliers >1.5 times the interquartile range away from the upper and lower quartiles, n = 12. Panel (C): a, b show H&E stained sections of an untreated 965 xenograft tumor; c,d show H&E stained sections of a 965 xenograft tumor taken from a mouse treated with TMZ (100 μg/Kg)- see materials and methods section for details of the TMZ treatment(s).
Figure 3
Figure 3
Effects of doxorubicin (DOX) treatment(s) on cell viability, tumor size and caspase or Ki67 expression in glioblastoma and non-tumor (normal) spheroid tissues. Cleaved caspase 3 and Ki67 expression in tumour cells were measured by first segmentation in the GFP channel followed by thresholding in the caspase/ki67 channel facilitating the measurement of these end-points (both expressed as percentages of caspase/Ki67 positive cells relative to total cells) differentially in tumour cells and normal neuronal cells (see materials and methods section 4.8). Panel (A) shows effects of DOX treatments (0.025–0.5 uM) on cell viability (relative to control) as measured by the resazurin assay. Results are means ± SD, n = 3. Panel (B) shows effects of DOX treatment (0.025–0.3 uM) on the total number of non-tumor (normal neuronal) cells and the total number of glioblastoma cells in the gBS spheroid sections. Values are expressed as percentages of the total numbers of cells (normal + tumour) in the spheroid sections. Panel (C) shows effects of DOX treatment (0.025–0.3 uM) on the number of cleaved caspase 3 positive glioblastoma cells and non-tumor (normal neuronal) cells in representative dual immunofluorescence (GFP/cleaved caspase 3) stained gBS sections from the microTMA: a - control; b - 0.025 uM DOX; c - 0.05 uM DOX; d - 0.1 uM DOX; e - 0.3 uM DOX. Panel (D) shows the number of cleaved caspase 3 positive tumour (glioblastoma) cells and non-tumour cells in the spheroid sections expressed as a percentage of the total cells in the spheroid section. Values are expressed as percentages of the total numbers of glioblastoma or normal neuronal cells in the spheroid sections. Panel (E) shows effects of doxorubicin (DOX) treatment (0.025–0.3 uM) on the number of Ki67 positive glioblastoma cells and neuronal cells in representative dual immunofluorescence (GFP/Ki67) stained gBS sections from the microTMA: a - control; b - 0.025 uM DOX; c - 0.05 uM DOX; d - 0.1 uM DOX; e - 0.3 uM DOX. Panel (F) shows the number of Ki67 positive tumor cells and normal cells in the spheroid sections. Values are expressed as percentages of the total numbers of cells (normal + tumour) in the spheroid sections. Box plots show the median (black horizontal line) and the upper and lower quartiles (ends of the boxes); n = 32. Open circles show outliers >1.5 times the interquartile range away from the upper and lower quartiles. ***Significantly different from control p < 0.0001 by t test, n = 32.
Figure 4
Figure 4
Measurement of the effect of doxorubicin (DOX) treatment (0.025–0.3 uM) on the number of glioblastoma cells made in three different microTMAsections of the same array. (ac) Manual image acquisition (Zeiss LSM confocal); (df) automated image acquisition and analysis (Perkin Elmer Polaris/Inform software). Values are expressed as percentages of the total numbers of glioblastoma cells in the spheroid sections relative to the total number of glioblastoma and normal neuronal cells. ***Significantly different from control p < 0.0001 by t test, n = 16.
Figure 5
Figure 5
Construction and image analysis of a spheroid tissue microarray (microTMA). Fixed spheroids were embedded in a microTMA mold prior to paraffin embedding and sectioning on a microtome. TMA sections were then immunofluorescence stained and imaged using confocal scanning followed by dearraying of the spheroid images and image analysis (see material and methods section 4.8). (A) Schematic showing construction and processing of the microTMA for high-throughput histology and image analysis of 3D spheroid cultures. (B) Image analysis process to quantify tumor size and the number of cleaved caspase 3 or Ki67 positive tumor cells or normal neuronal cells. a dual immunofluorescence stained section showing GFP positive cells (red) and Ki67 positive cells (green), blue cells (DAPI) are normal (non-tumor cells); b - spectral unmixing and removal of autofluorescence; c, d - segmentation in the red channel to define tumor and normal cells/areas; e–h: segmentation in the green channel to define Ki67 or caspase positive cells, which are then counted in the tumor- and normal-cell regions.

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References

    1. Waring MJ, et al. An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nature reviews. Drug discovery. 2015;14:475–486. doi: 10.1038/nrd4609. - DOI - PubMed
    1. Kaitin KI. Deconstructing the drug development process: the new face of innovation. Clinical pharmacology and therapeutics. 2010;87:356–361. doi: 10.1038/clpt.2009.293. - DOI - PMC - PubMed
    1. DiMasi JA, Grabowski HG, Hansen RW. Innovation in the pharmaceutical industry: New estimates of R&D costs. Journal of health economics. 2016;47:20–33. doi: 10.1016/j.jhealeco.2016.01.012. - DOI - PubMed
    1. DiMasi JA, Feldman L, Seckler A, Wilson A. Trends in risks associated with new drug development: success rates for investigational drugs. Clinical pharmacology and therapeutics. 2010;87:272–277. doi: 10.1038/clpt.2009.295. - DOI - PubMed
    1. Taylor K, Gordon N, Langley G, Higgins W. Estimates for worldwide laboratory animal use in 2005. Alternatives to laboratory animals: ATLA. 2008;36:327–342. - PubMed
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