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, 26 (12), 3203-3211.e5

Modeling Patient-Derived Glioblastoma With Cerebral Organoids

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Modeling Patient-Derived Glioblastoma With Cerebral Organoids

Amanda Linkous et al. Cell Rep.

Abstract

The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

Keywords: brain tumors; cancer stem cells; cerebral organoids; glioblastoma; glioma; glioma stem cells; human embryonic stem cells; stem-cell-based disease models; tissue engineering; tumor microtubes.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Patient-Derived GSCs Form Infiltrative Tumors in Cerebral Organoids
(A) Tumor formation from GFP-expressing 827 GSCs (left) 1 week after co-culture; scale bar, 400 μm; n = 6 organoids per cell line. (B) Historical development of patient-derived GLICOs from six patient tumor specimens. (C) Diffuse GSC infiltration (blue arrow indicates tumor cells) of the organoid as evident by morphological (H&E) analysis of control organoids (top) and GFP-expressing 923 GLICO tumors (bottom) at day 14; scale bars, 1,000 μm (left) and 100 μm (right); six organoids were analyzed per group. Tumors exhibit necrosis (black arrow). (D) Representative image and bar graph showing quantification of Ki67+/GFP+ cells in GFP+ 923 GLICO tumors after 2 weeks of growth; green, GFP; red, Ki67; blue, DAPI; n = 3 organoids; scale bar, 50 μm.
Figure 2.
Figure 2.. GLICO Tumor Growth Is Supported by a Network of Tumor Microtubes
(A) Formation of 923 GSC-derived tumor micro-tubes in vitro after 12 days in 2D culture (arrows); scale bar, 200 μm (top) and 100 μm (bottom). (B) Two-photon microscopy of tumor microtubes (arrows) in GLICO tumor from GFP-positive 923 GSCs; day 10; 3D rendering of GLICO tumor, top; individual z stack images of microtubes, bottom; scale bar, 80 μm; n = 3 organoids. (C) Electron micrographs of peroxidase-based immunolabeling of connexin-43-positive gap junctions (top) and desmosomes (bottom) in GFP-positive 923 GLICO tumors; scale bars, 500 nm (top) and 100 nm (bottom); n = 5 organoids. (D) Electron micrographs showing tube-like connections between 923 GLICO tumor cells; arrows indicate peroxidase labeling for connexin-43; scale bars, 2 μm (left) and 500 nm (right). (E) Cytoplasmic fusion between two 0728 GLICO tumor cells (left) and a cytoplasmic fusion between a neuron and 0728 GLICO tumor cell (right); scale bars, 500 nm (left) and 2 mm (right); m, mitochondria; N, nucleus; N1, neuronal nucleus; N2, 0728 tumor cell nucleus; n = 5 organoids. (F) Time-lapse image of calcium waves (Rhod-2AM) traveling along 923 GSC microtubes; scale bar, 50 μm. (G) Two-photon microscopy of tumor microtube (arrows) in GLICO tumor from GFP- and RFP-positive 827 GSCs; day 10; n = 3 organoids; scale bar, 80 μm.
Figure 3.
Figure 3.. GLICO Tumors Exhibit Differential Response to Therapy and Preserve Key Genetic Changes of Parental Tumor
(A) Representative fluorescent microscopic images (left) and quantification of secreted luciferase activity (right) for 827 and 923 GLICOs treated with temozolomide (TMZ) or bis-chloroethylnitrosourea (BCNU); n = 4–6 organoids per group; **p < 0.01 and ***p < 0.001; scale bar, 500 μm. (B) Representative fluorescent microscopic images (left) and quantification of secreted luciferase activity (right) for 923 GLICOs treated with a single dose of 0, 5, or 10 Gy of ionizing radiation; n = 4–6 organoids per group; scale bar, 500 μm. (C) Quantification of EGFR copy number variation (left) in 2D or GLICO samples from patients with EGFR amplification; ****p < 0.0001. Gender-mismatched 0607 GLICOs or 0810 GLICOS were generated; 0607 (from a female patient) was co-cultured with H1 (from a male embryo)-derived organoids, and 0810 (from a male patient) was co-cultured with H9 (from a female embryo)-derived organoids. Representative images of DNA FISH for EGFR/Cen7/ChrY (Y chromosome) in GLICOs are shown (right); red, EGFR; orange, Cen7; green, ChrY; white arrow indicates tumor cell; n = 5 organoid sections per patient line.
Figure 4.
Figure 4.. GLICOs Exhibit Similar Growth Rates in iPSC-Derived Organoids and in Organoids of Different Ages
(A) Immunofluorescence staining for GFAP (green) and DAPI (blue) in 6- or 20-week-old H1-derived cerebral organoids; n = 4 organoids per time point; scale bars, 200 μm. (B) Representative images of GLICO tumor growth (top) and quantification of secreted luciferase activity (bottom) from 923 GLICO tumors grown in either 1- or 4-month-old H1-derived cerebral organoids; n = 6 organoids per group; scale bar, 500 μm. (C) Representative images of GLICO tumor growth (top) and quantification of secreted luciferase activity (bottom) from 923 GLICO tumors grown in either 1-month-old hESC-derived (H1) or iPSC-derived (H6) organoids; n = 6 organoids per group; scale bar, 500 μm.

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