2018 Apr 24
Glioblastoma Model Using Human Cerebral Organoids
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Glioblastoma Model Using Human Cerebral Organoids
We have developed a cancer model of gliomas in human cerebral organoids that allows direct observation of tumor initiation as well as continuous microscopic observations. We used CRISPR/Cas9 technology to target an HRas
G12V-IRES-tdTomato construct by homologous recombination into the TP53 locus. Results show that transformed cells rapidly become invasive and destroy surrounding organoid structures, overwhelming the entire organoid. Tumor cells in the organoids can be orthotopically xenografted into immunodeficient NOD/SCID IL2RG -/- animals, exhibiting an invasive phenotype. Organoid-generated putative tumor cells show gene expression profiles consistent with mesenchymal subtype human glioblastoma. We further demonstrate that human-organoid-derived tumor cell lines or primary human-patient-derived glioblastoma cell lines can be transplanted into human cerebral organoids to establish invasive tumor-like structures. Our results show potential for the use of organoids as a platform to test human cancer phenotypes that recapitulate key aspects of malignancy.
CRISPR/Cas9; cerebral organoid; glioblastoma; glioma.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Figure 1. Generation and Transformation of Human Cerebral Organoids Using the CRISPR/Cas9 System
(A) Schematic representation of the CRISPR/Cas9-mediated homologous recombination strategy. CRISPR/Cas9 TP53 sgRNA plasmid was co-electroporated with HRas
G12V-expressing homologous recombination donor vector (HRas G12V-tdTomato p53 exon HA), which is also targeted and cleaved by the TP53 sgRNA (orange). Two homologous regions (HA1 and HA2, purple) mediate integration within the TP53 gene locus. (B) Co-localization of CRISPR/Cas9 (GFP) and HRas G12V (tdTomato) observed 2 weeks after transfection. (C) Ki-67 immunostaining shows HRas G12V-transduced cells are highly proliferative compared to controls. (D) Time-lapse imaging of HRas G12V-transduced cells showing invasion from 4 weeks on after electroporation. (a) High-magnification view of the invasive edges of a 13-week transduced organoid. Scale bars: 100 mm in (B), (C), and (D-a) and 1 mm in (D).
Figure 2. Molecular Architecture of Invasive Tumor in Organoids
(A) Organoid images and FACS profile of tdTomato+ transduced cells in organoids at 8 and 16 weeks after electroporation. FACS analysis shows that tdTomato+ cells are 5.7% of the organoid at 8 weeks, whereas at 16 weeks, they are (86.8%) of the organoid. Arrowheads show abnormal budding regions within the organoid (arrowhead). (B–E) Immunostaining at 16 weeks for post-electroporation organoids. Transduced cells exhibit large numbers of OLIG2+ (B), GFAP+ (C), and SOX2+ (D), and cells are highly proliferative as seen in Ki-67 expression (E). (F) RNA-seq analysis of FACS-sorted tdTomato+ (POS) and tdTomato− (NON) fractions. Samples were classified using the Verhaak four GBM subtypes: mesenchymal (orange), proneural (purple), classical (green), and neural (gray) subtypes. Upregulated (red) or dowregulated (blue) genes of each subtype are highlighted along the left side. Relative expression is plotted for each organoid sample (red indicates up, and blue indicates down). A two-tailed Welch’s t test was used to determine significance of differential enrichment/depletion between POS and NON organoids of average log-normalized fragments per kilobase of transcript per million mapped reads (FPKM) values (Figure S4). (G) Fraction of POS and NON genes in each of the eight Verhaak gene sets (binomial test; NS, not significant). Scale bars: 1 mm in (A) and (B) and 200 mm in (C)–(E). See also Figure S4.
Figure 3. Tumors from Organoids Show Tumorigenic Behavior and Invasiveness
(A) Organoid tumor explants were propagated either on Matrigel for 2D culture or as neurospheres. (B) Tumor explants on 2D culture shows co-expression of GFAP and SATB2 (left), NeuN and SOX2 (middle), and OLIG2 with TuJ1 (right). (C) CRISPR-mediated integration analysis. Southern probe design for tdTomato and the endogenous TP53 site. (D) Sanger sequencing of the integration loci. (E) Gross appearance and fluorescence signal of xenografted mouse brain. The dotted area shows a slightly pinkish appearance from tdTomato expression. Right panel indicates the fluorescence signal with a red fluorescent protein (RFP) filter. (F) Kaplan-Meier survival curve of xenografted mice with mean survivals of 90 and 110 days. (G) H&E staining of xenografted mouse brain coronal sections. Highly invasive phenotypes of the tumors. (H) H&E staining shows a high degree of microscopic invasiveness. The tumor is diffuse (arrow) and perivascular (arrowhead). (I–K) Higher magnification shows perivascular invasion (I; arrowhead indicates blood vessel), nuclear pleomorphism (J; arrowhead indicates giant cell, and arrows indicate hyperchromatic nuclei), and necrotic areas (K; arrow). (L) Immunostaining with anti-CD31 antibody visualizes the endothelium and microvascular angiogenesis (upper panel). Organoid-derived tumors show high Ki-67 and are positive for SOX2 and GFAP. Scale bars: 100 mm in (A), 20 mm in (B), and 100 mm in (H)–(L).
Figure 4. Organoids as Platforms for Tumor Transplantation
(A) Tumorsphere of the transformed organoid-derived cells attaches and spreads through an untransduced mature organoid showing an invasive phenotype (arrowheads). Tumor cells are highly proliferative (Ki-67) and show high expression of SOX2 and are GFAP positive as they invade the organoid. (B) Kaplan-Meier survival curve of mice injected with SK2176 and SK429 (n = 3; mean survival for SK2176 xenografts: 34 days). SK429 xenografts show a lack of lethality in mice. (C and D) SK2176 cell-line-generated tumorspheres attach and invade mature cerebral organoids over the course of 21 days (C, arrowheads). SK429 and SK2176 both express proliferation marker Ki-67, but only SK2176 displayed an invasive phenotype (D). Scale bars: 1 mm in (A, top panel) and (C) and 100 mm in (A, lower panel) and (D).
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