Abstract
Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.
Highlights
Introduction of cancerassociated genetic alterationsClonal selection DifferentiationIn vivo transformation /progressionEdited human pluripotent stem cells (hiPSCs) cloneDifferentiated progeny with genomic editsOrthotopic engraftment Re TumorWildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9974 bp 140 bp 140 bp 974 bp 974 bp 2733 bp 2733 bp 160 bp 2733 bp 2733 bp 2507 bp 2507 bp 2507 bp 608 bp 608 bp 3468 bp 3468 bp 3468 bp 3468 bp 181 bp b PTENExons sgRNA target sites
A second combination of deletions targeted TP53 and exons 8 and 9 of PDGFRA (PDGFRAΔ8–9). This creates a constitutively active truncating PDGFRA mutation observed in 40% of PDGFRA amplified GBM20, resulting in a genotype commonly found in the proneural subtype of isocitrate dehydrogenase-wildtype GBM18,19
Edited induced pluripotent stem cells (iPSCs) clones with desired mutations were differentiated into neural progenitor cells (NPCs), using a small molecule protocol[21] and differentiation status was confirmed by downregulation of pluripotency markers, Nanog and Oct[4], and corresponding upregulation of NPC markers, Pax[6], Nestin, and Sox[1] (Fig. 1e)
Summary
Introduction of cancerassociated genetic alterationsClonal selection DifferentiationIn vivo transformation /progressionEdited hiPSC cloneDifferentiated progeny with genomic editsOrthotopic engraftment Re TumorWildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9974 bp 140 bp 140 bp 974 bp 974 bp 2733 bp 2733 bp 160 bp 2733 bp 2733 bp 2507 bp 2507 bp 2507 bp 608 bp 608 bp 3468 bp 3468 bp 3468 bp 3468 bp 181 bp b PTENExons sgRNA target sites. Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9 Wildtype PTEN–/– PTEN–/–;NF1–/– TP53–/– TP53–/–;PDGFRAΔ8–9. 974 bp 140 bp 140 bp 974 bp 974 bp 2733 bp 2733 bp 160 bp 2733 bp 2733 bp 2507 bp 2507 bp 2507 bp 608 bp 608 bp 3468 bp 3468 bp 3468 bp 3468 bp 181 bp b PTEN
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