Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin

Abstract

Retinoblastoma (Rb) is the most prevalent intraocular malignancy in children, with a worldwide survival rate <30%. We have developed a cancerous model of Rb in retinal organoids derived from genetically engineered human embryonic stem cells (hESCs) with a biallelic mutagenesis of the RB1 gene. These organoid Rbs exhibit properties highly consistent with Rb tumorigenesis, transcriptome, and genome-wide methylation. Single-cell sequencing analysis suggests that Rb originated from ARR3-positive maturing cone precursors during development, which was further validated by immunostaining. Notably, we found that the PI3K-Akt pathway was aberrantly deregulated and its activator spleen tyrosine kinase (SYK) was significantly up-regulated. In addition, SYK inhibitors led to remarkable cell apoptosis in cancerous organoids. In conclusion, we have established an organoid Rb model derived from genetically engineered hESCs in a dish that has enabled us to trace the cell of origin and to test novel candidate therapeutic agents for human Rb, shedding light on the development and therapeutics of other malignancies.

Dissecting the Transcriptional and Chromatin Accessibility Heterogeneity of Proliferating Cone Precursors in Human Retinoblastoma Tumors by Single Cell Sequencing—Opening Pathways to New Therapeutic Strategies?

“…In parallel, pluripotent stem cell derived organoid retinoblastoma models have recently been generated, corroborating the Arrestin 3 + maturing cone precursors as cell of origin for Rb. 14 Next-generation sequencing methods have enabled detection of mutations in peripheral blood samples at a low level of mosaicism that would have been missed with the traditional sequencing methods. Notwithstanding, it has not been possible to assess the full spectrum of clonal types within the tumor or the molecular changes occurring at the cells of origin, which are likely to be characterized by a different transcriptional and epigenetic profile.…”

In vitro Modeling of Embryonal Tumors

“…Single cell transcriptome profiling of tumors and developing tissues has proven to be a promising tool to reveal such processes, which could potentially serve as therapeutic targets (Filbin et al, 2018;Zhang et al, 2019). Similar methods can also be applied to in vitro models recapitulating embryonal tumorigenesis, as demonstrated for the retinoblastoma organoid model generated by Liu H. et al (2020), which has the advantage that it allows for a direct comparison of normal and tumor development. Although many in vitro embryonic cell-derived tumor models have been established over the years, the spectrum is biased toward ectoderm-derived tumors.…”

“…In addition, transplantation of RB1-null organoids into immune-deficient mice did not result in retinoblastoma formation (Zheng et al, 2020). In contrast, Liu H. et al (2020) utilized an alternative hESC-derived retinal organoid model, in which RB1 depletion did successfully generate tumors upon xenografting and better resembled patient retinoblastoma. These findings illustrate that the finetuning of retinal organoid establishment can affect the outcome of RB1 depletion, possibly due to differences in cellular composition and the presence or absence of the cell-of-origin.…”

“…Recently, the organoid technology was also successfully applied to several pediatric cancers, including embryonal tumors such as MRT and Wilms tumors (Schutgens et al, 2019;Calandrini et al, 2020). The efficient establishment and cryopreservation of tumor organoid models from primary patient tissue allows for the generation of large patient cohorts MYCN overexpression in mouse primary NCCs (Olsen et al, 2017) MYCN/ALK-F1174L overexpression in a mouse NC cell-line (Schulte et al, 2013) Mouse-human chimeras with MYCN overexpression in iPSC-derived hNCCs (Cohen et al, 2020) Engineering human 1p36 deletions in mouse NCCs (García-López et al, 2020) Retinoic acid treatment (Lone et al, 2016;Westerlund et al, 2017) HDAC inhibitors (Hahn et al, 2008;Frumm et al, 2013) EZH2 inhibitors (Chen et al, 2018) MRT Neural crest cells (NCCs) SMARCB1 knockout in iPSCs (Terada et al, 2019) SMARCB1 knockdown in ESCs (Langer et al, 2019) SMARCB1 knockout in cerebellar organoids (Parisian et al, 2020) HDAC inhibitors (Muscat et al, 2016) EZH2 inhibitors (Knutson et al, 2013) Medulloblastoma Neural progenitor cells c-MYC overexpression in cerebellar organoids (Ballabio et al, 2020) MYCN overexpression in neuroepithelial stem cells (Huang et al, 2019) Retinoic acid treatment (Patties et al, 2016) EZH2 inhibitors (Cheng et al, 2020) SHH inhibitors (Ocasio et al, 2019) BET-bromodomain inhibitors (Bandopadhayay et al, 2019) DIPG Oligodendrocyte precursor cells H3K27M mutations in hESC derived NPCs (Funato et al, 2014) ACVR1 mutations in neurospheres (Hoeman et al, 2019) HDAC inhibitors (Anastas et al, 2019) BET-bromodomain inhibitors (Mohammad et al, 2017) Retinoblastoma Cone precursor cells RB1 depletion in fetal retinal cell cultures (Xu et al, 2014) RB1 depletion in hESC derived retinal organoids (Liu H. et al, 2020;…”

Circular RNAs in the Central Nervous System

“…Investigators further assessed the possible mechanism of this circRNA through RPE cell lines and found that circNR3C1 protected RPE functions via the circNR3C1-miR-382-5p-PTEN network ( Figure 4B). Retinoblastoma (Rb) is an important cause of blindness in early childhood (Liu et al, 2020). CircRNAs have also been shown to be dysregulated in RB and some RB cell lines.…”