Purpose
Retinoblastomas are thought to result from the inactivation of RB1. We previously found that retinoblastoma has properties of a cone precursor tumor and depends on cone-related signaling proteins. Here, we tested whether human cone precursors are uniquely sensitive to Rb inactivation, and provide direct evidence for cell origin of retinoblastoma.
Methods
Lentivirus expressing RB1 or control shRNA was injected into the subretinal space and vitreous of gestational week 20 human fetal eyes and retinal cell proliferation was evaluated by Ki67 staining after d14 of in vitro culture. We combined CD133 and CD44 immunostaining and cell size to separate cone precursors (medium+large-CD133highCD44- at 98% purity), rod precursors (small-CD133highCD44-), and retinal progenitors and glia (CD133lowCD44+). We knocked down RB1 either in purified or mixed human fetal retinal cells, either with or without co-knockdown of cone or Rb-related proteins, and examined proliferation of different cell types by labeling and co-staining for EdU, Ki67, and retinal cell type-specific markers. These cone precursors with RB1 knockdown (KD) were injected into subretinal space of nude mice to check the tumor formation.
Results
In situ RB1 KD in whole human fetal retina caused significant cone proliferation, especially in the fovea region (Fig. A). RB1 KD strongly induced proliferation and expression of Ki67 in purified cone arrestin+ and Crx+ cone precursors (Fig. B) and apoptosis of Muller glia and progenitors, but did not induce Ki67 expression in other retinal cell types. Co-KD of RB1 with TRbeta2, MDM2, MycN, SKP2, or p107 blocked the cone cell proliferation. Co-knockdown of RB1 with p130 promoted cone proliferation and cell line formation. All of the proliferating cones had failed to label with EdU labeled prior to RB1 KD, indicating that they are derived from non-proliferating cells. Xenograft of cone precursors with RB1-KD caused growth of an ocular mass in nude mice (Fig. C).
Conclusions
These results suggest that Rb is required to suppress the proliferation of cone precursors but not other retinal types. The Rb-deficient cone precursor proliferation depended upon cone related factors TRbeta2, N-Myc, and MDM2. Notably, p130 suppressed whereas p107 was required for cone precursor proliferation after Rb deletion. These data provide direct support for a cone precursor origin of retinoblastoma.
Keywords: 703 retinoblastoma •
744 tumors •
648 photoreceptors