Retinoblastoma (RB) is the most common primary intraocular malignancy of childhood. It is known that the tumor microenvironment (TME) regulates tumorigenesis and metastasis. However, how the malignant progression in RB is determined by the heterogeneity of tumor cells and TME remains uncharacterized. Here, we conducted integrative single-cell transcriptome and whole-exome sequencing analysis of RB patients with detailed pathological and clinical measurements. By single-cell transcriptomic sequencing, we profiled around 70,000 cells from tumor samples of seven RB patients. We identified that the major cell types in RB were cone precursor-like (CP-like) and MKI67+ cone precursor (MKI67+ CP) cells. By integrating copy number variation (CNV) analysis, we found that RB samples had large clonal heterogeneity, where the malignant MKI67+ CP cells had significantly larger copy number changes. Enrichment analysis revealed that the conversion of CP-like to MKI67+ CP resulted in the loss of photoreceptor function and increased cell proliferation ability. The TME in RB was composed of tumor-associated macrophages (TAMs), astrocyte-like, and cancer-associated fibroblasts (CAFs). Particularly, during the invasion process, TAMs created an immunosuppressive environment, in which the proportion of TAMs decreased, M1-type macrophage was lost, and the TAMs-related immune functions were depressed. Finally, we identified that TAMs regulated tumor cells through GRN and MIF signaling pathways, while TAMs self-regulated through inhibition of CCL and GALECTIN signaling pathways during the invasion process. Altogether, our study creates a detailed transcriptomic map of RB with single-cell characterization of malignant phenotypes and provides novel molecular insights into the occurrence and progression of RB.

In terrestrial animals, the lacrimal drainage apparatus evolved to serve as conduits for tear flow; however, little is known about the ontogenesis of this system. Here, we defined the anatomy of the fully formed tear duct (TD) in mice, characterized crucial morphogenetic events for the development of tear duct components, and identified the site for primordial tear duct (PTD) initiation. We report that the PTD originates from the orbital lacrimal lamina (LL), a junction formed by the epithelia of the maxillary (mxp) and lateral nasal processes (lnp). We demonstrate that Prickle 1, a key component of planar cell polarity (PCP) signaling, is expressed in progenitors of the PTD and throughout tear duct morphogenesis. Disruption of Prickle 1 stalls tear duct elongation; in particular, the loss of basement membrane (BM) deposition and aberrant cytoplasmic accumulation of laminin are salient. Altered cell adhesion, cytoskeletal and vesicular transport systems, and cell axis orientation in Prickle 1 mutants support the role of Prickle 1 in PCP. Taken together, we highlight a crucial role of Prickle 1-mediated polarized BM secretion and deposition in PTD elongation.

The study demonstrates a spatiotemporal requirement for Prickle 1-mediated PCP signaling during eyelid morphogenesis and homeostasis. The study links Prickle 1-mediated PCP signaling to existing networks, and provides a useful animal model for studying congenital ocular surface diseases.

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