Single-cell characterization of malignant phenotypes and microenvironment alteration in retinoblastoma

Abstract

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.

Spatial transcriptomic profiling of human retinoblastoma

“…To study the genotype of the RB sample, we inferred copy number variations (CNVs) for each cell by averaging relative expression levels across intervals of the genome and calculated the CNV mean square in the control retina and RB samples 15,16 . We found various CNV types in the RB sample, including gain of chromosome (Chr) 1q, 6p and loss of Chr 16 (Figure 1D), consistent with previous studies in human RB 5 . Also, CNV patterns were also conserved across all ten RB clusters.…”

“…While a majority of children with RB cases stem from biallelic RB1 inactivation, for a minor portion, MYCN amplification is exhibited without RB1 alterations 2,3 . Next-generation sequencing studies have also revealed additional genetic abnormalities beyond RB1 inactivation that are associated with aggressive histopathological features 4,5 , highlighting the complexity of the genetic lesions causing the disease.…”

“…Recent studies using human pluripotent stem cells and retinal organoids provided a valuable in vitro model to study the cellular abnormalities caused by biallelic mutations of the RB1 9,10 . In addition, to study the cellular heterogeneity of RB, a number of recent studies have profiled primary RB tumors using single cell RNAseq (scRNA-seq) 5,11,12 , advancing our understanding of the cellular origin of RB and the pathological mechanisms driving RB. However, these single cell transcriptomic datasets do not retain structural information which would be important to study the collective tissue architecture of RB.…”

Identification and immunological characterization of cuproptosis-related molecular clusters in retinoblastoma

“…Retinoblastoma is an aggressive intraocular tumor. The regulatory mechanisms of m6A [23], tumor microenvironment [24] and methylation [25] in retinoblastoma have been extensively reported in literature, and the prognosis of retinoblastoma has been predicted. Despite the bioinformatics analysis of retinoblastoma, the existing cuproptosis-related retinoblastoma research is not abundant.…”

Retinoblastoma

“…It is estimated to account for about 4% of all forms of cancer in children under age of 15. It predominantly impacts infants and young children, and clinical signs are uncommon [4]. A white colour in the pupil can be seen in dim light or when taking a picture with flash named cat's eye reflex or Leukocoria; crossed eyes, the eyes that seem to be looking in different directions termed strabismus, that can end up causing squinting; alter in iris colour; Poor sight, eye strain, reddening, and inflammation, Infection of the eyelids, larger eyeball than normal, the coloured portion of the eye, and pupil apparently cloudy.…”