Abstract
Intratumor heterogeneity has been recognized in numerous cancers as a major source of metastatic dissemination. In uveal melanomas, the existence and identity of specific subpopulations, their biological function and their contribution to metastasis remain unknown. Here, in multiscale analyses using single-cell RNA sequencing of six different primary uveal melanomas, we uncover an intratumoral heterogeneity at the genomic and transcriptomic level. We identify distinct transcriptional cell states and diverse tumor-associated populations in a subset of the samples. We also decipher a gene regulatory network underlying an invasive and poor prognosis state driven in part by the transcription factor HES6. HES6 heterogenous expression has been validated by RNAscope assays within primary human uveal melanomas, which further unveils the existence of these cells conveying a dismal prognosis in tumors diagnosed with a favorable outcome using bulk analyses. Depletion of HES6 impairs proliferation, migration and metastatic dissemination in vitro and in vivo using the chick chorioallantoic membrane assay, demonstrating the essential role of HES6 in uveal melanomas. Thus, single-cell analysis offers an unprecedented view of primary uveal melanoma heterogeneity, identifies bona fide biomarkers for metastatic cells in the primary tumor, and reveals targetable modules driving growth and metastasis formation. Significantly, our findings demonstrate that HES6 is a valid target to stop uveal melanoma progression.
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Data availability
The experimental data from single-cell RNA sequencing, whole exome sequencing and array-CGH have been deposited in the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) under the SuperSeries GSE138665.
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Acknowledgements
We thank the Single Cell Core Facility at IPMC and the imaging facility of C3M. The authors thank all patients with primary uveal melanoma for participating as well as Frédéric Reinier for the bioinformatics analysis at the beginning of the project. We also thank Dr. M. Jager (Leiden, The Netherlands) for providing the primary uveal melanoma cell lines.
Funding
This work was supported by the French government, INSERM, La Ligue Nationale contre le cancer, INCA PLBio to CB and PB (INCA-12824), Conseil Départemental des Alpes Maritimes (2016-294DGADSH-CV), the National Research Agency under the Investments for the Futur program UCAJEDI «ANR-15-IDEX-01», National Infrastructure France Génomique «ANR-10-INBS-09-03» and «ANR-10-INBS-09-02», Canceropole PACA, Club Francophone des spécialistes de la rétine (CFSR), La Ville de Nice, Mickaël Fulci, La Fondation ARC to CB (20171206312) and La Fondation pour la Recherche Médicale for CP and GG. ID is an ‘équipe labélisée’ of the French League against Cancer.
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CB and RB designed and supervised the study, prepared the figures and wrote the manuscript. VM carried out scRNA-seq experiments. KLB and NN performed the computational analyses and assisted in data analysis in discussions with PB who also critically reviewed the manuscript. CP, TS, YC and GB performed patient sample processing and functional experiments. KB, CH, MD and MI provided technical assistance. GG and ID carried out the RNAscope assays. SL and PH coordinated patient sample collection, maintained IRB approval and performed histological analysis. FP provided cytogenetic analyses. AM, SNE, CM, JPC and SB gathered patients’ consent, provided the samples and clinical data.
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The study was approved by the hospital ethics committee (Nice Hospital Center and University of Nice Côte d’Azur). The study was performed in accordance with the Declaration of Helsinki.
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Pandiani, C., Strub, T., Nottet, N. et al. Single-cell RNA sequencing reveals intratumoral heterogeneity in primary uveal melanomas and identifies HES6 as a driver of the metastatic disease. Cell Death Differ 28, 1990–2000 (2021). https://doi.org/10.1038/s41418-020-00730-7
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DOI: https://doi.org/10.1038/s41418-020-00730-7
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