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Milroy disease is an autosomal dominant congenital primary lymphedema, which manifests as chronic swelling of lower extremities. Heterozygous pathogenic variants in lymphangiogenic receptor tyrosine kinase VEGFR3 and its ligand VEGFC, cause Milroy (VEGFR3) or Milroy-like lymphedema (Gordon Syndrome) (VEGFC). Here, we report RPL10a encoding a component of 60S ribosomal subunit as a new causative gene for Milroy-like lymphedema.

in silico analysis of RPL10a variants structure, plasmid transfection, lentivirus transduction, gene knockdown, western blotting, immunofluorescent staining

We identified frameshift, nonsense, and start-loss heterozygous variants in RPL10a in seven families of patients with Milroy-like lymphedema. All variants led to C-terminally truncated RPL10a protein. Molecular modeling revealed that even the variant with the longest remaining C-terminus does not fold properly, preventing RPL10a interaction with 28S rRNA and incorporation into ribosomes. By overexpressing this mutant in lymphatic endothelial cells, we showed its exclusion from nucleoli and cytoplasm, the sites of ribosome assembly and activity, respectively, validating our in silico predictions.

While ribosomes are ubiquitously used in all cell types, the association of heterozygous loss-of-function pathogenic variants in RPL10a with congenital lymphatic vascular defects provides a first example of vascular-related ribosomopathy and suggests an unrecognized novel function of RPL10a in lymphangiogenesis.

Tumors can be classified as “hot” or “cold” based on their immune contexture, particularly CD8⁺ T cell infiltration. Hot tumors are T cell-inflamed with elevated adhesion molecule and chemokine expression in tumor-associated (TA) blood vessels, promoting immune cell entry. Cold tumors are immunologically inert and lack these features. Whether TA lymphatic vessels (LVs) show similar changes and influence leukocyte egress remains unclear. This study investigates lymphatic endothelial cells (LECs) in hot and cold tumor microenvironments (TMEs) using single-cell RNA sequencing (scRNA-seq) to identify differentially expressed genes that regulate leukocyte migration. Adhesion molecules ICAM-1 and VCAM-1 were identified as promising candidates for further functional analysis.

We used the YUMM1.7 (cold) and YUMMER1.7 (hot) melanoma cell lines to grow syngeneic tumors in photoconvertible Kikume Green-Red mice. 24 hours after photoconversion by UV illumination, LECs from normal skin or tumors, and photoconverted leukocytes that remained in the tumor or migrated to draining lymph nodes, were isolated by FACS and subjected to scRNA-seq. In parallel, candidate gene expression and function were validated using FACS and immunofluorescence. Additionally, an LEC-specific conditional ICAM-1 & VCAM-1 knockout mouse model was established and validated for gene deletion efficiency.

YUMM and YUMMER tumors grew successfully and were photoconverted with 75–90% efficiency. scRNA-seq confirmed LEC purity and revealed 8 LEC clusters reflecting the spatial and functional heterogeneity of LECs within the TME. ICAM-1 and VCAM-1 were enriched in Collector and Capillary 1 clusters and FACS analysis confirmed significantly higher precentage of ICAM-1⁺ and VCAM-1⁺ LECs in YUMMER tumors compared to YUMM tumors. To functionally dissect the roles of these adhesion molecules we have generated Prox1-CreERT2x VCAM-1fl/flxICAM-1fl/fl mice and validated the LEC-specific deletion of these genes at the mRNA and protein levels in lymph node LECs from double-knockout mice. This model will now be used to study leukocyte trafficking via TA-LVs and their impact on anti-tumor immunity.

This study reveals how tumor immunogenicity alters LEC gene expression and leukocyte dynamics. It establishes a foundation for identifying immunotherapeutic targets that regulate leukocyte egress through afferent lymphatics.