CXCR4-Mediated Chemokine Signaling Orchestrates the Progression of Conjunctival Melanocytic Lesions

Purpose: The biological and microenvironmental differences between benign and malignant conjunctival melanocytic lesions remain poorly understood. This study aims to characterize their microenvironment and reveal the role of chemokine signaling in lesion progression.

Methods: Bulk RNA Sequencing and single-cell RNA Sequencing were used to delineate the microenvironment of conjunctival melanocytic lesions and identify divergent molecular pathways. CXCR4 expression was validated in PIG1 melanocytes and the conjunctival melanoma (CoM) cell lines (CRMM1, CRMM2, and CM2005.1) using quantitative Real-Time PCR and Western blotting. Tissue specimens were further analyzed for CXCR4 and CXCL12 expression via immunohistochemistry and immunofluorescence. To assess functional relevance, CoM cell lines were treated with the CXCR4 Antagonist AMD3100, followed by analysis of cell viability and downstream PI3K/Akt and MEK/ERK phosphorylation using Western blotting. Additionally, the antitumor efficacy of AMD3100 was validated in vivo.

Results: Transcriptomic profiling revealed marked microenvironmental heterogeneity between benign and malignant lesions. A significant upregulation of chemokine signaling pathway, along with notably elevated CXCR4 expression, was observed in malignant lesions compared to the benign. Exogenous CXCL12 stimulation significantly enhanced the proliferation of CoM cells, whereas AMD3100 treatment induced cytotoxic effects and reduced phosphorylation of Akt and ERK. Additionally, in vivo experiments confirmed the antitumor efficacy of AMD3100.

Conclusions: CXCR4-mediated chemokine signaling pathway is significantly upregulated in CoM. Pharmacological blockade of CXCR4 with AMD3100 exerts cytotoxic effects on CoM cells through inhibition of PI3K/Akt and MEK/ERK pathways, indicating CXCR4 as a promising translational target that warrants further investigation and clinical validation.