NEFL⁺NEFM⁺ myeloid-reprogrammed cells promote ccRCC progression through CX3CL1-CX3CR1-mediated Tregs chemotaxis.

Tumor-associated myeloid cells (TAMCs) fuel immune evasion and progression, yet their roles in clear cell renal cell carcinoma (ccRCC) remain unclear. Single-cell transcriptomic analyses have suggested the emergence of myeloid cells expressing neuronal structural genes such as NEFL and NEFM, implying a neuron-like reprogrammed state. In addition, CX3CL1, a chemokine involved in immune-cell recruitment, has been implicated in tumor immune regulation. However, the existence and function of NEFL/NEFM-associated neuron-like myeloid cells and their potential link to CX3CL1 signaling in ccRCC remain unclear. We analyzed a publicly available single-cell RNA sequencing dataset of human ccRCC tumors and identified a distinct subpopulation of myeloid cells co-expressing canonical myeloid markers and neuronal structural genes, NEFL and NEFM. Functional assays were conducted using ccRCC-conditioned THP-1 cells with or without NEFL/NEFM knockdown or CX3CL1 neutralization. T cell co-culture, ELISA, Transwell assays, and tumor cell phenotyping were applied to investigate immunomodulatory and pro-tumorigenic functions. Single-cell results showed that NEFL⁺NEFM⁺ neuron-like myeloid cells emerged as a reprogrammed TAMC state in ccRCC and were characterized by elevated CX3CL1 expression without CX3CR1 co-expression. Conditioned medium from 786-O ccRCC cells induced THP-1 cells to acquire NEFL⁺NEFM⁺CD68⁺ phenotype and secrete CX3CL1. Genetic silencing of NEFL/NEFM suppressed CX3CL1 secretion. Functionally, NEFL/NEFM-induced reprogrammed cells suppressed T cell proliferation, activation, and cytokine secretion, while enhancing Tregs recruitment via the CX3CL1-CX3CR1 axis. Furthermore, these reprogrammed cells promoted ccRCC cell proliferation, migration, invasion, and EMT, which were mitigated by NEFL/NEFM knockdown or CX3CL1 blockade. Our study identifies NEFL/NEFM-driven myeloid reprogramming in ccRCC, producing neuron-like cells that secrete CX3CL1 to recruit Tregs and foster tumor progression.