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3D-tumor sphere (SPH) cultures enriched in CSCs were generated from intrahepatic CCA (iCCA) cell lines and CCA patient-derived organoids (PDOs) were employed. Circulating monocytes were differentiated into M2c MØs and recombinant Gas-6, a MerTK ligand, was used to activate MerTK. MERTK expression in human CCA tissues was analyzed at mRNA level in public database (n=78) and confirmed by immunohistochemistry (IMH) (n=74). Single-cell RNA sequencing of CD45+ sorted cells was performed in paired non-tumoral and tumoral specimens from intrahepatic CCA patients (n=6). Conditioned media (CM) of iCCA SPH induced higher MerTK expression in macrophages. Conversely, soluble mediators released by Gas-6-stimulated M2c MØs, which express MerTK at high levels, increased sphere number and volume, expression of stem-like genes and drug-resistance in iCCA cells. Moreover, the exposure to CM of M2c MØs induced an increase in the cell viability of organoids CCA PDOs, which was further increased when macrophages were stimulated with Gas-6. These effects were reduced following treatment of macrophages with UNC2025, a small molecule inhibitor of MerTK. Transcriptomic analysis of laser-captured, micro-dissected epithelium and stroma from 23 iCCA patients showed that MerTK mRNA expression is significantly higher in intratumoral stroma. These data were further confirmed in a public iCCA dataset showing that high MerTK levels are associated with immunologically hot iCCA. Single-cell RNA sequencing of CD45+ cells from non-tumoral and tumoral areas in iCCA patients showed that MerTK is predominantly expressed at the level of myeloid cells. Further reclustering showed MerTK expression in ID3 MØs, corresponding to Kupffer cells in tumor tissue. Notably, expression of MerTK correlated with greater tumor size, tumor grade, microvascular invasion, and risk of recurrence in iCCA patients. These data indicate that a cross-talk between MerTK-expressing cells in the stroma and iCCA cells results in increased malignant features. [ABSTRACT FROM AUTHOR]