Background and Aims: Cholangiocarcinoma (CCA) is a highly malignant and extremely heterogeneous adenocarcinoma arising from epithelial cells of bile ducts. CCA is currently associated with poor clinical outcome and, together with hepatocellular carcinoma (HCC), is the major primitive liver cancer in adults. Severity of CCA, lack of good diagnostic markers and frustrating benefit of current therapeutic strategies has rendered this disease a major challenge. Therapeutically challenging subset, termed cancer stem cells (CSCs) has been proposed as a driving force of tumor initiation, dissemination and drug-resistance, including in liver cancer. CSCs could be responsible for CCA wide multi-layered heterogeneity and clinical severity. Although it has already been shown that HCC progression is driven by CSCs, little is known about the presence of CSCs in human CCA. Similar to normal stem cells, CSCs are believed to reside in a specialized microenvironment (?CSC-niche?) within tumor-context that supports self-renewal and drug-resistance. Among various immune-subgroups within CSC-niche, tumor-associated macrophages (TAMs) represent a poor defined but very intriguing immune-subset, whose presence has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche, specifically CCA-associated macrophages. Methods: CCA cells were cultured in 3D-condition to generate spheres (SPH). CCA-SPH analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed as well as evaluation of drug responsiveness. In vitro and in vivo effect of CCA-SPH on macrophage-precursors was tested after culturing healthy donor CD14+ with CCA-SPH conditioned medium (CM). Evaluation of monocyte recruitment as well as macrophage markers? expression and presence of macrophage functional properties. CCA cells grown in adherence conditions as monolayer (MON) and matched CM used as control. Validation in human specimens. Results: CCA-SPHs engrafted 100% of transplanted mice, revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA-SPHs were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. CCA-SPH showed also a higher resistance to common chemotherapeutic drugs compared to MON. Analysis of CD14+ chemotaxis revealed that SPH-CM acted as a strong monocyte attractor. Next, fluorescence-activated cell sorting (FACS)-analysis showed that in presence of CCA-SPH-CM, CD14+ expressed key macrophage (M�) markers (CD68, CD115, HLA-DR, CD206) indicating that CCA-SPH-CM was a strong M�-activator. Gene expression profile of CCA-SPH activated M� (SPH M�) revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated M�s from CCA-resections recapitulated similar molecular phenotype of in vitro educated-M�s. Consistently with invasive features, largest CD163+ set was found in tumor-front of human CCA specimens (n=23) and correlated with high level of serum CA19.9 (n=17). Among mediators released by CCA-SPHs, only IL13, IL34 and Osteoactivin (OA) were detected and further confirmed in CCA patient sera (n=12). Surprisingly, significant association of IL13, IL34 and OA with SPH stem-like genes was provided by CCA database (n=104). In vitro combination of IL13, IL34, OA was responsible for M�-differentiation and invasion as well as for in vivo tumor-promoting effect. Conclusion: CCA-CSCs molded a specific subset of stem-like associated-M�s, thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.