BACKGROUND AND AIMS: Therapeutically challenging subset, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche.

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. In vitro and in vivo effect of CCA-SPH on macrophage-precursors was tested after culturing healthy donor CD14+ with CCA-SPH conditioned medium .

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. Next, FACS-analysis showed that in presence of CCA-SPH-CM, CD14+ expressed key macrophage (MØ) markers (CD68, CD115, HLA-DR, CD206) indicating that CCA-SPH- conditioned medium was a strong MØ-activator. Gene expression profile of CCA-SPH activated MØ (SPH MØ) revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated MØs from CCA-resections recapitulated similar molecular phenotype of in vitro educated-MØs. 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 were detected and further confirmed in CCA patient sera (n=12). Surprisingly, significant association of IL13, IL34 and osteoactivin with SPH stem-like genes was provided by CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for MØ-differentiation and invasion as well as for in vivo tumor-promoting effect.

CONCLUSION: CCA-CSCs molded a specific subset of stem-like associated-MØs thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.

LAY SUMMARY: Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of key deregulated immune subtype responsible to cooperate in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.