Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages

Chiara Raggi (1) , Margherita Correnti (1) , Antonio Sica (1) , Jesper B. Andersen (2) , Vincenzo Cardinale (3) , Domenico Alvaro (3) , Giovanna Chiorino (4) , Elisa Forti (1) , Shannon S. Glaser (5) , Gianfranco Alpini (5) , Annarita Destro (1) , Francesca Sozio (1) , Luca Di Tommaso (1) , Massimo Roncalli (1) , Jesus Banales (6) , Cédric Coulouarn (7) , Luis Bujanda (6) , Guido Torzilli (1) , Pietro Invernizzi (8, 1)

1 IRCCS Milan - Istituto Clinico Humanitas [Milan]
2 UCPH - University of Copenhagen = Københavns Universitet
3 UNIROMA - Università degli Studi di Roma "La Sapienza" = Sapienza University [Rome]
4 Cancer Genomics Laboratory, Fondazione “Edo ed Elvo Tempia Valenta”
5 Texas AandM Health Science Center
6 Hospital Donostia. CIBERehd
7 Foie, métabolismes et cancer
8 UNIMIB - Università degli Studi di Milano-Bicocca = University of Milano-Bicocca

Background & Aims. A therapeutically challenging subset of cells, 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 conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium.Results. CCA spheres engrafted in 100% of transplanted mice and 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 spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n = 23) and correlated with a high level of serum cancer antigen 19.9 (n = 17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n = 12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n = 104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect.Conclusion.CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.

Mots clés: Cholangiocarcinoma; Cancer stem cells; Tumor-associated macrophages

Domaines: Cancer; Hépatologie et Gastroentérologie

Chiara Raggi, Margherita Correnti, Antonio Sica, Jesper B. Andersen, Vincenzo Cardinale, et al.. Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages. Journal of Hepatology, 2017, 66 (1), pp.102-115. ⟨10.1016/j.jhep.2016.08.012⟩. ⟨hal-01414693⟩