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



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.



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.



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



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 the key dysregulated immune subtypes responsible for cooperating 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 in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.
Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy. Electronic address: chiara.raggi@humanitasresearch.it.
Chiara
C
Raggi
Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy.
Margherita
M
Correnti
Laboratory of Molecular Immunology, Humanitas Clinical and Research Center, Rozzano, Italy; Department of Pharmaceutical Sciences, University of Piemonte Orientale "Amedeo Avogadro" Novara, Italy.
Antonio
A
Sica
Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.
Jesper B
JB
Andersen
Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy.
Vincenzo
V
Cardinale
Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy.
Domenico
D
Alvaro
Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia, Biella, Italy.
Giovanna
G
Chiorino
Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy.
Elisa
E
Forti
Research, Central Texas Veterans Health Care System, Scott & White Digestive Disease Research Center, Scott & White, Department of Medicine, Texas A&M Health Science Center, College of Medicine, Temple, TX, United States.
Shannon
S
Glaser
Research, Central Texas Veterans Health Care System, Scott & White Digestive Disease Research Center, Scott & White, Department of Medicine, Texas A&M Health Science Center, College of Medicine, Temple, TX, United States.
Gianfranco
G
Alpini
Pathology Unit, Humanitas Research Hospital, Rozzano, Italy.
Annarita
A
Destro
Leukocyte Migration Laboratory, Humanitas Clinical and Research Center, Rozzano, Italy; Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
Francesca
F
Sozio
Pathology Unit, Humanitas Research Hospital, Rozzano, Italy; University of Milan Medical School, Milan, Italy.
Luca
L
Di Tommaso
Pathology Unit, Humanitas Research Hospital, Rozzano, Italy; University of Milan Medical School, Milan, Italy.
Massimo
M
Roncalli
Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), CIBERehd, Ikerbasque, San Sebastián, Spain.
Jesus M
JM
Banales
Inserm U991, Hôpital Pontchaillou, 35033 Rennes cedex, France.
Cédric
C
Coulouarn
Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), CIBERehd, Ikerbasque, San Sebastián, Spain.
Luis
L
Bujanda
Department of Hepatobiliary and General Surgery, Humanitas Research Hospital, Rozzano, Italy.
Guido
G
Torzilli
Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy; Program for Autoimmune Liver Diseases, International Center for Digestive Health, Department of Medicine and Surgery, University of Milan-Bicocca, Italy. Electronic address: pietro.invernizzi@unimib.it.
Pietro
P
Invernizzi
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001650:Bile Duct Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D063646:Carcinogenesis; D002273:Carcinogens; D018281:Cholangiocarcinoma / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007378:Interleukins / Q000032:analysis; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D014411:Neoplastic Stem Cells / Q000502:physiology*
D015703:Antigens, CD; D002273:Carcinogens; C503042:GPNMB protein, human; D007378:Interleukins; D008562:Membrane Glycoproteins
Cancer stem cells; Cholangiocarcinoma; Tumor-associated macrophages