Chiara Raggi1, Margherita Correnti2, Antonio Sica3, Jesper B Andersen4, Vincenzo Cardinale5, Domenico Alvaro5, Giovanna Chiorino6, Elisa Forti2, Shannon Glaser7, Gianfranco Alpini7, Annarita Destro8, Francesca Sozio9, Luca Di Tommaso10, Massimo Roncalli10, Jesus M Banales11, Cédric Coulouarn12, Luis Bujanda11, Guido Torzilli13, Pietro Invernizzi14. 1. Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy. Electronic address: chiara.raggi@humanitasresearch.it. 2. Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy. 3. Laboratory of Molecular Immunology, Humanitas Clinical and Research Center, Rozzano, Italy; Department of Pharmaceutical Sciences, University of Piemonte Orientale "Amedeo Avogadro" Novara, Italy. 4. Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark. 5. Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy. 6. Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia, Biella, Italy. 7. 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. 8. Pathology Unit, Humanitas Research Hospital, Rozzano, Italy. 9. Leukocyte Migration Laboratory, Humanitas Clinical and Research Center, Rozzano, Italy; Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy. 10. Pathology Unit, Humanitas Research Hospital, Rozzano, Italy; University of Milan Medical School, Milan, Italy. 11. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), CIBERehd, Ikerbasque, San Sebastián, Spain. 12. Inserm U991, Hôpital Pontchaillou, 35033 Rennes cedex, France. 13. Department of Hepatobiliary and General Surgery, Humanitas Research Hospital, Rozzano, Italy. 14. 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.
Abstract
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. 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 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. Copyright Â
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 humanCCA 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 CCApatient 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. 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 the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that humancholangiocarcinoma 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. Copyright Â
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