Cholangiocarcinoma progression depends on the uptake and metabolization of extracellular lipids.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with a dismal prognosis. We investigated if lipid metabolism is disrupted in CCA and its role in tumor proliferation. APPROACH AND RESULTS: The in vitro and in vivo tumorigenic capacity of five human CCA cell lines was analyzed. Proteome, lipid content, and metabolic fluxes were evaluated in CCA cells and compared with normal human cholangiocytes (NHC). The Akt1/NOTCH1 intracellular cytoplasmic domain (Nicd1)-driven CCA mouse model was also evaluated. The proteome of CCA cells was enriched in pathways involved in lipid and lipoprotein metabolism. The EGI1 CCA cell line presented the highest tumorigenic capacity. Metabolic studies in high (EGI1) versus low (HUCCT1) proliferative CCA cells in vitro showed that both EGI1 and HUCCT1 incorporated more fatty acids (FA) than NHC, leading to increased triglyceride storage, also observed in Akt1/Nicd1-driven CCA mouse model. The highly proliferative EGI1 CCA cells showed greater uptake of very-low-density and HDLs than NHC and HUCCT1 CCA cells and increased cholesteryl ester content. The FA oxidation (FAO) and related proteome enrichment were specifically up-regulated in EGI1, and consequently, pharmacological blockade of FAO induced more pronounced inhibition of their tumorigenic capacity compared with HUCCT1. The expression of acyl-CoA dehydrogenase ACADM, the first enzyme involved in FAO, was increased in human CCA tissues and correlated with the proliferation marker PCNA. CONCLUSIONS: Highly proliferative human CCA cells rely on lipid and lipoprotein uptake to fuel FA catabolism, suggesting that inhibition of FAO and/or lipid uptake could represent a therapeutic strategy for this CCA subclass.

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.

DCLK1, a Putative Stem Cell Marker in Human Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). APPROACH AND RESULTS: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine-rich repeat-containing G protein-coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real-time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real-time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme-linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared with unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in a dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls. CONCLUSIONS: DCLK1 characterizes a specific CSC subpopulation of iCCACD133+ and pCCALGR5+ , and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.

Peribiliary Gland Niche Participates in Biliary Tree Regeneration in Mouse and in Human Primary Sclerosing Cholangitis.

BACKGROUND AND AIMS: Mechanisms underlying the repair of extrahepatic biliary tree (EHBT) after injury have been scarcely explored. The aims of this study were to evaluate, by using a lineage tracing approach, the contribution of peribiliary gland (PBG) niche in the regeneration of EHBT after damage and to evaluate, in vivo and in vitro, the signaling pathways involved. APPROACH AND RESULTS: Bile duct injury was induced by the administration of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 14 days to Krt19Cre TdTomatoLSL mice. Human biliary tree stem/progenitor cells (BTSC) within PBGs were isolated from EHBT obtained from liver donors. Hepatic duct samples (n = 10) were obtained from patients affected by primary sclerosing cholangitis (PSC). Samples were analyzed by histology, immunohistochemistry, western blotting, and polymerase chain reaction. DDC administration causes hyperplasia of PBGs and periductal fibrosis in EHBT. A PBG cell population (Cytokeratin19- /SOX9+ ) is involved in the renewal of surface epithelium in injured EHBT. The Wnt signaling pathway triggers human BTSC proliferation in vitro and influences PBG hyperplasia in vivo in the DDC-mediated mouse biliary injury model. The Notch signaling pathway activation induces BTSC differentiation in vitro toward mature cholangiocytes and is associated with PBG activation in the DDC model. In human PSC, inflammatory and stromal cells trigger PBG activation through the up-regulation of the Wnt and Notch signaling pathways. CONCLUSIONS: We demonstrated the involvement of PBG cells in regenerating the injured biliary epithelium and identified the signaling pathways driving BTSC activation. These results could have relevant implications on the pathophysiology and treatment of cholangiopathies.

Neoplastic Transformation of the Peribiliary Stem Cell Niche in Cholangiocarcinoma Arisen in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory cholangiopathy frequently complicated by cholangiocarcinoma (CCA). Massive proliferation of biliary tree stem/progenitor cells (BTSCs), expansion of peribiliary glands (PBGs), and dysplasia were observed in PSC. The aims of the present study were to evaluate the involvement of PBGs and BTSCs in CCA which emerged in PSC patients. Specimens from normal liver (n = 5), PSC (n = 20), and PSC-associated CCA (n = 20) were included. Samples were processed for histology, immunohistochemistry, and immunofluorescence. In vitro experiments were performed on human BTSCs, human mucinous primary CCA cell cultures, and human cholangiocyte cell lines (H69). Our results indicated that all CCAs emerging in PSC patients were mucin-producing tumors characterized by PBG involvement and a high expression of stem/progenitor cell markers. Ducts with neoplastic lesions showed higher inflammation, wall thickness, and PBG activation compared to nonneoplastic PSC-affected ducts. CCA showed higher microvascular density and higher expression of nuclear factor kappa B, interleukin-6, interleukin-8, transforming growth factor ß, and vascular endothelial growth factor-1 compared to nonneoplastic ducts. CCA cells were characterized by a higher expression of epithelial-to-mesenchymal transition (EMT) traits and by the absence of primary cilia compared to bile ducts and PBG cells in controls and patients with PSC. Our in vitro study demonstrated that lipopolysaccharide and oxysterols (PSC-related stressors) induced the expression of EMT traits, the nuclear factor kappa B pathway, autophagy, and the loss of primary cilia in human BTSCs. Conclusion: CCA arising in patients with PSC is characterized by extensive PBG involvement and by activation of the BTSC niche in these patients, the presence of duct lesions at different stages suggests a progressive tumorigenesis.

Matrisome analysis of intrahepatic cholangiocarcinoma unveils a peculiar cancer-associated extracellular matrix structure.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a malignancy that arises from the intrahepatic biliary tree, showing high mortality rates due to its late clinical presentation and limited treatment options. iCCA is characterized by a dense, reactive desmoplastic stroma marked by a dramatic accumulation of extracellular matrix (ECM). Although recent results strongly suggest a relationship between increasing desmoplastic stroma and the enhanced malignant behaviour of iCCA, the importance of ECM proteins in the pathogenesis of iCCA still have to be addressed. METHODS: iCCA ECM fibrillar structural organization was characterized by histological analysis. ECM proteome profiles from decellularized iCCA and surrounding noncancerous tissues were analysed by nLC coupled to MALDI-TOF/TOF analysis. RESULTS: iCCA tissues displayed high levels of collagen fibers and low abundance of reticular and elastic fibers, suggesting stiffness and loss of polarity. The ECM proteome profiles of iCCA samples, when compared to those obtained from the surrounding noncancerous tissues showed a dismantling of the basement membrane, a reduced angiogenesis and a downregulation of oncosuppressive activity. In particular, we focused on the effects of the overexpression of collagen type III alpha 1 chain (COL3A1) in iCCA, thus providing evidences that COL3A1 promotes iCCA cells migration and is a component of tumor-associated aligned collagen. CONCLUSIONS: Overall, this study contributes to the understanding of molecular basis underlying desmoplasia in iCCA and indicates the type III collagen as a promising therapeutic target.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Background: Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. Methods: We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. Results: We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. Conclusions: NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

The roles of different forms of IL-15 in human melanoma progression.

Background: Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. Methods: The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Results: Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Conclusions: Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

Neuroblastoma Tumor-Associated Mesenchymal Stromal Cells Regulate the Cytolytic Functions of NK Cells.

Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been thoroughly investigated. Herein, we study the immune-regulatory potential of six primary young and senescent NB-TA-MSC on NK cell function. Young cells display a phenotype (CD105+/CD90+/CD73+/CD29+/CD146+) typical of MSC cells and, in addition, express high levels of immunomodulatory molecules (MHC-I, PDL-1 and PDL-2 and transcriptional-co-activator WWTR1), able to hinder NK cell activity. Notably, four of them express the neuroblastoma marker GD2, the most common target for NB immunotherapy. From a functional point of view, young NB-TA-MSC, contrary to the senescent ones, are resistant to activated NK cell-mediated lysis, but this behavior is overcome using anti-CD105 antibody TRC105 that activates antibody-dependent cell-mediated cytotoxicity. In addition, proliferating NB-TA-MSC, but not the senescent ones, after six days of co-culture, inhibit proliferation, expression of activating receptors and cytolytic activity of freshly isolated NK. Inhibitors of the soluble immunosuppressive factors L-kynurenine and prostaglandin E2 efficiently counteract this latter effect. Our data highlight the presence of phenotypically heterogeneous NB-TA-MSC displaying potent immunoregulatory properties towards NK cells, whose inhibition could be mandatory to improve the antitumor efficacy of targeted immunotherapy.

Therapeutic effects of dexamethasone-loaded hyaluronan nanogels in the experimental cholestasis.

A major function of the intrahepatic biliary epithelium is bicarbonate excretion in bile. Recent reports indicate that budesonide, a corticosteroid with high receptor affinity and hepatic first pass clearance, increases the efficacy of ursodeoxycholic acid, a choleretic agent, in primary biliary cholangitis patients. We have previously reported that bile ducts isolated from rats treated with dexamethasone or budesonide showed an enhanced activity of the Na+/H+ exchanger isoform 1 (NHE1) and Cl-/HCO3- exchanger protein 2 (AE2) . Increasing the delivery of steroids to the liver may result in three beneficial effects: increase in the choleresis, treatment of the autoimmune or inflammatory liver injury and reduction of steroids' systemic harmful effects. In this study, the steroid dexamethasone was loaded into nanohydrogels (or nanogels, NHs), in order to investigate corticosteroid-induced increased activities of transport processes driving bicarbonate excretion in the biliary epithelium (NHE-1 isoform) and to evaluate the effects of dexamethasone-loaded NHs (NHs/dex) on liver injury induced by experimental cholestatis. Our results showed that NHs and NHs/dex do not reduce cell viability in vitro in human cholangiocyte cell lines. Primary and immortalized human cholangiocytes treated with NHs/dex show an increase in the functional marker expression of NHE1 cholangiocytes compared to control groups. A mouse model of cholangiopathy treated with NHs/dex shows a reduction in markers of hepatocellular injury compared to control groups (NHs, dex, or sham group). In conclusion, we believe that the NHs/dex formulation is a suitable candidate to be investigated in preclinical models of cholangiopathies.

Pediatric Tumors-Mediated Inhibitory Effect on NK Cells: The Case of Neuroblastoma and Wilms' Tumors.

Natural killer (NK) cells play a key role in the control of cancer development, progression and metastatic dissemination. However, tumor cells develop an array of strategies capable of impairing the activation and function of the immune system, including NK cells. In this context, a major event is represented by the establishment of an immunosuppressive tumor microenvironment (TME) composed of stromal cells, myeloid-derived suppressor cells, tumor-associated macrophages, regulatory T cells and cancer cells themselves. The different immunoregulatory cells infiltrating the TME, through the release of several immunosuppressive molecules or by cell-to-cell interactions, cause an impairment of the recruitment of NK cells and other lymphocytes with effector functions. The different mechanisms by which stromal and tumor cells impair NK cell function have been particularly explored in adult solid tumors and, in less depth, investigated and discussed in a pediatric setting. In this review, we will compare pediatric and adult solid malignancies concerning the respective mechanisms of NK cell inhibition, highlighting novel key data in neuroblastoma and Wilms' tumor, two of the most frequent pediatric extracranial solid tumors. Indeed, both tumors are characterized by the presence of stromal cells acting through the release of immunosuppressive molecules. In addition, specific tumor cell subsets inhibit NK cell cytotoxic function by cell-to-cell contact mechanisms likely controlled by the transcriptional coactivator TAZ. These findings could lead to a more performant diagnostic approach and to the development of novel immunotherapeutic strategies targeting the identified cellular and molecular targets.

Identification of neuroblastoma cell lines with uncommon TAZ+/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells.

BACKGROUND: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells. METHODS: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset. RESULTS: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105+/CD90+/CD73+/CD29+/CD146+/GD2+/TAZ+). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105+/CD73+/TAZ+). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells. CONCLUSIONS: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Interleukin-15 and cancer: some solved and many unsolved questions.

Soluble interleukin (IL)-15 exists under two forms: as monomer (sIL-15) or as heterodimeric complex in association with sIL-15Rα (sIL-15/IL-15Rα). Both forms have been successfully tested in experimental tumor murine models and are currently undergoing investigation in phase I/II clinical trials. Despite more than 20 years research on IL-15, some controversial issues remain to be addressed. A first point concerns the detection of the sIL-15/IL-15Rα in plasma of healthy donors or patients with cancer and its biological significance. The second and third unsolved question regards the protumorigenic role of the IL-15/IL-15Rα complex in human cancer and the detrimental immunological consequences associated to prolonged exposure of natural killer (NK) cells to both forms of soluble IL-15, respectively. Data suggest that in vivo prolonged or repeated exposure to monomeric sIL-15 or the soluble complex may lead to NK hypo-responsiveness through the expansion of the CD8+/CD44+ T cell subset that would suppress NK cell functions. In vitro experiments indicate that soluble complex and monomeric IL-15 may cause NK hyporesponsiveness through a direct effect caused by their prolonged stimulation, suggesting that this mechanism could also be effective in vivo. Therefore, a better knowledge of IL-15 and a more appropriate use of both its soluble forms, in terms of concentrations and time of exposure, are essential in order to improve their therapeutic use. In cancer, the overproduction of sIL-15/IL-15Rα could represent a novel mechanism of immune escape. The soluble complex may act as a decoy cytokine unable to efficiently foster NK cells, or could induce NK hyporesponsiveness through an excessive and prolonged stimulation depending on the type of IL-15Rα isoforms associated. All these unsolved questions are not merely limited to the knowledge of IL-15 pathophysiology, but are crucial also for the therapeutic use of this cytokine. Therefore, in this review, we will discuss key unanswered issues on the heterogeneity and biological significance of IL-15 isoforms, analyzing both their cancer-related biological functions and their therapeutic implications.

Hyaluronan-Based Grafting Strategies for Liver Stem Cell Therapy and Tracking Methods.

Cell adhesion is essential for survival, it plays important roles in physiological cell functions, and it is an innovative target in regenerative medicine. Among the molecular interactions and the pathways triggered during cell adhesion, the binding of cluster of differentiation 44 (CD44), a cell-surface glycoprotein involved in cell-cell interactions, to hyaluronic acid (HA), a major component of the extracellular matrix, is a crucial step. Cell therapy has emerged as a promising treatment for advanced liver diseases; however, so far, it has led to low cell engraftment and limited cell repopulation of the target tissue. Currently, different strategies are under investigation to improve cell grafting in the liver, including the use of organic and inorganic biomatrices that mimic the microenvironment of the extracellular matrix. Hyaluronans, major components of stem cell niches, are attractive candidates for coating stem cells since they improve viability, proliferation, and engraftment in damaged livers. In this review, we will discuss the new strategies that have been adopted to improve cell grafting and track cells after transplantation.

Cholest-4,6-Dien-3-One Promote Epithelial-To-Mesenchymal Transition (EMT) in Biliary Tree Stem/Progenitor Cell Cultures In Vitro.

Human biliary tree stem/progenitor cells (hBTSCs), reside in peribiliary glands, are mainly stimulated by primary sclerosing cholangitis (PSC) and cholangiocarcinoma. In these pathologies, hBTSCs displayed epithelial-to-mesenchymal transition (EMT), senescence characteristics, and impaired differentiation. Here, we investigated the effects of cholest-4,6-dien-3-one, an oxysterol involved in cholangiopathies, on hBTSCs biology. hBTSCs were isolated from donor organs, cultured in self-renewal control conditions, differentiated in mature cholangiocytes by specifically tailored medium, or exposed for 10 days to concentration of cholest-4,6-dien-3-one (0.14 mM). Viability, proliferation, senescence, EMT genes expression, telomerase activity, interleukin 6 (IL6) secretion, differentiation capacity, and HDAC6 gene expression were analyzed. Although the effect of cholest-4,6-dien-3-one was not detected on hBTSCs viability, we found a significant increase in cell proliferation, senescence, and IL6 secretion. Interestingly, cholest-4.6-dien-3-one impaired differentiation in mature cholangiocytes and, simultaneously, induced the EMT markers, significantly reduced the telomerase activity, and induced HDAC6 gene expression. Moreover, cholest-4,6-dien-3-one enhanced bone morphogenic protein 4 (Bmp-4) and sonic hedgehog (Shh) pathways in hBTSCs. The same pathways activated by human recombinant proteins induced the expression of EMT markers in hBTSCs. In conclusion, we demonstrated that chronic exposition of cholest-4,6-dien-3-one induced cell proliferation, EMT markers, and senescence in hBTSC, and also impaired the differentiation in mature cholangiocytes.

Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells.

Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota's Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs.

CXCR7 contributes to the aggressive phenotype of cholangiocarcinoma cells.

Development of cholangiocarcinoma (CCA) is dependent on a cross-talk with stromal cells, which release different chemokines including CXCL12, that interacts with two different receptors, CXCR4 and CXCR7. The aim of the present study was to investigate the role of CXCR7 in CCA cells. CXCR7 is overexpressed by different CCA cell lines and in human CCA specimens. Knock-down of CXCR7 in HuCCT-1 cells reduced migration, invasion, and CXCL12-induced adhesion to collagen I. Survival of CCA was also reduced in CXCR7-silenced cells. The ability of CXCL12 to induce cell migration and survival was also blocked by CCX733, a CXCR7 antagonist. Similar effects of CXCR7 activation were observed in CCLP-1 cells and in primary iCCA cells. Enrichment of tumor stem-like cells by a 3D culture system resulted in increased CXCR7 expression compared to cells grown in monolayers, and genetic knockdown of CXCR7 robustly reduced sphere formation both in HuCCT-1 and in CCLP-1 cells. In HuCCT-1 cells CXCR7 was found to interact with ß-arrestin 2, which was necessary to mediate CXCL12-induced migration, but not survival. In conclusion, CXCR7 is widely expressed in CCA, and contributes to the aggressive phenotype of CCA cells, inducing cell migration, invasion, adhesion, survival, growth and stem cell-like features. Cell migration induced by CXCR7 requires interaction with ß-arrestin 2.

Hyaluronan coating improves liver engraftment of transplanted human biliary tree stem/progenitor cells.

BACKGROUND: Cell therapy of liver diseases with human biliary tree stem cells (hBTSCs) is biased by low engraftment efficiency. Coating the hBTSCs with hyaluronans (HAs), the primary constituents of all stem cell niches, could facilitate cell survival, proliferation, and, specifically, liver engraftment given that HAs are cleared selectively by the liver. METHODS: We developed a fast and easy method to coat hBTSCs with HA and assessed the effects of HA-coating on cell properties in vitro and in vivo. RESULTS: The HA coating markedly improved the viability, colony formation, and population doubling of hBTSCs in primary cultures, and resulted in a higher expression of integrins that mediate cell attachment to matrix components. When HA-coated hBTSCs were transplanted via the spleen into the liver of immunocompromised mice, the engraftment efficiency increased to 11% with respect to 3% of uncoated cells. Notably, HA-coated hBTSC transplantation in mice resulted in a 10-fold increase of human albumin gene expression in the liver and in a 2-fold increase of human albumin serum levels with respect to uncoated cells. Studies in distant organs showed minimal ectopic cell distribution without differences between HA-coated and uncoated hBTSCs and, specifically, cell seeding in the kidney was excluded. CONCLUSIONS: A ready and economical procedure of HA cell coating greatly enhanced the liver engraftment of transplanted hBTSCs and improved their differentiation toward mature hepatocytes. HA coating could improve outcomes of stem cell therapies of liver diseases and could be immediately translated into the clinic given that GMP-grade HAs are already available for clinical use.

Cryopreservation protocol for human biliary tree stem/progenitors, hepatic and pancreatic precursors.

Human biliary tree stem/progenitor cells (hBTSCs) are being used for cell therapies of patients with liver cirrhosis. A cryopreservation method was established to optimize sourcing of hBTSCs for these clinical programs and that comprises serum-free Kubota's Medium (KM) supplemented with 10% dimethyl sulfoxide (DMSO), 15% human serum albumin (HSA) and 0.1% hyaluronans. Cryopreserved versus freshly isolated hBTSCs were similar in vitro with respect to self-replication, stemness traits, and multipotency. They were able to differentiate to functional hepatocytes,cholangiocytes or pancreatic islets, yielding similar levels of secretion of albumin or of glucose-inducible levels of insulin. Cryopreserved versus freshly isolated hBTSCs were equally able to engraft into immunocompromised mice yielding cells with human-specific gene expression and human albumin levels in murine serum that were higher for cryopreserved than for freshly isolated hBTSCs. The successful cryopreservation of hBTSCs facilitates establishment of hBTSCs cell banking offering logistical advantages for clinical programs for treatment of liver diseases.