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.

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.

Cholangiocarcinoma (CCA) is an aggressive cancer with high resistance to chemotherapeutics. CCA is enriched in cancer stem cells, which correlate with aggressiveness and prognosis. FXR, a member of the metabolic nuclear receptor family, is markedly down-regulated in human CCA. Our aim was to evaluate, in primary cultures of human intrahepatic CCA (iCCA), the effects of the FXR agonist obeticholic acid (OCA), a semisynthetic bile acid derivative, on their cancerogenic potential. Primary human iCCA cell cultures were prepared from surgical specimens of mucinous or mixed iCCA subtypes. Increasing concentrations (0–2.5 μM) of OCA were added to culture media and, after 3–10 days, effects on proliferation (MTS assay, cell population doubling time), apoptosis (annexin V-FITC/propidium iodide), cell migration and invasion (wound healing response and Matrigel invasion assay), and cancerogenic potential (spheroid formation, clonogenic assay, colony formation capacity) were evaluated. Results: FXR gene expression was downregulated (RT-qPCR) in iCCA cells vs normal human biliary tree stem cells (p < 0.05) and in mucinous iCCA vs mixed iCCA cells (p < 0.05) but was upregulated by addition of OCA. OCA significantly (p < 0.05) inhibited proliferation of both mucinous and mixed iCCA cells, starting at a concentration as low as 0.05 μM. Also, CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with its different affinity for FXR. OCA significantly induced apoptosis of both iCCA subtypes and decreased their in vitro cancerogenic potential, as evaluated by impairment of colony and spheroid formation capacity and delayed wound healing and Matrigel invasion. In general, these effects were more evident in mixed than mucinous iCCA cells. When tested together with Gemcitabine and Cisplatin, OCA potentiated the anti-proliferative and pro-apoptotic effects of these chemotherapeutics, but mainly in mixed iCCA cells. OCA abolished the capacity of both mucinous and mixed iCCA cells to form colonies when administered together with Gemcitabine and Cisplatin. In subcutaneous xenografts of mixed iCCA cells, OCA alone or combined with Gemcitabine or Cisplatin markedly reduced the tumor size after 5 weeks of treatment by inducing necrosis of tumor mass and inhibiting cell proliferation. In conclusion, FXR is down-regulated in iCCA cells, and its activation by OCA results in anti-cancerogenic effects against mucinous and mixed iCCA cells, both in vitro and in vivo. The effects of OCA predominated in mixed iCCA cells, consistent with the lower aggressiveness and the higher FXR expression in this CCA subtype. These results, showing the FXR-mediated capacity of OCA to inhibit cholangiocarcinogenesis, represent the basis for testing OCA in clinical trials of CCA patients.

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 pCCA^LGR5+ and in iCCA^CD133+ 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 iCCA^CD133+ and pCCA^LGR5+, 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.