Background: Intrahepatic cholangiocarcinoma (CCA) is a highly treatment-resistant malignancy of biliary epithelium with increasing global mortality. Histologically, CCA is characterised by a pronounced inflammatory stroma of tumour-associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix (ECM). In other solid cancers, the stroma plays a tumour promoting role. The functional role of the stroma in CCA remains unclear. The origin and the proportional contribution to the stroma by haematopoietic and mesenchymal bone marrow (BM) -derived cells is not known in CCA. Intriguingly, reports suggest that mesenchymal stem cells (MSCs) may contribute to the epithelial compartment of malignant tumours. Furthermore, the Notch signalling pathway is known to play oncogenic and tumour suppressive roles in diverse neoplasms but its role in CCA remains unclear. Aims and Methods: The functional role of myofibroblasts and macrophages in the tumour stroma of CCA was investigated together with an analysis of the origin and contribution of BM-derived cells to the stromal and epithelial compartments of CCA. The Notch signalling pathway was studied as a potential signalling mechanism through which the stroma and malignant epithelial compartments of CCA may interact. Results: The thioacetamide rat model of CCA was optimised and found to display excellent histological congruence with human lesions. The tumour cellular microenvironment comprised of myofibroblasts, migratory macrophages and immune cells. During cholangiocarcinogenesis, progressive intrahepatic accumulation of inflammatory cells and proliferation of bipotential progenitor cells preceded the development of invasive CCA. In vitro, CCA lines were identified to contain a side population of stem cells. Adoptive transfer of BM from Enhanced Green Fluorescent Protein (EGFP) transgenic rats to wild type rats to establish chimeras was undertaken. In transplanted rats, persistent EGFP+ chimerism of both haematopoietic and mesenchymal stem cell compartments was established. In tumours, macrophages and neutrophils were overwhelmingly EGFP+ve, whereas myofibroblasts, fibroblasts and benign and malignant bile ducts were EGFP-ve. There was no evidence of cell fusion or EGFP silencing. These findings were confirmed in spontaneous breast, skin and colon tumours in EGFP+ chimeric rats not treated with TAA. In vitro studies to recapitulate the cellular and extracellular elements of the tumour niche identified that ECM components induce characteristic cell proliferation patterns dependent on the matrix component but do not appear to affect chemosensitivity. Bidirectional interaction between CCA cells and hepatic stellate cells (mediated by soluble factors) was identified. Furthermore, in direct co-culture, M2 polarised macrophages appear to enhance CCA cell proliferation compared to M1 macrophages. In considering the Notch pathway, Notch signalling components (particularly Notch3) and target genes were upregulated in human CCA specimens. Immunohistochemical analysis identified apparent distribution of Notch ligand on tumour stroma and Notch receptor subtypes on malignant epithelia. Although direct co-culture of CCA cells with myofibroblasts and M1/M2 polarised macrophages did not clearly demonstrate stromal:epithelial Notch pathway activation, this may have been a function of in vitro experimental limitations. Gamma-secretase inhibition downregulated the Notch pathway, reduced proliferation and appeared to enhance chemosensitivity of CCA cells in vitro. Conclusions: A stereotypical niche forms around CCA in developing and malignant lesions. There was no evidence of a BM-derived stem cell contribution to the epithelial component of CCA, breast, colon or skin malignancies. Haematopoietic but not mesenchymal components of the tumour stroma were of BM origin. Notch signalling is upregulated in CCA and appears to play a tumour promoting role in CCA; pathway inhibition represents a potential therapeutic target.

Acute Lymphoblastic Leukemia (ALL) is an aggressive hematologic tumor and is the most common malignancy in children (Horton and Steuber 2014). This disease is characterized by the infiltration of bone marrow by malignant immature lymphoid progenitor cells and is invariably fatal without treatment. Although multi-agent combination chemotherapy is curative in a significant fraction of ALL patients, treatment currently fails in approximately 20% of children and up to 50% of adults with ALL, making relapse and drug resistance the most substantial challenge in the treatment of this disease(Fielding, Richards et al. 2007, Aster and DeAngelo 2013). Understanding what causes treatment failure is of great medical importance as second line therapies also fail in the majority of relapse T-cell ALL (TALL) patients (Fielding, Richards et al. 2007, Aster and DeAngelo 2013). Using next-generation sequencing to compare the genomes of tumors before and after therapy, mutations in gene cytosolic 5’-nucleotidase II (NT5C2) were discovered in 19% of pediatric samples with relapsed T-ALL(Tzoneva, Carpenter et al. 2013). Preliminary structure function analysis and subsequent in vitro experimental nucleotidase activity assays confirmed that these mutations lead to hyperactive NT5C2 protein. Furthermore, NT5C2 mutant proteins conferred resistance to 6-mercaptopurine and 6-thioguanine chemotherapy drugs when expressed in ALL lymphoblasts, suggesting NT5C2 is responsible for the inactivation of nucleoside-analog chemotherapy drugs. In order to assess the ability of these mutations to lead to novel inhibitor schemes, the functional impact of each mutation was analyzed through robust structure function methods. The result of this in silico analysis, is the identification of a potential allosteric regulatory mechanism of negative feedback inhibition never before described. Most notably, the majority of NT5C2 mutations identified have characteristics that suggest they abrogate the function of this proposed mechanism, yielding a novel viable target for the development of allosteric inhibitors specific for constitutively active NT5C2 mutant proteins. Overall these findings support a prominent role for activating mutations in NT5C2 and chemotherapy resistance in ALL, and highlight new avenues for relapsed ALL therapy development in the future.

The principle of trained immunity represents innate immune memory due to sustained, mainly epigenetic, changes triggered by endogenous or exogenous stimuli in bone marrow (BM) progenitors (central trained immunity) and their innate immune cell progeny, thereby triggering elevated responsiveness against secondary stimuli. BM progenitors can respond to microbial and sterile signals, thereby possibly acquiring trained immunity-mediated long-lasting alterations that may shape the fate and function of their progeny, for example, neutrophils. Neutrophils, the most abundant innate immune cell population, are produced in the BM from committed progenitor cells in a process designated granulopoiesis. Neutrophils are the first responders against infectious or inflammatory challenges and have versatile functions in immunity. Together with other innate immune cells, neutrophils are effectors of peripheral trained immunity. However, given the short lifetime of neutrophils, their ability to acquire immunological memory may lie in the central training of their BM progenitors resulting in generation of reprogrammed, that is, “trained”, neutrophils. Although trained immunity may have beneficial effects in infection or cancer, it may also mediate detrimental outcomes in chronic inflammation. Here, we review the emerging research area of trained immunity with a particular emphasis on the role of neutrophils and granulopoiesis.