Btc3 Cells

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BTC is an aggressive disease exacerbated by inflammation and immune suppression. Expansion of immunosuppressive cells occurs in biliary tract cancer (BTC), yet the role of BTC-derived cytokines in this process is unclear.

Biliary tract cancers (BTCs) comprise a rare, heterogeneous group of malignancies with an average 5-year survival rate ranging from 8 to 10% for intrahepatic and extrahepatic bile duct cancer, respectively.1 In the United States, >7000 new cases of BTC are diagnosed annually.2 Recent evidence suggests the anatomic origin of BTC, specifically intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), and hilar cholangiocarcinoma, categorises tumours into distinct subtypes.3,4,5 Despite advances in targeting genomic aberrations such as IDH and FGFR fusions in BTC,6,7,8 there has been limited investigation into targetable immune signatures within BTC.3 We suspect that BTC tumours may have distinct immune features that can be leveraged for nuanced treatment. In particular, we hypothesised that the inflammatory nature of this disease may promote suppressive myeloid cell expansion that acts to limit lymphocyte responses to BTC.

Inflammation of the bile duct, autoimmune disorders, parasitic infections and exposure to alcohol or toxins contributes to BTC pathogenesis.9,14 These inflammatory conditions upregulate cytokines such as interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-β (TGF-β), yet specific mechanisms by which these cytokines influence tumour development and progression in BTC have yet to be described. A number of reports suggest that IL-6 may act in an autocrine or paracrine manner to enhance BTC growth and survival.15,16 In models beyond BTC, IL-6 acts with other tumour or stromal factors to expand immunosuppressive cells. In particular, GM-CSF enhances expansion of myeloid-derived suppressor cells (MDSCs),17,18,19 while TGF-β can expand T regulatory cells (Tregs)20,21 and promote T helper type 17 (Th17) differentiation, both of which can mediate immune suppression.22,23 MDSCs are of particular interest, given their capability to limit T and natural killer cell function through production of reactive oxygen or nitrogen intermediates and depletion of key amino acids.24,25,26 In addition, investigations of MDSCs have revealed significant impacts of these populations on disease progression and metastasis, whereby MDSCs actually precede neoplastic cells to sites of metastasis and provide a hospitable environment for cancer growth.27,28,29,30,31,32 Characterisation of these soluble factors and cellular interactions may reveal viable targets for future immunotherapy strategies.

The Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway is an important mediator in the inflammatory response.15 STAT proteins are transcription factors that promote expression of distinct genes that differentially regulate cell growth, survival, and inflammation. STAT1 is typically associated with growth arrest and apoptosis. In contrast, STAT3 and STAT5 are associated with proliferation, resistance to apoptosis, and avoidance of antitumour immune responses. Constitutive STAT3 or STAT5 activation occurs in many tumours and is implicated in malignant progression.33 A limited number of studies confirmed that nuclear localisation of STAT3 was detectable in BTC patient tumours34 and associated with shorter survival.35 In myeloid compartments, STAT3/5 signalling regulates a phenotypic switch to promote immunologic sequelae, including expansion of MDSCs, M2 macrophages, and a shift in the balance of Treg/Th17 cells.36,37,38 We postulate cytokine-mediated STAT3/5 activation in BTC may lead to expansion of immune-suppressive cell populations and disease progression.

In the present study, we hypothesise that BTC-derived cytokines contribute to immunosuppression through distinct signalling pathways. We demonstrate human BTC cells produce a unique profile of soluble cytokines, capable of inducing in vitro expansion of functional MDSCs. IL-6 and GM-CSF excreted from BTC cells contribute but likely act in concert with other factors to facilitate these changes in myeloid cells. Within human BTC tissue samples, we demonstrate elevated IL-6 and GM-CSF are associated with higher infiltration of CD33+S100a9+ myeloid cells. In addition, increased percentages of CD33+S100a9+ cells in BTC tumour tissue correlated with higher tumour grade, the presence of satellite lesions, and more poorly differentiated tumours. Taken together, our studies indicate a dynamic tumour promoting interaction between BTC and MDSCs, by which tumour cells drive MDSC expansion and contribute to aggressive disease characteristics. These data provide novel insight into the role for myeloid cells in resectable BTC and a greater understanding of cytokine-regulated mechanisms contributing to the ability of BTC to escape immune recognition.

Immunofluorescent images of TMAs were analysed using FIJI (NIH) and CellProfiler.44 For IL-6 and GM-CSF images, a MaxEntropy threshold for DAPI, IL-6 (AlexaFluor 488) and GM-CSF (AlexaFluor568) was determined using FIJI. Total area of particles within the threshold was measured and area of IL-6 and GM-CSF was normalised to DAPI for each image. Utilisation of thresholding allowed for exclusion of background signal. Max projection images of CD33+S100a9+ cells were analysed using CellProfiler. Thresholding was performed for intensity and size. DAPI, S100a9, and CD33 were identified as primary objects and related using DAPI as the parent object, with S100a9 and CD33 as children. DAPI parent objects with both S100a9 and CD33 children were counted as dual positive cells and represent a phenotype consistent with MDSCs as described by Ortiz.43 Counts for CD33+S100a9+ cells were normalised to total cell counts per image as determined by DAPI object identification. Each sample was imaged as a single 20 image for IL-6 and GM-CSF and two 40 sets of Z-stacks for MDSCs.

The profile of 18 cytokines and chemokines secreted from a collection of human BTC cell lines derived from intrahepatic (HuCCT1, HuH28), extrahepatic (WITT/Sk-Cha-1, SNU-245), gallbladder (Mz-ChA-1), or ampullary tumours (SNU-478) was characterised via multiplex analysis in culture supernatants (Fig. 1a). Cytokines involved in MDSC expansion and migration or T cell biology were our area of focus. Cell lines demonstrated variable levels of cytokine secretion. Overall, there was limited secretion (

Several redundant mechanisms limit immune recognition of tumours in patients with advanced BTC.46 This report explored the unique contribution of the cytokines IL-6 and GM-CSF and their relationship to signalling and myeloid cells in the context of BTC. We demonstrate that these BTC-derived factors expand functionally suppressive MDSCs in vitro and may have clinical implications when present in resectable patient tumours. These data are novel in the setting of BTC and aligned with their role in other gastrointestinal malignancies.24,47 Our results suggest that IL-6 and GM-CSF deserve investigation as therapeutic targets in BTC due to their ability to activate Jak/STAT signalling across tumour or immune cell types and their correlation with phenotypically defined myeloid cells in tumours from BTC patients.

This study lends credence to the concept that BTC has the capacity to exploit immune cells by secreting soluble factors that act in trans to drive signalling events, thereby influencing cell phenotype and function. Although IL-6 and GM-CSF emerged as the focus of this study, our data clearly point out that these factors are part of a larger imbalance encompassing multiple immune and metabolic factors in BTC. Certainly these factors can act in concert to facilitate MDSC expansion, along with other cell types including Tregs and Th17 cells.24,48,49 Further investigation of other BTC-derived factors may reveal cytokine/chemokine-induced expansion or migration of other immune subsets within the tumour microenvironment. Of particular interest for future studies are MCP-1, M-CSF, G-CSF, and SDF-1, all of which were secreted in abundance by BTC cells.

Considering our data, it is likely that IL-6, GM-CSF, or other cytokines in BTC are derived from multiple cell compartments. Indeed, fibroblasts, myeloid cells, and T cells can produce these cytokines and other immunomodulatory factors.24,50,51 In addition, the feedforward nature of the IL-6/Jak/STAT3 pathway can exacerbate cytokine production among any combination of these cellular subsets.24,52,53,54 This study also did not investigate Stromal Derived Factors known to be present in BTCs such as prostaglandin E2 and cyclooxygenase-2. This latter phospholipase has a key role in suppressing T cell-driven immune responses to cancer and driving MDSC expansion and promoting biliary cancer tumorigenesis.55,56 Given the dynamic relationship observed in the study between MDSCs and BTC, other targets such as these deserve further consideration. Here, we have specifically focussed on contributions of intratumoural IL-6 and GM-CSF, but further study is needed to assess tissue levels of other BTC-derived cytokines. Our data demonstrating activation of STAT3/5 in BTC cells and in PBMCs suggest a dual role for Jak/STAT signalling in promoting cancer cell proliferation and MDSC expansion. Further characterising the signalling dynamics in response to cytokines secreted by tumour cells may reveal novel targets for BTC that can be leveraged in combination with immunotherapy.

The analysis of patient tumours further established clinical relevance for IL-6, GM-CSF, and MDSC in BTC. Of note was the heterogeneous expression of these factors, and the fact that their expression was strongly correlated. The significant positive correlation between IL-6 and GM-CSF with infiltration of CD33+S100a9+ cells indicates that these cytokines contribute to progression by modulating the immune composition of the tumour. Of particular future interest is the significant correlation between the presence of MDSCs in pathologic specimens with aggressive tumour characteristics. The role of MDSC in establishing a pre-metastatic niche in various organs, particularly the liver, is an area of growing research with significant findings demonstrating mechanisms by which MDSCs shift local tissue microenvironments.28,32,57,58 The correlation observed here between worse tumour grade, poorly differentiated tumours, and the presence of satellite lesions demonstrates a relationship between aggressive tumour development and MDSCs. This correlation has also been observed in renal cell carcinoma, hepatocellular carcinoma, and other solid cancers.27,31,59,60 Given these data and the growing body of evidence suggesting that MDSCs play an integral role in patient responses to immune checkpoint blockade, further efforts to target the suppressive mechanisms of these cells in BTC should be made.29,30,61,62 Although these data are provocative, one limitation of this study is that the data are limited to a single phenotypic definition of CD33+S100a9+ cells.43 This is unfortunately due to lack of robust, validated immunohistochemical methods for MDSC phenotypic subsets in paraffin-embedded tissues. We are also aware that this population can encompass myeloid cells which may not functionally suppress antitumour immunity in BTC. It should also be noted that MDSC infiltration and cytokine staining in the tumours of these patients seem to independently correlate with clinicopathological features. Thus, while there is a direct correlation between increased staining for these cytokines and MDSC infiltration, the manner in which IL-6, GM-CSF, and MDSCs contribute to disease severity is likely complicated and influenced by other soluble mediators. One other factor to consider is the temporal aspect of this process that cannot be captured by a single time-point biopsies.

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