Immune evasion on the nanoscale: Small extracellular vesicles in pancreatic ductal adenocarcinoma immunity.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer alarmingly expanding in our modern societies that is still proving to be very challenging to counteract. This disease constitutes a quintessential example of the multiple interactions existing between the tumour and its surrounding microenvironment. In particular, PDAC is characterized by a very immunosuppressive environment that favours cancer growth and makes this cancer type very resistant to immunotherapy. The primary tumour releases many factors that influence both the microenvironment and the immune landscape. Extracellular vesicles (EVs), recently identified as indispensable entities ensuring cell-to-cell communication in both physiological and pathological processes, seem to play a pivotal function in ensuring the delivery of these factors to the tumour-surrounding tissues. In this review, we summarize the present understanding on the crosstalk among tumour cells and the cellular immune microenvironment emphasizing the pro-malignant role played by extracellular vesicles. We also discuss how a greater knowledge of the roles of EVs in tumour immune escape could be translated into clinical applications.

Adenosine 2A Receptor Activation Amplifies Ibrutinib Antiplatelet Effect; Implications in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia patients have an increased bleeding risk with the introduction of Bruton tyrosine kinase (BTK) inhibitors. BTK is a signaling effector downstream of the platelet GPVI receptor. Innate platelet dysfunction in CLL patients and the contribution of the leukemia microenvironment to the anti-platelet effect of BTK inhibitors are still not well defined. Herein, we investigated platelet function in stable, untreated CLL patients in comparison to age-matched healthy subjects as control. Secondly, we proposed a novel mechanism of platelet dysfunction via the adenosinergic pathway during BTK inhibitor therapy. Our data indicate that the nucleotidase that produces adenosine, CD73, was expressed on one-third of B-cells in CLL patients. Inhibition of CD73 improved platelet response to ADP in the blood of CLL patients ex vivo. Using healthy platelets, we show that adenosine 2A (A2A) receptor activation amplifies the anti-platelet effect of ibrutinib (10 nM). Ibrutinib plus an A2A agonist-but not ibrutinib as a single agent-significantly inhibited collagen (10 µg/mL)-induced platelet aggregation. Mechanistically, A2A activation attenuated collagen-mediated inhibition of p-VASP and synergized with ibrutinib to inhibit the phosphorylation of AKT, ERK and SYK kinases. This manuscript highlights the potential role of adenosine generated by the microenvironment in ibrutinib-associated bleeding in CLL patients.

Dissecting lipid metabolism alterations in SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that has infected over a hundred million people globally. There have been more than two million deaths recorded worldwide, with no end in sight until a widespread vaccination will be achieved. Current research has centred on different aspects of the virus interaction with cell surface receptors, but more needs to be done to further understand its mechanism of action in order to develop a targeted therapy and a method to control the spread of the virus. Lipids play a crucial role throughout the viral life cycle, and viruses are known to exploit lipid signalling and synthesis to affect host cell lipidome. Emerging studies using untargeted metabolomic and lipidomic approaches are providing new insight into the host response to COVID-19 infection. Indeed, metabolomic and lipidomic approaches have identified numerous circulating lipids that directly correlate to the severity of the disease, making lipid metabolism a potential therapeutic target. Circulating lipids play a key function in the pathogenesis of the virus and exert an inflammatory response. A better knowledge of lipid metabolism in the host-pathogen interaction will provide valuable insights into viral pathogenesis and to the development of novel therapeutic targets.

Antiplatelet Drug Ticagrelor Enhances Chemotherapeutic Efficacy by Targeting the Novel P2Y12-AKT Pathway in Pancreatic Cancer Cells.

Background: Extensive research has reported that extracellular ADP in the tumour microenvironment can stimulate platelets through interaction with the platelet receptor P2Y12. In turn, activated platelets release biological factors supporting cancer progression. Experimental data suggest that the tumour microenvironment components, of which platelets are integral, can promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Thus, overcoming chemoresistance requires combining multiple inhibitors that simultaneously target intrinsic pathways in cancer cells and extrinsic factors related to the tumour microenvironment. We aimed to determine whether ticagrelor, an inhibitor of the ADP-P2Y12 axis and a well-known antiplatelet drug, could be a therapeutic option for PDAC. Methods: We investigated a functional P2Y12 receptor and its downstream signalling in a panel of PDAC cell lines and non-cancer pancreatic cells termed hTERT-HPNE. We tested the synergistic effect of ticagrelor, a P2Y12 inhibitor, in combination with chemotherapeutic drugs (gemcitabine, paclitaxel and cisplatin), in vitro and in vivo. Results: Knockdown studies revealed that P2Y12 contributed to epidermal growth factor receptor (EGFR) activation and the expression of SLUG and ZEB1, which are transcriptional factors implicated in metastasis and chemoresistance. Studies using genetic and pharmacological inhibitors showed that the P2Y12-EGFR crosstalk enhanced cancer cell proliferation. Inhibition of P2Y12 signalling significantly reduced EGF-dependent AKT activation and promoted the anticancer activity of anti-EGFR treatment. Importantly, ticagrelor significantly decreased the proliferative capacity of cancer but not normal pancreatic cells. In vitro, synergism was observed when ticagrelor was combined with several chemodrugs. In vivo, a combination of ticagrelor with gemcitabine significantly reduced tumour growth, whereas gemcitabine or ticagrelor alone had a minimal effect. Conclusions: These findings uncover a novel effect and mechanism of action of the antiplatelet drug ticagrelor in PDAC cells and suggest a multi-functional role for ADP-P2Y12 signalling in the tumour microenvironment.

CD8+ cytotoxic T cell responses to dominant tumor-associated antigens are profoundly weakened by aging yet subdominant responses retain functionality and expand in response to chemotherapy.

Increasing life expectancy is associated with increased cancer incidence, yet the effect of cancer and anti-cancer treatment on elderly patients and their immune systems is not well understood. Declining T cell function with aging in response to infection and vaccination is well documented, however little is known about aged T cell responses to tumor antigens during cancer progression or how these responses are modulated by standard chemotherapy. We examined T cell responses to cancer in aged mice using AE17sOVA mesothelioma in which ovalbumin (OVA) becomes a 'spy' tumor antigen containing one dominant (SIINFEKL) and two subdominant (KVVRFDKL and NAIVFKGL) epitopes. Faster progressing tumors in elderly (22-24 months, cf. 60-70 human years) relative to young (2-3 months, human 15-18 years) mice were associated with increased pro-inflammatory cytokines and worsened cancer cachexia. Pentamer staining and an in-vivo cytotoxic T lymphocyte (CTL) assay showed that whilst elderly mice generated a greater number of CD8+ T cells recognizing all epitopes, they exhibited a profound loss of function in their ability to lyse targets expressing the dominant, but not subdominant, epitopes compared to young mice. Chemotherapy was less effective and more toxic in elderly mice however, similar to young mice, chemotherapy expanded CTLs recognizing at least one subdominant epitope in tumors and draining lymph nodes, yet treatment efficacy still required CD8+ T cells. Given the significant dysfunction associated with elderly CTLs recognizing dominant epitopes, our data suggest that responses to subdominant tumor epitopes may become important when elderly hosts with cancer are treated with chemotherapy.

The History of Armand Trousseau and Cancer-Associated Thrombosis.

"Je suis perdu; une phlegmatia qui vient de se déclarer cette nuit, ne me laisse aucun doute sur nature de mon mal [...].

Cancer-Associated Thrombosis: An Overview of Mechanisms, Risk Factors, and Treatment.

Cancer-associated thrombosis is a major cause of mortality in cancer patients, the most common type being venous thromboembolism (VTE). Several risk factors for developing VTE also coexist with cancer patients, such as chemotherapy and immobilisation, contributing to the increased risk cancer patients have of developing VTE compared with non-cancer patients. Cancer cells are capable of activating the coagulation cascade and other prothrombotic properties of host cells, and many anticancer treatments themselves are being described as additional mechanisms for promoting VTE. This review will give an overview of the main thrombotic complications in cancer patients and outline the risk factors for cancer patients developing cancer-associated thrombosis, focusing on VTE as it is the most common complication observed in cancer patients. The multiple mechanisms involved in cancer-associated thrombosis, including the role of anticancer drugs, and a brief outline of the current treatment for cancer-associated thrombosis will also be discussed.

Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones.

BACKGROUND: The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response. METHODS: Isolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation. RESULTS: Cell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y12 and αIIbß3 receptors. CONCLUSIONS: In vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation.

Aged neutrophils accumulate in lymphoid tissues from healthy elderly mice and infiltrate T- and B-cell zones.

The average age of the human population is rising, leading to an increasing burden of age-related diseases, including increased susceptibility to infection. However, immune function can decrease with age which could impact on processes that require a functional immune system. Aging is also characterized by chronic low-grade inflammation which could further impact immune cell function. While changes to neutrophils in blood during aging have been described, little is known in aging lymphoid organs. This study used female C57BL/6J mice comparing bone marrow (BM), spleen and lymph nodes from young mice aged 2-3 months (equivalent to 18 human years) with healthy elderly mice aged 22-24 months (equivalent to 60-70 human years). Neutrophil proportions increased in BM and secondary lymphoid organs of elderly mice relative to their younger counterparts and presented an atypical phenotype. Interestingly, neutrophils from elderly spleen and lymph nodes were long lived (with decreased apoptosis via Annexin V staining and increased proportion of BrdUneg mature cells) with splenic neutrophils also demonstrating a hypersegmented morphology. Furthermore, splenic neutrophils of elderly mice expressed a mixed phenotype with increased expression of activation markers, CD11b and ICAM-1, increased proinflammatory TNFα, yet increased anti-inflammatory transforming growth factor-beta. Elderly splenic architecture was compromised, as the marginal zone (required for clearing infections) was contracted. Moreover, neutrophils from elderly but not young mice accumulated in lymph node and splenic T- and B-cell zones. Overall, the expansion of functionally compromised neutrophils could contribute to increased susceptibility to infection observed in the elderly.

Molecular and cellular mechanisms of chemoresistance in pancreatic cancer.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most chemoresistant cancers, and current therapies targeting cancer-associated molecular pathways have not given satisfactory results, owing in part to rapid upregulation of alternative compensatory pathways. Most of the available treatments are palliative, focussing on improving the quality of life. At present, available options are surgery, embolization, radiation, chemotherapy, immunotherapy and use of other more targeted drugs. In this review, we describe the cellular and molecular effects of current chemotherapy drugs such as gemcitabine, FOLFIRINOX (5-fluorouracil [5-FU], oxaliplatin, irinotecan, and leucovorin) and ABRAXANE (nab-Paclitaxel), which have shown a survival benefit, although modest, for pancreatic cancer patients. Nevertheless, gemcitabine remains the standard first-line option for advanced-stage pancreatic cancer patients and, as resistance to the drug has attracted an increasing scientific interest, we deliberate on the main intracellular processes and proteins vital in acquired chemoresistance to gemcitabine. Lastly, our review examines various microenvironmental factors capable of instigating PDAC to develop resistance to chemotherapeutic drugs.

The Role of Platelet-Derived ADP and ATP in Promoting Pancreatic Cancer Cell Survival and Gemcitabine Resistance.

Platelets have been demonstrated to be vital in cancer epithelial-mesenchymal transition (EMT), an important step in metastasis. Markers of EMT are associated with chemotherapy resistance. However, the association between the development of chemoresistance, EMT, and the contribution of platelets to the process, is still unclear. Here we report that platelets regulate the expression of (1) human equilibrative nucleoside transporter 1 (hENT1) and (2) cytidine deaminase (CDD), markers of gemcitabine resistance in pancreatic cancer. Human ENT1 (hENT1) is known to enable cellular uptake of gemcitabine while CDD deactivates gemcitabine. Knockdown experiments demonstrate that Slug, a mesenchymal transcriptional factor known to be upregulated during EMT, regulates the expression of hENT1 and CDD. Furthermore, we demonstrate that platelet-derived ADP and ATP regulate Slug and CDD expression in pancreatic cancer cells. Finally, we demonstrate that pancreatic cancer cells express the purinergic receptor P2Y12, an ADP receptor found mainly on platelets. Thus ticagrelor, a P2Y12 inhibitor, was used to examine the potential therapeutic effect of an ADP receptor antagonist on cancer cells. Our data indicate that ticagrelor negated the survival signals initiated in cancer cells by platelet-derived ADP and ATP. In conclusion, our results demonstrate a novel role of platelets in modulating chemoresistance in pancreatic cancer. Moreover, we propose ADP/ATP receptors as additional potential drug targets for treatment of pancreatic cancer.

Targeting Platelets for the Treatment of Cancer.

The majority of cancer-associated mortality results from the ability of tumour cells to metastasise leading to multifunctional organ failure and death. Disseminated tumour cells in the blood circulation are faced with major challenges such as rheological shear stresses and cell-mediated cytotoxicity mediated by natural killer cells. Nevertheless, circulating tumour cells with metastatic ability appear equipped to exploit host cells to aid their survival. Despite the long interest in targeting tumour-associated host cells such as platelets for cancer treatment, the clinical benefit of this strategy is still under question. In this review, we provide a summary of the latest mechanistic and clinical evidence to evaluate the validity of targeting platelets in cancer.

Aging and cancer: The role of macrophages and neutrophils.

Impaired immune function has been implicated in the declining health and higher incidence of cancer in the elderly. However, age-related changes to immunity are not completely understood. Neutrophils and macrophages represent the first line of defence yet their ability to phagocytose pathogens decrease with aging. Cytotoxic T lymphocytes are critical in eliminating tumors, but T cell function is also compromised with aging. T cell responses can be regulated by macrophages and may depend on the functional phenotype macrophages adopt in response to microenvironmental signals. This can range from pro-inflammatory, anti-tumorigenic M1 to anti-inflammatory, pro-tumorigenic M2 macrophages. Macrophages in healthy elderly adipose and hepatic tissue exhibit a more pro-inflammatory M1 phenotype compared to young hosts whilst immunosuppressive M2 macrophages increase in elderly lymphoid tissues, lung and muscle. These M2-like macrophages demonstrate altered responses to stimuli. Recent studies suggest that neutrophils also regulate T cell function and, like macrophages, neutrophil function is modulated with aging. It is possible that age-modified tissue-specific macrophages and neutrophils contribute to chronic low-grade inflammation that is associated with dysregulated macrophage-mediated immunosuppression, which together are responsible for development of multiple pathologies, including cancer. This review discusses recent advances in macrophage and neutrophil biology in healthy aging and cancer.

Pancreatic Cancer-Induced Neutrophil Extracellular Traps: A Potential Contributor to Cancer-Associated Thrombosis.

Pancreatic cancer (PaCa) is a highly metastatic cancer, and patients are at high risk of developing venous thromboembolism (VTE). Neutrophil extracellular traps (NETs) have been associated with cancer metastasis and cancer-associated thrombosis, but the ability of cancer to stimulate NET release is not known. The release of NETs has been shown to be a slow process and requires reactive oxygen species (ROS) production. Studies suggest that activated platelets are important mediators in the release. Here, we show that PaCa cells can stimulate the rapid release of NETs, independently of ROS production. We further assessed the role of platelets in PaCa-induced NETs and observed a trend of increased the NET release by PaCa-primed platelets. Additionally, NETs promoted thrombus formation under venous shear stress ex vivo. Taken together, our results suggest that PaCa-induced NETs can contribute to the high risk of venous thromboembolism development in PaCa patients, and reveal NETs as a potential therapeutic target.

Epithelial-mesenchymal transition as a therapeutic target for overcoming chemoresistance in pancreatic cancer.

Pancreatic cancer has one of the worst prognoses among all cancers due to the late manifestation of identifiable symptoms and high resistance to chemo- and radiation therapies. In recent years, a cancer development phase termed epithelial-mesenchymal transition (EMT) has gained increasing research focus. The process is implicated in tumour metastasis, and emerging evidence suggests EMT also contributes or induces chemoresistance in several cancers. Nevertheless, the applicability of therapeutic targeting of EMT faces many challenges. In this mini-review, we summarise the evidence supporting the role of EMT in pancreatic cancer progression, focusing particularly on its association with chemoresistance.

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα.

BACKGROUND: Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated. OBJECTIVE: To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation. METHODS AND RESULTS: Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins. CONCLUSIONS: Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.

COMP: an endogenous thrombin inhibitor.

In this issue of Blood, Liang and colleagues demonstrate that cartilage oligomeric matrix protein (COMP) acts as a major endogenous plasma- and platelet-derived inhibitor of thrombin activity in vitro and in vivo.

Current state and novel approaches of antiplatelet therapy.

An unresolved problem with clinical use of antiplatelet therapy is that a significant number of individuals either still get thrombosis or run the risk of life-threatening bleeding. Antiplatelet drugs are widely used clinically, either chronically for people at risk of athero/thrombotic disease or to prevent thrombus formation during surgery. However, a subpopulation may be resistant to standard doses, while the platelet targets of these drugs are also critical for the normal hemostatic function of platelets. In this review, we will briefly examine current antiplatelet therapy and existing targets while focusing on new potential approaches for antiplatelet therapy and improved monitoring of effects on platelet reactivity in individuals, ultimately to improve antithrombosis with minimal bleeding. Primary platelet adhesion-signaling receptors, glycoprotein (GP)Ib-IX-V and GPVI, that bind von Willebrand factor/collagen and other prothrombotic factors are not targeted by drugs in clinical use, but they are of particular interest because of their key role in thrombus formation at pathological shear.

The functional role of platelets in the regulation of angiogenesis.

Functionally, platelets are primarily recognized as key regulators of thrombosis and hemostasis. Upon vessel injury, the typically quiescent platelet interacts with subendothelial matrix to regulate platelet adhesion, activation and aggregation, with subsequent induction of the coagulation cascade forming a thrombus. Recently, however, newly described roles for platelets in the regulation of angiogenesis have emerged. Platelets possess an armory of pro- and anti-angiogenic proteins, which are actively sequestered and highly organized in α-granule populations. Platelet activation facilitates their release, eliciting potent angiogenic responses through mechanisms that appear to be tightly regulated. In conjunction, the release of platelet-derived phospholipids and microparticles has also earned merit as synergistic regulators of angiogenesis. Consequently, platelets have been functionally implicated in a range of angiogenesis-dependent processes, including physiological roles in wound healing, vascular development and blood/lymphatic vessel separation, whilst facilitating aberrant angiogenesis in a range of diseases including cancer, atherosclerosis and diabetic retinopathy. Whilst the underlying mechanisms are only starting to be elucidated, significant insights have been established, suggesting that platelets represent a promising therapeutic strategy in diseases requiring angiogenic modulation. Moreover, anti-platelet therapies targeting thrombotic complications also exert protective effects in disorders characterized by persistent angiogenesis.

Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

BACKGROUND: We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI. AIMS: To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway. METHODS AND RESULTS: Human and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P) surface expression) and integrin αIIbß3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton's tyrosine kinase (Btk) and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbß3 activation. CONCLUSION: Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.