Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Impact of Recombinant Granulocyte Colony-Stimulating Factor During Neoadjuvant Therapy on Outcomes of Resected Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by chronic inflammation and a tolerogenic immune response. The granulocyte colony-stimulating factor (G-CSF)-neutrophil axis promotes oncogenesis and progression of PDAC. Despite frequent use of recombinant G-CSF in the management and prevention of chemotherapy-induced neutropenia, its impact on oncologic outcomes of patients with resected PDAC is unclear. PATIENTS AND METHODS: This cohort study assessing the impact of G-CSF administration was conducted on 351 patients with PDAC treated with neoadjuvant therapy (NAT) and pancreatic resection at a high-volume tertiary care academic center from 2014 to 2019. Participants were identified from a prospectively maintained database and had a median follow-up of 45.8 months. RESULTS: Patients receiving G-CSF (n=138; 39.3%) were younger (64.0 vs 66.7 years; P=.008), had lower body mass index (26.5 vs 27.9; P=.021), and were more likely to receive 5-FU-based chemotherapy (42.0% vs 28.2%; P<.0001). No differences were observed in baseline or clinical tumor staging. Patients receiving G-CSF were more likely to have an elevated (>5.53) post-NAT neutrophil-to-lymphocyte ratio (45.0% vs 29.6%; P=.004). G-CSF recipients also demonstrated higher circulating levels of neutrophil extracellular traps (+709 vs -619 pg/mL; P=.006). On multivariate analysis, G-CSF treatment was associated with perineural invasion (hazard ratio [HR], 2.65; 95% CI, 1.16-6.03; P=.021) and margin-positive resection (HR, 1.67; 95% CI, 1.01-2.77; P=.046). Patients receiving G-CSF had decreased overall survival (OS) compared with nonrecipients (median OS, 29.2 vs 38.7 months; P=.001). G-CSF administration was a negative independent predictor of OS (HR, 2.02; 95% CI, 1.45-2.79; P<.0001). In the inverse probability weighted analysis of 301 matched patients, neoadjuvant G-CSF administration was associated with reduced OS. CONCLUSIONS: In patients with localized PDAC receiving NAT prior to surgical extirpation, G-CSF administration may be associated with worse oncologic outcomes and should be further evaluated.

AllergoOncology: Danger signals in allergology and oncology: A European Academy of Allergy and Clinical Immunology (EAACI) Position Paper.

The immune system interacts with many nominal 'danger' signals, endogenous danger-associated (DAMP), exogenous pathogen (PAMP) and allergen (AAMP)-associated molecular patterns. The immune context under which these are received can promote or prevent immune activating or inflammatory mechanisms and may orchestrate diverse immune responses in allergy and cancer. Each can act either by favouring a respective pathology or by supporting the immune response to confer protective effects, depending on acuity or chronicity. In this Position Paper under the collective term danger signals or DAMPs, PAMPs and AAMPs, we consider their diverse roles in allergy and cancer and the connection between these in AllergoOncology. We focus on their interactions with different immune cells of the innate and adaptive immune system and how these promote immune responses with juxtaposing clinical outcomes in allergy and cancer. While danger signals present potential targets to overcome inflammatory responses in allergy, these may be reconsidered in relation to a history of allergy, chronic inflammation and autoimmunity linked to the risk of developing cancer, and with regard to clinical responses to anti-cancer immune and targeted therapies. Cross-disciplinary insights in AllergoOncology derived from dissecting clinical phenotypes of common danger signal pathways may improve allergy and cancer clinical outcomes.

Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells.

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.

Fighting Fire With Fire: Oncolytic Virotherapy for Thoracic Malignancies.

Thoracic malignancies are associated with high mortality rates. Conventional therapy for many of the patients with thoracic malignancies is obviated by a high incidence of locoregional recurrence and distant metastasis. Fortunately, developments in immunotherapy provide effective strategies for both local and systemic treatments that have rapidly advanced during the last decade. One promising approach to cancer immunotherapy is to use oncolytic viruses, which have the advantages of relatively high tumor specificity, selective replication-mediated oncolysis, enhanced antigen presentation, and potential for delivery of immunogenic payloads such as cytokines, with subsequent elicitation of effective antitumor immunity. Several oncolytic viruses including adenovirus, coxsackievirus B3, herpes virus, measles virus, reovirus, and vaccinia virus have been developed and applied to thoracic cancers in preclinical murine studies and clinical trials. This review discusses the current state of oncolytic virotherapy in lung cancer, esophageal cancer, and metastatic malignant pleural effusions and considers its potential as an emergent therapeutic for these patients.

Outcomes of Neoadjuvant Chemotherapy Versus Chemoradiation in Localized Pancreatic Cancer: A Case-Control Matched Analysis.

BACKGROUND: Neoadjuvant therapy is increasingly used for patients with pancreatic ductal adenocarcinoma (PDAC). It is unknown whether neoadjuvant chemoradiotherapy is more effective than chemotherapy (NCRT vs. NAC). We aim to compare pathological and survival outcomes of NCRT and NAC in patients with PDAC. PATIENTS AND METHODS: Single-center analysis of PDAC patients treated with NCRT or NAC followed by resection between December 2008 and December 2018 was performed. Average treatment effect (ATE) was estimated after case-control matching using Mahalanobis distance nearest-neighbor matching. Inverse probability weighted estimates (IPWE)-based ATE was estimated for disease-free survival (DFS) and overall survival (OS). RESULTS: Among the 418 patients (mean age 66.8 years, 51% female) included in the study, 327 received NAC and 91 received NCRT. NCRT patients had higher rates of locally advanced disease, number of neoadjuvant chemotherapy cycles, more chemotherapy regimen crossover (gemcitabine and 5-FU based), and were more likely to undergo open surgical procedures and/or vascular resection (all p < 0.05). After matched analysis, NCRT was associated with a significant reduction in lymph node positive disease [ATE = (-)0.24, p = 0.007] and lymphovascular invasion [ATE = (-)0.20, p = 0.02]. While NCRT was associated with significantly improved DFS by 9.5 months (p = 0.006), it did not affect OS by IPWE-based ATE after adjusting for adjuvant therapy (ATE = 5.5 months; p = 0.32). CONCLUSION: Compared with NAC alone, NCRT is associated with improved pathologic surrogates and disease-free survival, but not overall survival in patients with PDAC.

Boning up: amino-bisphophonates as immunostimulants and endosomal disruptors of dendritic cell in SARS-CoV-2 infection.

Amino-bisphosphonates such as zoledronic acid (ZA) can possibly ameliorate or prevent severe COVID-19 disease by at least three distinct mechanisms: (1) as immunostimulants which could boost γδ T cell expansion, important in the acute response in the lung; (2) as DC modulators, limiting their ability to only partially activate T cells; and (3) as prenylation inhibitors of small GTPases in the endosomal pathway of the DC to prevent expulsion of lysosomes containing SARS-CoV-2 virions. Use of ZA or other amino-bisphosphonates as modulators of COVID-19 disease should be considered.

Prognostic Value of the Systemic Immune-Inflammation Index (SII) After Neoadjuvant Therapy for Patients with Resected Pancreatic Cancer.

BACKGROUND: The systemic immune-inflammation index (SII), calculated using absolute platelet, neutrophil, and lymphocyte counts, has recently emerged as a predictor of survival for patients with pancreatic ductal adenocarcinoma (PDAC) when assessed at diagnosis. Neoadjuvant therapy (NAT) is increasingly used in the treatment of PDAC. However, biomarkers of response are lacking. This study aimed to determine the prognostic significance of SII before and after NAT and its association with the pancreatic tumor biomarker carbohydrate-antigen 19-9 (CA 19-9). METHODS: This study retrospectively analyzed all PDAC patients treated with NAT before pancreatic resection at a single institution between 2007 and 2017. Pre- and post-NAT lab values were collected to calculate SII. Absolute pre-NAT, post-NAT, and change in SII after NAT were evaluated for their association with clinical outcomes. RESULTS: The study analyzed 419 patients and found no significant correlation between pre-NAT SII and clinical outcomes. Elevated post-NAT SII was an independent, negative predictor of overall survival (OS) when assessed as a continuous variable (hazard ratio [HR], 1.0001; 95% confidence interval [CI] 1.00003-1.00014; p = 0.006). Patients with a post-NAT SII greater than 900 had a shorter median OS (31.9 vs 26.1 months; p = 0.050), and a post-NAT SII greater than 900 also was an independent negative predictor of OS (HR, 1.369; 95% CI 1.019-1.838; p = 0.037). An 80% reduction in SII independently predicted a CA 19-9 response after NAT (HR, 4.22; 95% CI 1.209-14.750; p = 0.024). CONCLUSION: Post-treatment SII may be a useful prognostic marker in PDAC patients receiving NAT.

Characteristics of Malignant Pleural Effusion Resident CD8+ T Cells from a Heterogeneous Collection of Tumors.

While T cell-based cancer immunotherapies have shown great promise, there remains a need to understand how individual metastatic tumor environments impart local T cell dysfunction. At advanced stages, cancers that metastasize to the pleural space can result in a malignant pleural effusion (MPE) that harbors abundant tumor and immune cells, often exceeding 108 leukocytes per liter. Unlike other metastatic sites, MPEs are readily and repeatedly accessible via indwelling catheters, providing an opportunity to study the interface between tumor dynamics and immunity. In the current study, we examined CD8+ T cells within MPEs collected from patients with heterogeneous primary tumors and at various stages in treatment to determine (1) if these cells possess anti-tumor activity following removal from the MPE, (2) factors in the MPE that may contribute to their dysfunction, and (3) the phenotypic changes in T cell populations that occur following ex vivo expansion. Co-cultures of CD8+ T cells with autologous CD45- tumor containing cells demonstrated cytotoxicity (p = 0.030) and IFNγ production (p = 0.003) that inversely correlated with percent of myeloid derived suppressor cells, lactate, and lactate dehydrogenase (LDH) within the MPE. Ex vivo expansion of CD8+ T cells resulted in progressive differentiation marked by distinct populations expressing decreased CD45RA, CCR7, CD127, and increased inhibitory receptors. These findings suggest that MPEs may be a source of tumor-reactive T cells and that the cellular and acellular components suppress optimal function.

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice.

BACKGROUND & AIMS: Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. METHODS: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. RESULTS: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. CONCLUSIONS: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia.

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3-/-) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1ß (IL-1ß), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3-/- mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.

High mobility group protein B1 controls liver cancer initiation through yes-associated protein -dependent aerobic glycolysis.

Emerging studies have suggested that the Hippo pathway is involved in the tumorigenesis of hepatocellular carcinoma (HCC). However, the key regulator of the Hippo pathway in liver tumor metabolic reprogramming remains elusive. Here, we provide evidence that high mobility group box 1 (HMGB1), a chromosomal protein, plays a role in the regulation of the Hippo pathway during liver tumorigenesis. Cre/loxP recombination-mediated HMGB1 depletion in hepatocytes blocks diethylnitrosamine-induced liver cancer initiation in mice, whereas short hairpin RNA-mediated gene silencing of HMGB1 inhibits HCC cell proliferation. Mechanistically, the binding of HMGB1 to GA-binding protein alpha promotes the expression of yes-associated protein (YAP), a major downstream effector of the Hippo pathway that contributes to liver tumorigenesis by inducing hypoxia-inducible factor 1α (HIF1α)-dependent aerobic glycolysis. Like wild-type YAP-complementary DNA, YAP-5SA-S94A can restore HIF1α DNA binding activity, glycolysis-associated gene expression, and HIF1α-YAP complex formation in YAP-knockdown HCC cell lines. In contrast, verteporfin, a reagent targeting the interface between YAP and TEA domain transcription factor, has the ability to block YAP-HIF1α complex formation. Notably, genetic or pharmacologic inhibition of the HMGB1-YAP-HIF1α pathway confers protection against excessive glycolysis and tumor growth in mice. CONCLUSION: These findings demonstrate that HMGB1 plays a novel role in modulating the YAP-dependent HIF1α pathway and shed light on the development of metabolism-targeting therapeutics for HCC chemoprevention. (Hepatology 2018;67:1823-1841).

The platelet NLRP3 inflammasome is upregulated in a murine model of pancreatic cancer and promotes platelet aggregation and tumor growth.

Platelets are activated in solid cancers, including pancreatic ductal adenocarcinoma (PDA), a highly aggressive malignancy with a devastating prognosis and limited therapeutic options. The mechanisms by which activated platelets regulate tumor progression are poorly understood. The nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) inflammasome is a key inflammatory mechanism recently identified in platelets, which controls platelet activation and aggregation. In an orthotopic PDA mouse model involving surgical implantation of Panc02 murine cancer cells into the tail of the pancreas, we show that the NLRP3 inflammasome in circulating platelets is upregulated in pancreatic cancer. Pharmacological inhibition or genetic ablation of NLRP3 in platelets resulted in decreased platelet activation, platelet aggregation, and tumor progression. Moreover, interfering with platelet NLRP3 signaling significantly improved survival of tumor-bearing mice. Hence, the platelet NLRP3 inflammasome plays a critical role in PDA and might represent a novel therapeutic target.

Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells: an in vitro experimental study.

PURPOSE: The loss of nutrient supply is a suspected contributor of intervertebral disc degeneration. However, the extent to which low nutrition affects disc annulus fibrosus (AF) cells is unknown as nutrient deprivation has mainly been investigated in disc nucleus pulposus cells. Hence, an experimental study was designed to clarify the effects of limited nutrients on disc AF cell fate, including autophagy, the process by which cells recycle their own damaged components. METHODS: Rabbit disc AF cells were cultured in different media with varying serum concentrations under 5% oxygen. Cellular responses to changes in serum and nutrient concentrations were determined by measuring proliferation and metabolic activity. Autophagic flux in AF cells was longitudinally monitored using imaging cytometry and Western blotting for LC3, HMGB1, and p62/SQSTM1. Apoptosis (TUNEL staining and cleaved caspase-3 immunodetection) and cellular senescence (senescence-associated ß-galactosidase assay and p16/INK4A immunodetection) were measured. RESULTS: Markers of apoptosis and senescence increased, while cell proliferation and metabolic activity decreased under the withdrawal of serum and of nutrients other than oxygen, confirming cellular stress. Time-dependent increases in autophagy markers, including LC3 puncta number per cell, LC3-II expression, and cytoplasmic HMGB1, were observed under conditions of reduced nutrition, while an autophagy substrate, p62/SQSTM1, decreased over time. Collectively, these findings suggest increased autophagic flux in disc AF cells under serum and nutrient deprivation. CONCLUSION: Disc AF cells exhibit distinct responses to serum and nutrient deprivation. Cellular responses include cell death and quiescence in addition to reduced proliferation and metabolic activity, as well as activation of autophagy under conditions of nutritional stress. These slides can be retrieved under Electronic Supplementary Material.

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps.

BACKGROUND: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. METHODS: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated. RESULTS: The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE. CONCLUSION: NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer. TRIAL REGISTRATION: This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Extracellular DNA promotes colorectal tumor cell survival after cytotoxic chemotherapy.

BACKGROUND: Inflammation promotes the growth and survival of malignant cells. Inflammation within the tumor microenvironment is a result of damage-associated molecular patterns released by dead or dying cells that provide survival signals to the surrounding cells. It has been proposed that extracellular DNA can act as a damage-associated molecular pattern given the association between circulating DNA and autoimmune diseases. Herein, we demonstrate a novel role for genomic extracellular DNA binding to the Toll-like receptor (TLR)-9 on tumor cells in response to cytotoxic insult. MATERIALS AND METHODS: The colorectal tumor cell line HCCT116 was used to study the role of DNA in tumor cell response to chemotherapy. Cell viability was assessed using CCK-8 assay. Cell death mechanisms were assessed by YOYO-1 and lactate dehydrogenase staining for necrosis and TUNEL staining for apoptosis. Autophagy was measured by LC3 punctate formation. TLR9-short hairpin RNA was used to knockdown TLR-9 and determine its role in tumor cell response to DNA. RESULTS: DNA is released from necrotic tumor cells after chemotherapy. Survival after cytotoxic insult is enhanced by the presence of extracellular DNA as a result of inhibition of apoptosis and enhanced autophagy. Knockdown of TLR-9 enhanced apoptosis, diminished autophagy, and decreased survival after cytotoxic insult in the presence or absence of extracellular DNA. CONCLUSIONS: DNA in the tumor microenvironment promotes survival through induction of autophagy via TLR-9 signaling. This work has important implications for targeting extracellular DNA, TLR-9, and autophagy during treatment with chemotherapy and enhances our understanding of the role of extracellular DNA in the tumor microenvironment.

The Receptor for Advanced Glycation End Products Activates the AIM2 Inflammasome in Acute Pancreatitis.

Severe acute pancreatitis (AP) is responsible for significant human morbidity and mortality worldwide. Currently, no specific treatments for AP exist, primarily due to the lack of a mechanistic understanding of sterile inflammation and the resultant multisystem organ dysfunction, the pathologic response of AP linked to early death. In this study, we demonstrate that the class III major histocompatibility region III receptor for advanced glycation end products (RAGE) contributes to AP by modulating inflammasome activation in macrophages. RAGE mediated nucleosome-induced absent in melanoma 2 (but not NLRP3) inflammasome activation by modulating dsRNA-dependent protein kinase phosphorylation in macrophages. Pharmacological and genetic inhibition of the RAGE-dsRNA-dependent protein kinase pathway attenuated the release of inflammasome-dependent exosomal leaderless cytokines (e.g., IL-1ß and high-mobility group box 1) in vitro. RAGE or absent in melanoma 2 depletion in mice limited tissue injury, reduced systemic inflammation, and protected against AP induced by l-arginine or cerulein in experimental animal models. These findings define a novel role for RAGE in the propagation of the innate immune response with activation of the nucleosome-mediated inflammasome and will help guide future development of therapeutic strategies to treat AP.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells.

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a "high" density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs. At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NKIL-12) produced high amounts of IFNγ. The addition of a low number of A-NKIL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24-48h after adoptive transfer of A-NKIL-12 cells. OT-I-CTLs and A-NKIL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NKIL-12 cells and OT-I-CTLs lead to a significant prolongation of survival. Thus, an important function of tumor-infiltrating NK cells may be to increase tumor cell expression of MHC class-I through secretion of IFNγ, to prepare them for recognition by tumor-specific CTLs.

The NLRP3 inflammasome and bruton's tyrosine kinase in platelets co-regulate platelet activation, aggregation, and in vitro thrombus formation.

Cleavage of interleukin-1ß (IL-1ß) is a key inflammatory event in immune cells and platelets, which is mediated by nucleotide-binding domain leucine rich repeat containing protein (NLRP3)-dependent activation of caspase-1. In immune cells, NLRP3 and caspase-1 form inflammasome complexes with the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and bruton's tyrosine kinase (BTK). In platelets, however, the regulatory triggers and the functional effects of the NLRP3 inflammasome are unknown. Here, we show in vitro that the platelet NLRP3 inflammasome contributes to platelet activation, aggregation, and thrombus formation. NLRP3 activity, as monitored by caspase-1 activation and cleavage and secretion of IL-1ß, was upregulated in activated platelets, which was dependent on platelet BTK. Pharmacological inhibition or genetic ablation of BTK in platelets led to decreased platelet activation, aggregation, and in vitro thrombus formation. We identify a functionally relevant link between BTK and NLRP3 in platelets, with potential implications in disease states associated with abnormal coagulation and inflammation.