Jonathan Spicer

Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis

The majority of patients with cancer undergo at least one surgical procedure as part of their treatment. Severe postsurgical infection is associated with adverse oncologic outcomes; however, the mechanisms underlying this phenomenon are unclear. Emerging evidence suggests that neutrophils, which function as the first line of defense during infections, facilitate cancer progression. Neutrophil extracellular traps (NETs) are extracellular neutrophil-derived DNA webs released in response to inflammatory cues that trap and kill invading pathogens. The role of NETs in cancer progression is entirely unknown. We report that circulating tumor cells become trapped within NETs in vitro under static and dynamic conditions. In a murine model of infection using cecal ligation and puncture, we demonstrated microvascular NET deposition and consequent trapping of circulating lung carcinoma cells within DNA webs. NET trapping was associated with increased formation of hepatic micrometastases at 48 hours and gross metastatic disease burden at 2 weeks following tumor cell injection. These effects were abrogated by NET inhibition with DNAse or a neutrophil elastase inhibitor. These findings implicate NETs in the process of cancer metastasis in the context of systemic infection and identify NETs as potential therapeutic targets.

A systematic review of the effects of resident duty hour restrictions in surgery: impact on resident wellness, training, and patient outcomes.

Background:In 2003, the Accreditation Council for Graduate Medical Education (ACGME) mandated 80-hour resident duty limits. In 2011 the ACGME mandated 16-hour duty maximums for PGY1 (post graduate year) residents. The stated goals were to improve patient safety, resident well-being, and education. A systematic review and meta-analysis were performed to evaluate the impact of resident duty hours (RDH) on clinical and educational outcomes in surgery. Methods:A systematic review (1980–2013) was executed on CINAHL, Cochrane Database, Embase, Medline, and Scopus. Quality of articles was assessed using the GRADE guidelines. Sixteen-hour shifts and night float systems were analyzed separately. Articles that examined mortality data were combined in a random-effects meta-analysis to evaluate the impact of RDH on patient mortality. Results:A total of 135 articles met the inclusion criteria. Among these, 42% (N = 57) were considered moderate-high quality. There was no overall improvement in patient outcomes as a result of RDH; however, some studies suggest increased complication rates in high-acuity patients. There was no improvement in education related to RDH restrictions, and performance on certification examinations has declined in some specialties. Survey studies revealed a perception of worsened education and patient safety. There were improvements in resident wellness after the 80-hour workweek, but there was little improvement or negative effects on wellness after 16-hour duty maximums were implemented. Conclusions:Recent RDH changes are not consistently associated with improvements in resident well-being, and have negative impacts on patient outcomes and performance on certification examinations. Greater flexibility to accommodate resident training needs is required. Further erosion of training time should be considered with great caution.

Neutrophils promote liver metastasis via Mac-1 mediated interactions with circulating tumor cells.

Although circulating neutrophils are associated with distant metastasis and poor outcome in a number of epithelial malignancies, it remains unclear whether neutrophils play an active causal role in the metastatic cascade. Using in vivo models of metastasis, we found that neutrophils promote cancer cell adhesion within liver sinusoids and, thereby, influence metastasis. Neutrophil depletion before cancer cell inoculation resulted in a decreased number of gross metastases in an intrasplenic model of liver metastasis. This effect was reversed when inflamed neutrophils were co-inoculated with cancer cells. In addition, early adhesion within liver sinusoids was inhibited in the absence of neutrophils and partially restored with a short perfusion of isolated activated neutrophils. Intravital microscopy showed that cancer cells adhered directly on top of arrested neutrophils, indicating that neutrophils may act as a bridge to facilitate interactions between cancer cells and the liver parenchyma. The adhesion of lipopolysaccharide-activated neutrophils to cancer cells was mediated by neutrophil Mac-1/ICAM-1. Our findings, therefore, show a novel role for neutrophils in the early adhesive steps of liver metastasis.

LPS-Induced TLR4 Signaling in Human Colorectal Cancer Cells Increases β1 Integrin-Mediated Cell Adhesion and Liver Metastasis

Infectious complications resulting from resection of colorectal cancer (CRC) elevates the risk of cancer recurrence and metastasis, but the reason for this risk relationship is unknown. Defining the mechanisms responsible may offer opportunities to improve outcomes in a majority of patients whose tumors are resected as part of their therapy. The complex formed between Toll receptor TLR4 and myeloid differentiation factor MD2 defines a major cell surface receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen that has been implicated in infectious complications after CRC resection. As the TLR4/MD2 complex is expressed on CRC cells, we hypothesized that LPS may promote liver metastasis in CRC by stimulating TLR4 signaling. In support of this hypothesis, we report here that LPS enhances liver metastasis of human CRC cells that express TLR4/MD2 after intrasplenic graft of immunocompromised nude mice. Compared with TLR4 nonexpressing, nonmetastatic CRC cells, we observed increased in vitro adherence to different extracellular matrices and human umbilical vein endothelial cells (HUVEC). Furthermore, we observed an increased likelihood of in vivo capture within hepatic sinusoids after LPS treatment. No differences were apparent in phosphorylation of p38 and MAPK isoforms, but in metastatic CRC cells expressing surface TLR4 treatment with LPS increased Ser473 phosphorylation of AKT kinase. We showed that enhanced adherence elicited by LPS in these cells could be blocked at three different levels, using Eritoran (TLR4 small molecule antagonist), PI-103 (PI3K inhibitor), or anti-β1 integrin blocking antibodies. Taken together, the results indicate that stimulation of the TLR4/MD2 complex by LPS activates PI3K/AKT signaling and promotes downstream β1 integrin function, thereby increasing the adhesiveness and metastatic capacity of CRC cells. Our findings suggest that inhibiting LPS-induced TLR4 signaling could improve therapeutic outcomes by preventing cancer metastasis during the perioperative period of CRC resection.

Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP‐labeled Lewis lung carcinoma subline H‐59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide‐induced increase in H‐59 cell adhesion. Although direct selectin–selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver.

Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

ABSTRACT We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

Neutrophil extracellular traps in cancer progression.

Neutrophils are being increasingly recognized as an important element in tumor progression. They have been shown to exert important effects at nearly every stage of tumor progression with a number of studies demonstrating that their presence is critical to tumor development. Novel aspects of neutrophil biology have recently been elucidated and its contribution to tumorigenesis is only beginning to be appreciated. Neutrophil extracellular traps (NETs) are neutrophil-derived structures composed of DNA decorated with antimicrobial peptides. They have been shown to trap and kill microorganisms, playing a critical role in host defense. However, their contribution to tumor development and metastasis has recently been demonstrated in a number of studies highlighting NETs as a potentially important therapeutic target. Here, studies implicating NETs as facilitators of tumor progression and metastasis are reviewed. In addition, potential mechanisms by which NETs may exert these effects are explored. Finally, the ability to target NETs therapeutically in human neoplastic disease is highlighted.

Current Status of Management of Malignant Disease: Current Management of Esophageal Cancer

ObjectiveThe objective of this study is to outline the evidence regarding the surgical management of esophageal cancer and provide a single institutional outline regarding its implementation.BackgroundEsophageal cancer is a major cause of cancer-related morbidity and mortality worldwide. Surgery continues to play an important role in its management and offers the best chance for cure in localized and locally advanced disease. However, considerable controversy exists regarding the optimum treatment strategy in this patient population. Furthermore, despite advances in operative and perioperative care and the advent of minimally invasive approaches, the majority of patients succumb to distant metastases after curative intent resection. This failure highlights the importance of multimodal, stage-directed therapy in the management of patients with newly diagnosed esophageal tumors.MethodsHerein, we provide a comprehensive, evidence-based review of the diagnostic workup and locoregional and systemic treatment options available to esophageal cancer patients. The evidence supporting perioperative chemotherapy versus chemoradiotherapy is outlined and discussed. In addition, we highlight our institutional approach to the diagnostic evaluation, operative selection strategy, and perioperative treatment regimen selection based on the stage of presentation. Finally, we discuss the role of enhanced recovery in the postoperative management of this complex group of patients.ConclusionsEsophageal cancer remains a devastating disease with high mortality. Favorable outcomes mandate a multimodal, stage-directed treatment approach.

Neutrophil extracellular traps sequester circulating tumor cells via β1-integrin mediated interactions

Despite advances in cancer treatment, metastasis remains today the main cause of cancer death. Local control through complete surgical resection of the primary tumor continues to be a key principle in cancer treatment. However, surgical interventions themselves lead to adverse oncologic outcomes and are associated with significantly increased rates of metastasis. Neutrophils through release of neutrophil extracellular traps (NETs) in response to infections were shown to be able to capture circulating cancer cells, and in doing so, support the development of metastatic disease. To be able to intervene on this process, understanding the exact molecular nature of these mechanisms is crucial. We therefore hypothesize and demonstrate that β1‐integrin is an important factor mediating the interactions between circulating tumor cells and NETs. We show that β1‐integrin expression on both cancer cells and NETs is important for the adhesion of circulating tumor cells to NETs both in vitro and in vivo. Using a murine model of intra‐abdominal sepsis to mimic the postoperative inflammatory environment, we show that β1‐integrin expression is upregulated in the context of inflammation in vivo. Ultimately, we show that this increased early cancer cell adhesion to NETs in vivo and this effect is abrogated when mice are administered DNAse 1. Our data therefore sheds light on the first molecular mechanism by which NETs can trap circulating tumor cells (CTCs), broadening our understanding of this process.

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophils antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions. In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves. A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained. We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs.