Jessica Cedervall

Neutrophil Extracellular Traps Accumulate in Peripheral Blood Vessels and Compromise Organ Function in Tumor-Bearing Animals

Cancer produces a variety of collateral effects in patients beyond the malignancy itself, including threats to distal organ functions. However, the basis for such effects, associated with either primary or metastatic tumors, are generally poorly understood. In this study, we show how heart and kidney vascular function is impaired by neutrophils that accumulate in those tissues as a result of tumor formation in two different transgenic mouse models of cancer (RIP1-Tag2 model of insulinoma and MMTV-PyMT model of breast cancer). Neutrophil depletion by systemic administration of an anti-Gr1 antibody improved vascular perfusion and prevented vascular leakage in kidney vessels. We also observed the accumulation of platelet-neutrophil complexes, a signature of neutrophil extracellular traps (NET), in the kidneys of tumor-bearing mice that were completely absent from healthy nontumor-bearing littermates. NET accumulation in the vasculature was associated with upregulation of the proinflammatory adhesion molecules ICAM-1, VCAM-1, and E-selectin, as well as the proinflammatory cytokines IL1β, IL6, and the chemokine CXCL1. Administering DNase I to dissolve NETs, which have a high DNA content, restored perfusion in the kidney and heart to levels seen in nontumor-bearing mice, and also prevented vessel leakage in the blood vasculature of these organs. Taken together, our findings strongly suggest that NETs mediate the negative collateral effects of tumors on distal organs, acting to impair vascular function, and to heighten inflammation at these sites.

Tumor-Induced NETosis as a Risk Factor for Metastasis and Organ Failure

A large proportion of cancer-related deaths are caused by thrombosis and general organ failure. One example is acute renal failure, a major cause of morbidity and mortality in cancer patients. Surprisingly, however, little is known about the situation in organs that are not targets for metastasis or affected by the primary tumor. Recently, neutrophil extracellular traps (NET) were implicated in tumor-induced effects on distant organs unaffected by the actual tumor cells. Formation of NETs (NETosis) was identified a decade ago as a mechanism by which the innate immune system protects us from infections, especially in situations with sepsis. NETs are formed when neutrophils externalize their nuclear DNA together with antimicrobial granule proteins and form a web-like structure that can trap and kill microbes. It is now becoming increasingly clear that NETs also form under noninfectious inflammatory conditions like cancer, thrombosis, autoimmunity, and diabetes and significantly contribute to disease development. The existence of NET-dissolving drugs like heparin and DNase I, already in clinical use, and recent development of specific inhibitors of protein-arginine deiminase 4 (PAD4), an enzyme required for NET formation, should enable clinical targeting of NETosis. Preventing NETosis in cancer could provide a strategy to counteract tumor-induced thrombosis and organ failure as well as to suppress metastasis. Cancer Res; 76(15); 4311-5. ©2016 AACR.

Enhanced Platelet Activation Mediates the Accelerated Angiogenic Switch in Mice Lacking Histidine-Rich Glycoprotein

Background The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice. Methodology/Principal Findings To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG−/− mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG+/+ littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice. Conclusions Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.

Ras and TGF-β signaling enhance cancer progression by promoting the ΔNp63 transcriptional program

In tumors with mutant p53, both Ras and TGF-β promote metastatic disease by stimulating ΔNp63 activity. Metastatic convergence at ΔNp63 The p53 family of transcription factors, which includes p53, p63, and p73, is implicated in both tumor-suppressive and tumorigenic functions. Activation of the Ras and transforming growth factor–β (TGF-β) signaling pathways are similarly enigmatic with both tumor-suppressing and tumor-promoting activity. Deciphering their roles in various stages of tumor development is critical to developing targeted therapeutics. Vasilaki et al. found that these factors are all connected in the development of some cancers. Activation of Ras or TGF-β signaling stimulated the transcriptional activity of the isoform ΔNp63 in breast or squamous cancer cells by suppressing the abundance of the mutant form of p53, an inhibitor of ΔNp63 and also a feature implicated in early tumorigenesis. Increased abundance of ΔNp63 in breast cancer cells stimulated metastatic behaviors in culture and in mice and correlated with poor prognosis in patients with mutant p53–positive tumors. These findings provide some insight into dichotic, stage-specific signals in tumorigenesis. Identification of this downstream common effector could also offer new therapeutic opportunities for some advanced Ras- or TGF-β–driven tumors. The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context–dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal–truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor–β (TGF-β) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-β–induced or epidermal growth factor (which activates Ras)–induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-β signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program.

NETosis in Cancer - Platelet-Neutrophil Crosstalk Promotes Tumor-Associated Pathology.

It has become increasingly clear that circulating immune cells in the body have a major impact on cancer development, progression, and outcome. The role of both platelets and neutrophils as independent regulators of various processes in cancer has been known for long, but it has quite recently emerged that the platelet–neutrophil interplay is yet a critical component to take into account during malignant disease. It was reported a few years ago that neutrophils in mice with cancer have increased propensity to form neutrophil extracellular traps (NETs) – web-like structures formed by externalized chromatin and secreted proteases. The initial finding describing this as a cell death-associated process has been followed by reports of additional mechanisms for NET formation (NETosis), and it has been shown that similar structures can be formed also without lysis and neutrophil cell death as a consequence. Furthermore, presence of NETs in humans with cancer has been verified in a few recent studies, indicating that tumor-induced NETosis is clinically relevant. Several reports have also described that NETs contribute to cancer-associated pathology, by promoting processes responsible for cancer-related death such as thrombosis, systemic inflammation, and relapse of the disease. This review summarizes current knowledge about NETosis in cancer, including the role of platelets as regulators of tumor-induced NETosis. It has been shown that platelets can serve as inducers of NETosis, and the platelet–neutrophil interface can therefore be an important issue to consider when designing therapies targeting cancer-associated pathology in the future.

Tumor-Induced Local and Systemic Impact on Blood Vessel Function

Endothelial dysfunction plays a role in several processes that contribute to cancer-associated mortality. The vessel wall serves as a barrier for metastatic tumor cells, and the integrity and activation status of the endothelium serves as an important defense mechanism against metastasis. In addition, leukocytes, such as cytotoxic T-cells, have to travel across the vessel wall to enter the tumor tissue where they contribute to killing of cancer cells. Tumor cells can alter the characteristics of the endothelium by recruitment of leukocytes such as neutrophils and macrophages, which further stimulate inflammation and promote tumorigenesis. Recent findings also suggest that leukocyte-mediated effects on vascular function are not limited to the primary tumor or tissues that represent metastatic sites. Peripheral organs, such as kidney and heart, also display impaired vascular function in tumor-bearing individuals, potentially contributing to organ failure. Here, we discuss how vascular function is altered in malignant tissue and distant organs in individuals with cancer and how leukocytes function as potent mediators of these tumor-induced effects.

Vaccines targeting self-antigens: mechanisms and efficacy-determining parameters

We recently showed that it is possible to compromise tumor vessel function and, as a consequence, suppress growth of aggressive preclinical tumors by immunizing against the tumor vascular markers extra domain‐A (ED‐A) or ‐B (ED‐B) of fibronectin, using a fusion protein consisting of the ED‐A or ED‐B peptide fused to bacterial thioredoxin. To address the mechanism behind fusion protein‐induced immunization and the specific contribution of the different vaccine constituents to elicit an anti‐self‐antibody response, we immunized mice with modified or unmodified self‐antigens, combined with different adjuvant components, and analyzed antibody responses by ELISA in sera. Several essential requirements to circumvent tolerance were identified: (1) a potent pattern recognition receptor agonist like an oligonucleotide containing unmethylated cytosine and guanine dinucleotides (CpG); (2) a depot adjuvant to keep the CpG at the site of injection; and (3) the presence of foreign sequences in the vaccine protein. Lack of either of these factors abolished the anti‐self‐response (P= 0.008). In mice genetically deficient for type I IFN signaling, there was a 60% reduction in the anti‐self‐response compared with wild‐type (P= 0.011), demonstrating a key role of this pathway in CpG‐induced circumvention of self‐tolerance. Identification of these mechanistic requirements to generate a potent anti‐self‐immune response should significandy aid the design of efficient, specific, and safe therapeutic cancer vaccines.—Saupe, F., Huijbers, E. J. M., Hein, T., Femel, J., Cedervall, J., Olsson, A.‐K., Hellman, L. Vaccines targeting self‐antigens: mechanisms and efficacy‐determining parameters. FASEB J. 29, 3253‐3262 (2015). www.fasebj.org

Pharmacological targeting of peptidylarginine deiminase 4 prevents cancer-associated kidney injury in mice

ABSTRACT Renal insufficiency is a frequent cancer-associated problem affecting more than half of all cancer patients at the time of diagnosis. To minimize nephrotoxic effects the dosage of anticancer drugs are reduced in these patients, leading to sub-optimal treatment efficacy. Despite the severity of this cancer-associated pathology, the molecular mechanisms, as well as therapeutic options, are still largely lacking. We here show that formation of intravascular tumor-induced neutrophil extracellular traps (NETs) is a cause of kidney injury in tumor-bearing mice. Analysis of clinical biomarkers for kidney function revealed impaired creatinine clearance and elevated total protein levels in urine from tumor-bearing mice. Electron microscopy analysis of the kidneys from mice with cancer showed reversible pathological signs such as mesangial hypercellularity, while permanent damage such as fibrosis or necrosis was not observed. Removal of NETs by treatment with DNase I, or pharmacological inhibition of the enzyme peptidylarginine deiminase 4 (PAD4), was sufficient to restore renal function in mice with cancer. Tumor-induced systemic inflammation and impaired perfusion of peripheral vessels could be reverted by the PAD4 inhibitor. In conclusion, the current study identifies NETosis as a previously unknown cause of cancer-associated renal dysfunction and describes a novel promising approach to prevent renal failure in individuals with cancer.

Tropism of the in situ growth from biopsies of childhood neuroectodermal tumors following transplantation into experimental teratoma.

Experimental teratoma induced from human pluripotent stem cells with normal karyotype can be described as a failed embryonic process and includes besides advanced organoid development also large elements of tissue with a prolonged occurrence of immature neural components. Such immature components, although benign, exhibit strong morphological resemblance with tumors of embryonic neuroectodermal origin. Here, we demonstrate that biopsy material from childhood tumors of neural embryonic origin transplanted to mature experimental teratoma can show an exclusive preference for matching tissue. Tumor specimens from five children with; Supratentorial primitive neuroectodermal tumor (sPNET); Pilocytic astrocytoma of the brainstem; Classic medulloblastoma; peripheral primitive neuroectodermal tumor (pPNET) or neuroblastoma (NB), respectively, were transplanted. Analysis of up to 120 sections of each tumor revealed an engraftment for three of the transplanted tumors: pPNET, sPNET, and NB, with a protruding growth from the latter two that were selected for detailed examination. The histology revealed a strict tropism with a non‐random integration into what morphologically appeared as matched embryonic microenvironment recuperating the patient tumor histology. The findings suggest specific advantages over xenotransplantation and lead us to propose that transplantation to the human embryonic microenvironment in experimental teratoma can be a well‐needed complement for preclinical in vivo studies of childhood neuroectodermal tumors.

Platelet Regulation of Angiogenesis, Tumor Growth and Metastasis

Angiogenesis formation of new capillary blood vessels is essential during development and physiological conditions, such as wound healing and the reproductive cycle. Prolonged and excessive angiogenesis has been implicated in a number of pathological processes, for instance rheumatoid arthritis, retinopathy and tumor growth. The normal vasculature is tightly regulated by a balance between proand anti-angiogenic factors. The most well studied pro-angiogenic factor vascular endothelial growth factor-A(VEGF-A) – is required for development of a vascular system during embryogenesis and is also a central regulator of adult neovascularization (Olsson et al., 2006). Angiogenesis is a multistep process involving oxygen sensing, growth factor signaling, matrix degradation, endothelial cell proliferation, migration and differentiation into a functional blood vessel. This process formation of new blood vessels from pre-existing ones must take place without compromising blood flow. Platelets are central players in maintaining hemostasis of the blood. At sites of blood vessel injury, platelets are activated to induce blood coagulation and form aggregates at the site of the damaged endothelium to prevent hemorrhage and thereby protects us from fatal bleedings. Besides their role in hemostasis, platelets have been shown to contribute to nonhemostatic processes such as wound healing, immunity, angiogenesis, cardiovascular disease and tumor metastasis (Felding-Habermann et al., 1996; Jurk and Kehrel, 2005). A connection between platelets and malignant disease has been recognized since the end of the 19th century, when Armand Trousseau observed increased thrombotic events in patients that were later diagnosed with cancer (Trousseau, 1865). This enhanced tendency to form blood clots, or hypercoagulability, is named Trousseau’s syndrome (Varki, 2007) and is especially pronounced in certain forms of cancer such as pancreatic and lung cancer. Growth of solid tumors, like all expanding tissues, is dependent on angiogenesis for oxygen and nutrient supply, as well as for removal of waste products. The hypotheses that platelets contribute to tumor-induced angiogenesis was put forward by Pinedo and collegues in 1998 (Pinedo et al., 1998). During the last decade, this hypotheses has been experimentally supported by several independent research groups, demonstrating that platelets can regulate endothelial cell behavior and angiogenesis. Platelets are now recognized as the major source of VEGF-A in the body (Holmes et al., 2008; Peterson et al., 2010; Verheul et al., 1997). In addition to stimulation of tumor growth and angiogenesis, platelets have also been found to regulate metastasis. Possible explanations involve protection of the tumor cells