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Patients with Gorham-Stout disease (GSD) have lymphatic vessels in their bones and their bones gradually disappear. Here, we report that mice that overexpress VEGF-C in bone exhibit a phenotype that resembles GSD. To drive VEGF-C expression in bone, we generated Osx-tTA;TetO-Vegfc double-transgenic mice. In contrast to Osx-tTA mice, Osx-tTA;TetO-Vegfc mice developed lymphatics in their bones. We found that inhibition of VEGFR3, but not VEGFR2, prevented the formation of bone lymphatics in Osx-tTA;TetO-Vegfc mice. Radiological and histological analysis revealed that bones from Osx-tTA;TetO-Vegfc mice were more porous and had more osteoclasts than bones from Osx-tTA mice. Importantly, we found that bone loss in Osx-tTA; TetO-Vegfc mice could be attenuated by an osteoclast inhibitor. We also discovered that the mutant phenotype of Osx-tTA;TetO-Vegfc mice could be reversed by inhibiting the expression of VEGF-C. Taken together, our results indicate that expression of VEGF-C in bone is sufficient to induce the pathologic hallmarks of GSD in mice. © Hominick et al.

Roughly 10% - 20% of pancreatic cancer patients are candidates for curative intent surgical treatment. In the 2000s, many studies showed similar survival rates comparing pancreatic surgery with or without vein resection and reconstruction. The aim was to identify the best method of venous reconstruction.This was a retrospective cohort study. A total of 1 375 patients undergoing pancreatectomy between 2005 and 2018 were identified. Patients undergoing a combined pancreatic resection and venous reconstruction were included retrospectively. When tumour infiltration to the portal/superior mesenteric vein was detected, excision and reconstruction with tangential suturing/patch, end to end anastomosis, or a spiral graft from the great saphenous vein was performed. Next, 90 day and long term survival and outcomes across reconstruction techniques were analysed.Overall, 198 patients had venous involvement visible in pre-operative scans or detected during surgery, broken down as follows: 171 (86%) pancreaticoduodenectomy, 12 (6%) total pancreatectomy, and 15 (8%) distal pancreatectomy. In total, 69 (35%) spiral graft reconstructions, 77 (39%) end to end anastomoses, and 52 (26%) tangential/patch reconstructions were performed. Tumour histology revealed pancreatic adenocarcinomas in 162 (82%) patients, intraductal mucinous pancreatic neoplasia in 14 (7%), cholangiocarcinoma in five (3%), neuro-endocrine neoplasia in nine (5%), and eight other diagnoses. Overall, 183 (92%) were malignant and 15 (8%) benign. Two patients died within 90 days, one in hospital and one on post-operative day 38 due to thrombosis of the superior mesenteric vein and intestinal necrosis, a Clavien-Dindo grade 5 complication. In addition, 50 (23%) patients had Clavien-Dindo grade 3 - 4 complications. No differences in complications comparing vein reconstruction techniques or in the long term survival of pancreatectomy patients with or without venous reconstruction were detected.The spiral graft technique, used when more advanced venous infiltration occurs, does not increase complications, with outcomes mirroring those accompanying shorter venous resections.

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2-3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 x 10(5) PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.

Background and aims: Secretory phospholipase A(2) (PLA(2)) hydrolyzes LDL phospholipids generating modified LDL particles (PLA(2)-LDL) with increased atherogenic properties. Exocytosis of Weibel-Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation. Here, we investigated the effects of PLA(2)-LDL on exocytosis of WPBs. Methods: Human coronary artery endothelial cells (HCAECs) were stimulated with PLA(2)-LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA(2)-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. Results: Exposure of HCAECs to PLA(2)-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA(2)-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA(2)-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. Conclusions: Our studies reveal PLA(2)-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA(2)-LDL-dependent promotion of vascular inflammation and atherosclerosis.

Background: We studied the role of tumor-associated trypsin inhibitor (TATI) in serum and in tumor tissues among human papillomavirus (HPV)-positive and HPV-negative OPSCC patients. Materials and methods: The study cohort included 90 OPSCC patients treated at the Helsinki University Hospital (HUS), Helsinki, Finland, in 2012–2016. TATI serum concentrations (S-TATIs) were determined by an immunofluorometric assay. Immunostaining was used to assess tissue expression. HPV status was determined with a combination of p16 immunohistochemistry and HPV DNA PCR genotyping. The survival endpoints were overall survival (OS) and disease-specific survival (DSS). Results: A significant correlation was found between S-TATI positivity and poor OS (p &lt, 0.001) and DSS (p = 0.04) in all patients. In HPV-negative cases, S-TATI positivity was linked to poor OS (p = 0.01) and DSS (p = 0.05). In HPV-positive disease, S-TATI positivity correlated with poor DSS (p = 0.01). S-TATI positivity was strongly associated with HPV negativity. TATI serum was negatively linked to a lower cancer stage. TATI expression in peritumoral lymphocytes was associated with favorable OS (p &lt, 0.025) and HPV positivity. TATI expression in tumor and in peritumoral lymphocytes correlated with lower cancer stages. Conclusion: Our results suggest that S-TATI positivity may be a biomarker of poor prognosis in both HPV-positive and HPV-negative OPSCC.

We have previously shown that proto-oncoprotein c-Jun is activated in ornithine decarboxylase (ODC)- and RAS-transformed mouse fibroblasts, and that the transformed morphology of these cells can be reversed by expressing the transactivation domain deletion mutant of c-Jun (TAM67). Here, we found that lysyl oxidase (Lox), encoding an extracellular matrix-modifying enzyme, is downregulated in a c-Jun-dependent manner in ODC-transformed fibroblasts (Odc cells). In addition to Lox, the Lox family members Lox-like 1 and 3 (Loxl1 and Loxl3) were found to be downregulated in Odc as well as in RAS-transformed fibroblasts (E4), whereas Lox-like 4 (Loxl4) was upregulated in Odc and downregulated in E4 cells compared to normal N1 fibroblasts. Tetracycline-regulatable LOX re-expression in Odc cells led to inhibition of cell growth and invasion in three-dimensional Matrigel in an activity-independent manner. On the contrary, LOX and especially LOXL2, LOXL3, and LOXL4 were found to be upregulated in several human melanoma cell lines, and LOX inhibitor B-aminopropionitrile inhibited the invasive growth of these cells particularly when co-cultured with fibroblasts in Matrigel. Knocking down the expression of LOX and especially LOXL2 in melanoma cells almost completely abrogated the invasive growth capability. Further, LOXL2 was significantly upregulated in clinical human primary melanomas compared to benign nevi, and high expression of LOXL2 in primary melanomas was associated with formation of metastases and shorter survival of patients. Thus, our studies reveal that inactive pro-LOX (together with Lox propeptide) functions as a tumor suppressor in ODC- and RAS-transformed murine fibroblasts by inhibiting cell growth and invasion, and active LOX and LOXL2 as tumor promoters in human melanoma cells by promoting their invasive growth. Copyright: Kielosto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The development of new lymphatic vessels occurs in many cancerous and inflammatory diseases through the binding of VEGF-C to its receptors, VEGFR-2 and VEGFR-3. The regulation of VEGFR-2/VEGFR-3 heterodimerisation and its downstream signaling in lymphatic endothelial cells (LECs) remain poorly understood. Here, we identify the endocytic receptor, uPARAP, as a partner of VEGFR-2 and VEGFR-3 that regulates their heterodimerisation. Genetic ablation of uPARAP leads to hyperbranched lymphatic vasculatures in pathological conditions without affecting concomitant angiogenesis. In vitro, uPARAP controls LEC migration in response to VEGF-C but not VEGF-A or VEGF-CCys156Ser. uPARAP restricts VEGFR-2/VEGFR-3 heterodimerisation and subsequent VEGFR-2-mediated phosphorylation and inactivation of Crk-II adaptor. uPARAP promotes VEGFR-3 signaling through the Crk-II/JNK/paxillin/Rac1 pathway. Pharmacological Rac1 inhibition in uPARAP knockout mice restores the wild-type phenotype. In summary, our study identifies a molecular regulator of lymphangiogenesis, and uncovers novel molecular features of VEGFR-2/VEGFR-3 crosstalk and downstream signaling during VEGF-C-driven LEC sprouting in pathological conditions., info:eu-repo/semantics/published

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of similar to 291 nm in DLS and similar to 200-250 nm in SEM at 37 degrees C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in similar to 65% silencing of STAT3 mRNA in BT-13 gliospheres, while only similar to 20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.

Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-C C156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4 + and CD8 + T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-C C156S . Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Background: Malignant pleural mesothelioma (MPM) is a rare occupational cancer with a poor prognosis. Even with a multimodality treatment approach, the treatment outcomes remain unsatisfactory. The use of asbestos has been banned in most developed countries, but MPM continues to be a significant occupational disease also in these countries. Aim of this study is to identify modern epidemiology and assess equality in care.Methods: Our study cohort consists of 1010 patients diagnosed with MPM in Finland during 2000-2012. The data were collected from the Finnish Cancer Registry, the National Workers' Compensation Center Registry and the National Registry of Causes of Death, Statistics Finland.Results: Women were diagnosed a mean of 4.5 years later than males (p=.001), but survival did not differ (overall median survival 9.7 months). A workers' compensation claim was more common in males (OR 11.0 [95% CI 7.5-16.2]) and in regions with a major asbestos industry (OR 1.7 [95% CI 1.3-2.2]). One-year and three-year survivals did not differ regionally. Patients without chemotherapy treatment had an inferior survival (RR 1.8 [95% CI 1.5-2.0]). The initial survival benefit gained with pemetrexed was diluted at 51 months.Conclusions: MPM is a disease with a poor prognosis, although chemotherapy appears to improve survival time. Significant gender and regional variation exists among patients, with notable differences in diagnostic and treatment practices. Long-term outcomes with pemetrexed remain indeterminate.Impact: Emphasize centralized consult services for the diagnosis, treatment and support that patients receive for MPM, facilitating equal outcomes and compensation.

In metazoans, epithelial architecture provides a context that dynamically modulates most if not all epithelial cell responses to intrinsic and extrinsic signals, including growth or survival signalling and transforming oncogene action. Three-dimensional (3D) epithelial culture systems provide tractable models to interrogate the function of human genetic determinants in establishment of context-dependency. We performed an arrayed genetic shRNA screen in mammary epithelial 3D cultures to identify new determinants of epithelial architecture, finding that the key phenotype impacting shRNAs altered not only the data population average but even more noticeably the population distribution. The broad distributions were attributable to sporadic gene silencing actions by shRNA in unselected populations. We employed Maximum Mean Discrepancy concept to capture similar population distribution patterns and demonstrate here the feasibility of the test in identifying an impact of shRNA in populations of 3D structures. Integration of the clustered morphometric data with protein-protein interactions data enabled hypothesis generation of novel biological pathways underlying similar 3D phenotype alterations. The results present a new strategy for 3D phenotype-driven pathway analysis, which is expected to accelerate discovery of context-dependent gene functions in epithelial biology and tumorigenesis.

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

The aim of this work was to study the antifibrotic effect of pulmonary administration of tilorone to lung fibrosis. L-leucine coated tilorone particles were prepared and their aerosolization properties were analyzed using two dry powder inhalers (Easyhaler and Twister). In addition, the biological activity and cell monolayer permeation was tested. The antifibrotic effect of tilorone delivered by oropharyngeal aspiration was studied in vivo using a silica-induced model of pulmonary fibrosis in mice in a preventive setting. When delivered from the Easyhaler in an inhalation simulator, the emitted dose and fine particle fraction were independent from the pressure applied and showed dose repeatability. However, with Twister the aerosolization was pressure-dependent indicating poor compatibility between the device and the formulation. The formulation showed more consistent permeation through a differentiated Calu-3 cell monolayer compared to pristine tilorone. Tilorone decreased the histological fibrosis score in vivo in systemic and local administration, but only systemic administration decreased the mRNA expression of type I collagen. The difference was hypothesized to result from 40-fold higher drug concentration in tissue samples in the systemic administration group. These results show that tilorone can be formulated as inhalable dry powder and has potential as an oral and inhalable antifibrotic drug.

ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr(192) and Phe(229), but hypoxic activation at Tyr(192) only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr(192)-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium.

Background: Neoadjuvant therapy for pancreatic cancer remains controversial. Our aim was to assess differences in survival, disease recurrence and histopathological tumor characteristics between patients treated with neoadjuvant therapy followed by subsequent surgery and patients undergoing upfront surgery.Material and methods: Out of 399 consecutive pancreatic ductal adenocarcinoma (PDAC) patients operated at Helsinki University Hospital in 2000-2015, 75 borderline resectable patients were treated with neoadjuvant therapy. Resectable propensity scored patients (n=150) underwent upfront surgery. Neoadjuvant therapy consisted of folfirinox, single gemcitabine or combined with cisplatin, nab-paclitaxel or capecitabine with or without radiation. Survival was calculated with Kaplan-Meier and compared with the Breslow test. Survival was determined from the start of treatment, being the first day of treatment for patients treated with neoadjuvant therapy and the date of surgery for others.Results: Between 2000 and 2015 median disease-specific survival (DSS) [34 vs. 26 months, p=.016] and disease-free survival (DFS) [22 vs. 13 months, p=.001] were longer in patients treated with neoadjuvant therapy than in those undergoing upfront surgery. Survival differences were not significant in the 2000s but were, in turn, among patients treated in the 2010s with better survival for patients treated with neoadjuvant therapy [DSS 35 vs. 26 months, p=.008 and DFS 25 vs. 13 months, p=.001]. Especially patients with poorly differentiated G3 tumors [DSS 30 vs. 11 months, p=.004 and DFS 21 vs. 7 months, p=.001] and higher stage IIB-III [DSS 34 vs. 20 months, p=.006 and DFS 21 vs. 10 months, p=.001] had longer survival when treated with neoadjuvant therapy.Conclusions: PDAC patients treated with neoadjuvant therapy had longer DSS and DFS than those undergoing upfront surgery. Neoadjuvant therapy benefits especially borderline resectable patients with higher stage and poorly differentiated tumors.

Background: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. Methods: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. Results: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and alpha-1-antichymotrypsin, as well as complement C1q. Conclusions: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.

// Miao Yin 1, 2 , Mira Tissari 1, 2 , Jenni Tamminen 1, 2 , Irene Ylivinkka 1 , Mikko Ronty 3 , Pernilla von Nandelstadh 4 , Kaisa Lehti 4, 5, 6 , Marko Hyytiainen 4 , Marjukka Myllarniemi 7 and Katri Koli 1, 2 1 Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland 2 Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland 3 Department of Pathology, University of Helsinki and Fimlab Laboratories, Pathology, Tampere, Finland 4 Research Programs Unit, Genome Scale Biology, University of Helsinki, Helsinki, Finland 5 K. Albin Johansson Foundation, Finnish Cancer Institute, Helsinki, Finland 6 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Solna, Sweden 7 Department of Pulmonary Medicine, University of Helsinki and Helsinki University Hospital, Heart and Lung Center, Helsinki, Finland Correspondence to: Katri Koli, email: katri.koli@helsinki.fi Keywords: gremlin, mesothelioma, invasion Received: June 07, 2017 Accepted: September 21, 2017 Published: October 06, 2017 ABSTRACT Malignant mesothelioma originates from mesothelial cells and is a cancer type that aggressively invades into the surrounding tissue, has poor prognosis and no effective treatment. Gremlin-1 is a cysteine knot protein that functions by inhibiting BMP-pathway activity during development. BMP-independent functions have also been described for gremlin-1. We have previously shown high gremlin-1 expression in mesothelioma tumor tissue. Here, we investigated the functions of gremlin-1 in mesothelioma cell migration and invasive growth. Gremlin-1 promoted mesothelioma cell sprouting and invasion into three dimensional collagen and Matrigel matrices. The expression level of gremlin-1 was linked to changes in the expression of SNAI2, integrins, matrix metalloproteinases (MMP) and TGF-β family signaling - all previously associated with a mesenchymal invasive phenotype. Small molecule inhibitors of MMPs completely blocked mesothelioma cell invasive growth. In addition, inhibitors of TGF-β receptors significantly reduced invasive growth. This was associated with reduced expression of MMP2 but not SNAI2, indicating that gremlin-1 has both TGF-β pathway dependent and independent mechanisms of action. Results of in vivo mesothelioma xenograft experiments indicated that gremlin-1 overexpressing tumors were more vascular and had a tendency to send metastases. This suggests that by inducing a mesenchymal invasive cell phenotype together with enhanced tumor vascularization, gremlin-1 drives mesothelioma invasion and metastasis. These data identify gremlin-1 as a potential therapeutic target in mesothelioma.

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral Lymphatics and subpleural lymphatic plexuses that had incompetent Lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

It is of great clinical importance to identify simple prognostic markers from preoperative biopsies that could guide treatment planning. Here, we compared tumor budding (B), depth of invasion (D), and the combined scores (i.e., budding and depth of invasion (BD) histopathologic model) in preoperative biopsies and the corresponding postoperative specimens of oral tongue squamous cell carcinoma (OTSCC). Tumor budding and depth of invasion were evaluated in the pre- and postoperative samples from 100 patients treated for OTSCC. Sensitivity and specificity statistics were used. Our results showed statistically significant (P < 0.001) relationship between pre- and postoperative BD scores. There was an agreement between the pre- and postoperative BD model scores in 83 cases (83%) with 57.1% sensitivity (95% CI 39.4 to 73.7%) and 96.9% specificity (95% CI 89.3 to 99.6%). Our findings suggest that the BD model, analyzed from representative biopsies, could be used for the treatment planning of OTSCC.

Thyroid gland vasculature has a distinguishable characteristic of endothelial fenestrae, a critical component for proper molecular transport. However, the signaling pathway that critically governs the maintenance of thyroid vascular integrity, including endothelial fenestrae, is poorly understood. Here, we found profound and distinct expression of follicular epithelial VEGF-A and vascular VEGFR2 that were precisely regulated by circulating thyrotropin, while there were no meaningful expression of angiopoietin-Tie2 system in the thyroid gland. Our genetic depletion experiments revealed that VEGFR2, but not VEGFR3, is indispensable for maintenance of thyroid vascular integrity. Notably, blockade of VEGF-A or VEGFR2 not only abrogated vascular remodeling but also inhibited follicular hypertrophy, which led to the reduction of thyroid weights during goitrogenesis. Importantly, VEGFR2 blockade alone was sufficient to cause a reduction of endothelial fenestrae with decreases in thyrotropin-responsive genes in goitrogen-fed thyroids. Collectively, these findings establish follicular VEGF-Avascular VEGFR2 axis as a main regulator for thyrotropindependent thyroid angiofollicular remodeling and goitrogenesis.

Latent transforming growth factor beta binding protein 4 (LTBP4) belongs to the fibrillin/LTBP family of proteins and plays an important role as a structural component of extracellular matrix (ECM) and local regulator of TGF beta signaling. We have previously reported that Ltbp4S knock out mice (Ltbp4S-/-) develop centrilobular emphysema reminiscent of late stage COPD, which could be partially rescued by inactivating the antioxidant protein Sestrin 2 (Sesn2). More recent studies showed that Sesn2 knock out mice upregulate Pdgfr beta-controlled alveolar maintenance programs that protect against cigarette smoke induced pulmonary emphysema. Based on this, we hypothesized that the emphysema of Ltbp4S-/- mice is primarily caused by defective Pdgfr beta signaling. Here we show that LTBP4 induces Pdgfr beta signaling by inhibiting the antioxidant Nr12/Keap1 pathway in a TGF beta-dependent manner. Overall, our data identified Ltbp4 as a major player in lung remodeling and injury repair. (C) 2016 The Authors. Published by Elsevier B.V.

Summary Trafficking cells frequently transmigrate through epithelial and endothelial monolayers. How monolayers cooperate with the penetrating cells to support their transit is poorly understood. We studied dendritic cell (DC) entry into lymphatic capillaries as a model system for transendothelial migration. We find that the chemokine CCL21, which is the decisive guidance cue for intravasation, mainly localizes in the trans-Golgi network and intracellular vesicles of lymphatic endothelial cells. Upon DC transmigration, these Golgi deposits disperse and CCL21 becomes extracellularly enriched at the sites of endothelial cell-cell junctions. When we reconstitute the transmigration process in vitro, we find that secretion of CCL21-positive vesicles is triggered by a DC contact-induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. Altogether, our data demonstrate a chemokine-mediated feedback between DCs and lymphatic endothelium, which facilitates transendothelial migration., Graphical Abstract, Highlights • DC entry into lymphatic capillary induces CCL21 secretion to endothelial junctions • Chemokine CCL21 secretion is triggered by calcium fluxes • Direct contact by DC induces calcium signaling in LECs • Dynamic rather than pre-patterned chemokine CCL21 cues promote DC transmigration, Antigen-presenting dendritic cells approach and enter lymphatic capillaries in a chemokine CCL21-dependent manner. Vaahtomeri et al. show that migrating dendritic cells trigger chemokine-mediated feedback via direct contact with lymphatic endothelia, which locally release CCL21 and thus promote transmigration.

Parathyroid carcinoma (PC) is rare and diagnostically challenging. Reported outcomes are rather poor and the incidence might be increasing.We performed a nationwide study on all cases (n= 32) diagnosed in 2000-2011 in Finland, and compared clinical and histopathological characteristics and outcome to atypical parathyroid (APA; n= 28) and parathyroid adenomas (PA; n= 72). The incidence in years 1955-1999 was compared to that in 2000-2013.Preoperatively, calcium and parathyroid hormone concentrations were higher in PC compared to APA and PA (1.76, 1.56 and 1.44 mmol/l, p .001; and 989, 355 and 160 μmol/l, p .001, respectively). Calcium was ≤1.77 mmol/l for all PAs. Hospitalization (44% vs. 22% and 3%, respectively, p = .01), renal (50% vs. 48% vs. 22%, respectively, p = .01) and bone (47% vs. 15% vs. 38%, respectively p = .002) manifestations were more common. PC and APA tumors were larger than PA (p .001). Histopathological characteristics of PC compared to PA are increased mitotic activity (p= .001), chief cells (p = .003), diffuse growth pattern (p .001), higher Ki67 (p .001) and negative parafibromin (p .001). One PC (1/18) and one APA (1/16) patient had a CDC73 mutation. After 6.7 (2-13.9) years of follow-up, 9.4% of PC had residual, 21% recurrent disease and 12.5% died of disease. Overall mortality did not differ between subgroups (p = .094). Recurrent PC was characterized by vascular invasion, lymph node metastases, high mitotic activity, necrosis and negative parafibromin. Incidence increased from 1.42 (range 0.52-2.14) to 7.14 (range 3.42-10.38)/10.000.000/years; (p .001).PC associates with severe primary hyperparathyroidism and must be suspected if calcium ≥1.77 mmol/l. The prevalence of CDC73 germline mutations in PC and APA in Finland is 6%. PC has distinct histopathological characteristics and its incidence has increased over the past decades.

Background: The SLUG transcription factor has been linked with the KIT signalling pathway that is important for gastrointestinal stromal tumour (GIST) tumourigenesis. Its clinical significance in GIST is unknown. Methods: Influence of SLUG expression on cell proliferation and viability were investigated in GIST48 and GIST882 cell lines. The association between tumour SLUG expression in immunohistochemistry and recurrence-free survival (RFS) was studied in two clinical GIST series, one with 187 patients treated with surgery alone, and another one with 313 patients treated with surgery and adjuvant imatinib. Results: SLUG downregulation inhibited cell proliferation, induced cell death in both cell lines, and sensitised GIST882 cells to lower imatinib concentrations. SLUG was expressed in 125 (25.0%) of the 500 clinical GISTs evaluated, and expression was associated with several factors linked with unfavourable prognosis. SLUG expression was associated with unfavourable RFS both when patients were treated with surgery alone (HR = 3.40, 95% CI = 1.67-6.89, P = 0.001) and when treated with surgery plus adjuvant imatinib (HR = 1.83, 95% CI = 1.29-2.60, P = 0.001). Conclusions: GIST patients with high tumour SLUG expression have unfavourable RFS. SLUG may mediate pro-survival signalling in GISTs.

IMPORTANCE: Little is known about whether the duration of adjuvant imatinib influences the prognostic significance of KIT proto-oncogene receptor tyrosine kinase (KIT) and platelet-derived growth factor receptor α (PDGFRA) mutations. OBJECTIVE: To investigate the effect of KIT and PDGFRA mutations on recurrence-free survival (RFS) in patients with gastrointestinal stromal tumors (GISTs) treated with surgery and adjuvant imatinib. DESIGN, SETTING, AND PARTICIPANTS: This exploratory study is based on the Scandinavian Sarcoma Group VIII/Arbeitsgemeinschaft Internistische Onkologie (SSGXVIII/AIO) multicenter clinical trial. Between February 4, 2004, and September 29, 2008, 400 patients who had undergone surgery for GISTs with a high risk of recurrence were randomized to receive adjuvant imatinib for 1 or 3 years. Of the 397 patients who provided consent, 341 (85.9%) had centrally confirmed, localized GISTs with mutation analysis for KIT and PDGFRA performed centrally using conventional sequencing. During a median follow-up of 88 months (completed December 31, 2013), 142 patients had GIST recurrence. Data of the evaluable population were analyzed February 4, 2004, through December 31, 2013. MAIN OUTCOMES AND MEASURES: The main outcome was RFS. Mutations were grouped by the gene and exon. KIT exon 11 mutations were further grouped as deletion or insertion-deletion mutations, substitution mutations, insertion or duplication mutations, and mutations that involved codons 557 and/or 558. RESULTS: Of the 341 patients (175 men and 166 women; median age at study entry, 62 years) in the 1-year group and 60 years in the 3-year group), 274 (80.4%) had GISTs with a KIT mutation, 43 (12.6%) had GISTs that harbored a PDGFRA mutation, and 24 (7.0%) had GISTs that were wild type for these genes. PDGFRA mutations and KIT exon 11 insertion or duplication mutations were associated with favorable RFS, whereas KIT exon 9 mutations were associated with unfavorable outcome. Patients with KIT exon 11 deletion or insertion-deletion mutation had better RFS when allocated to the 3-year group compared with the 1-year group (5-year RFS, 71.0% vs 41.3%; P < .001), whereas no significant benefit from the 3-year treatment was found in the other mutational subgroups examined. KIT exon 11 deletion mutations, deletions that involved codons 557 and/or 558, and deletions that led to pTrp557-Lys558del were associated with poor RFS in the 1-year group but not in the 3-year group. Similarly, in the subset with KIT exon 11 deletion mutations, higher-than-the-median mitotic counts were associated with unfavorable RFS in the 1-year group but not in the 3-year group. CONCLUSIONS AND RELEVANCE: Patients with KIT exon 11 deletion mutations benefit most from the longer duration of adjuvant imatinib. The duration of adjuvant imatinib modifies the risk of GIST recurrence associated with some KIT mutations, including deletions that affect exon 11 codons 557 and/or 558.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4–5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Background Prognostic markers specific to a particular cancer type can assist in the evaluation of survival probability of patients and help clinicians to assess the available treatment modalities. Methods Gene expression data was analyzed from three independent colon cancer microarray gene expression data sets (N = 1052). Survival analysis was performed for the three data sets, stratified by the expression level of the LINE-1 type transposase domain containing 1 (L1TD1). Correlation analysis was performed to investigate the role of the interactome of L1TD1 in colon cancer patients. Results We found L1TD1 as a novel positive prognostic marker for colon cancer. Increased expression of L1TD1 associated with longer disease-free survival in all the three data sets. Our results were in contrast to a previous study on medulloblastoma, where high expression of L1TD1 was linked with poor prognosis. Notably, in medulloblastoma L1TD1 was co-expressed with its interaction partners, whereas our analysis revealed lack of co-expression of L1TD1 with its interaction partners in colon cancer. Conclusions Our results identify increased expression of L1TD1 as a prognostic marker predicting longer disease-free survival in colon cancer patients. Electronic supplementary material The online version of this article (10.1186/s12885-019-5952-2) contains supplementary material, which is available to authorized users.

Colon cancer represents one of the most common cancers in the world. Despite improved treatment, mortality remains high. In order to improve the assessment of prognosis for colon cancer patients, identifying new prognostic markers remains necessary. We analyzed preoperative serum samples from 148 colon cancer patients surgically treated at Helsinki University Hospital from 1998 through 2002 using a multiplex proximity extension assay (Oncology II panel, Olink Bioscience, Uppsala, Sweden), a panel constituting 92 immunological and oncological markers. We performed univariate and multivariate analyses on these patients and calculated the disease-specific survival among patients using the log-rank test for Kaplan–Meier estimates. In the univariate survival analysis of 92 biomarkers, 26 resulted in p

Background and objectivesEstrogen receptor signaling and cyclin D1 have a major role in tumor cell proliferation in breast cancer. Desmoid tumors are rare neoplasms that may respond to endocrine treatment. The present study aimed to investigate the expression levels and the clinical relevance of estrogen receptor beta (ER beta) and cyclin D1 in desmoid tumors. MethodsThis study consists of 83 patients with a surgically treated desmoid tumor. ER beta and cyclin D1 expression was examined by immunohistochemistry in tissue microarrays. Cyclin A and Ki67 were studied in our previous work. ResultsMedian ER beta expression was 10.8%. ER beta expression correlated with expression of the proliferation antigens Ki67 (r(p)=0.35, P=0.003), cyclin D1 (r(p)=0.34, P=0.004), and cyclin A (r(p)=0.34, P=0.004). ER beta immunoexpression showed a trend towards predictive impact for recurrence as a continuous variable. Further explorative analysis indicated that very high ER beta expression was related to high risk of relapse (hazard ratio [HR] 2.6; P=0.02).Median cyclin D1 expression was 15.6%. High cyclin D1 expression was associated with high Ki67 and cyclin A expression. Cyclin D1 was not associated with time to recurrence. ConclusionsER beta and cyclin D1 immunopositivity correlated with high proliferation in desmoid tumors. High ER beta expression might be predictive for postoperative recurrence.

Background Intraductal Papillary Mucinous Neoplasia (IPMN) are potentially malignant cystic tumors of the pancreas. IPMN can progress from low to moderate to high grade dysplasia and further to IPMN associated carcinoma. Often the difference between benign and malignant nature of the IPMN is not clear preoperatively. We aim to elucidate molecular expression patterns of various grades of IPMN and pancreatic carcinoma. Additionally we suggest potential novel biomarkers to differentiate IPMN from healthy individuals and pancreatic carcinoma to enable early detection as well as help in differential diagnosis in future. Methods We have performed retrospective label-free proteomic analysis of the serum samples from 44 patients with various grades of benign IPMN or IPMN associated carcinoma and 11 healthy controls. Proteomic data was further analyzed by various multivariate statistical methods. Four groups of samples (low-grade, high-grade IPMN, pancreatic carcinoma and age- and sex-matched healthy controls) were compared with ANOVA. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) modeling gave S-plot for feature selection. Stringently selected potential markers were further evaluated with ROC curve analysis and area under the curve was calculated. Differentially expressed proteins were used for pathway analysis. Linear trend analysis (Mann Kendall test) was used for identifying significant increasing or decreasing trends from healthy-low grade-high grade IPMN-pancreatic carcinoma. Results Based on protein expression (436 proteins quantified), PCA separated most sample groups from each other. S-Plot selected biomarker panels with moderate to very high AUC values for differentiating controls from Low-, High-Grade IPMN and carcinoma. Linear trend analysis identified 12 proteins which were consistently increasing or decreasing trend among the groups. We found potential biomarkers to differentiate healthy controls from different degrees of dysplasia and pancreatic carcinoma. These biomarkers can classify IPMN, carcinoma and healthy controls from each other which is an unmet clinical need. Data are available via ProteomeXchange with identifier PXD009139. Conclusion Kininogen-1 was able to differentiate healthy persons from low and high-grade IPMN. Retinol binding protein-4 could classify the low-grade IPMN from pancreatic carcinoma. Twelve proteins including apolipoproteins and complement proteins had significantly increasing or decreasing trends from healthy to low to high-grade IPMN to pancreatic carcinoma.

Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, for example in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D., eLife digest The lymphatic system is composed of networks of vessels that drain fluids from the body’s tissues and filter it back into the blood. Growing these vessels depends on a factor known as VEGF-C, which is released in an inactive form and must be cut by enzymes before it can work. One enzyme that is known to activate the VEGF-C signal when the early embryo is developing is ADAMTS3. If this signal fails to switch on this can result in a condition known as lymphedema – whereby problems in the lymphatic system cause tissues to swell due to insufficient drainage. However, it is unknown whether the VEGF-C signal can be activated by enzymes other than ADAMTS3. To investigate this Jha, Rauniyar et al. tested a specific family of proteins commonly found in the human prostate, which have previously been predicted to act on VEGF-C. This revealed that the lymphatic vessel growth factor can also be activated by an enzyme found in seminal fluid called prostate specific antigen, or PSA for short. To see if enzymes in other bodily fluids could switch on VEGF-C, different components of human saliva were separated and tested to see which could cut inactive VEGF-C. This showed that VEGF-C could be converted to an active form by another enzyme called cathepsin D. Unexpectedly, Jha, Rauniyar et al. found that VEGF-C was also present in semen. For conception to occur PSA must liquify the semen following ejaculation. It was discovered that PSA activates VEGF-C just as the semen starts to liquify, suggesting that the lymphatic vessel growth factor might also play an important role in reproduction. In addition to VEGF-C, both PSA and cathepsin D were found to activate another growth factor called VEGF-D, which has an unknown role in the human body. VEGF-C helps the spread of tumors, and blocking the two enzymes that activate this growth factor may be a new therapeutic approach for cancer. However, more work is needed to validate which types of tumor, if any, use these enzymes to activate VEGF-C. In addition, understanding the relationship between PSA and VEGF-C could help improve our knowledge of human reproduction.

A lacteal is a blunt-ended, long, tube-like lymphatic vessel located in the center of each intestinal villus that provides a unique route for drainage of absorbed lipids from the small intestine. However, key regulators for maintaining lacteal integrity are poorly understood. Here, we explore whether and how the gut microbiota regulates lacteal integrity. Germ depletion by antibiotic treatment triggers lacteal regression during adulthood and delays lacteal maturation during the postnatal period. In accordance with compromised lipid absorption, the button-like junction between lymphatic endothelial cells, which is ultrastructurally open to permit free entry of dietary lipids into lacteals, is significantly reduced in lacteals of germ-depleted mice. Lacteal defects are also found in germ-free mice, but conventionalization of germ-free mice leads to normalization of lacteals. Mechanistically, VEGF-C secreted from villus macrophages upon MyD88-dependent recognition of microbes and their products is a main factor in lacteal integrity. Collectively, we conclude that the gut microbiota is a crucial regulator for lacteal integrity by endowing its unique microenvironment and regulating villus macrophages in small intestine.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are neuroendocrine tumors that express somatostatin receptors (SSTRs), a phenomenon that constitutes a basis for tumor imaging and treatment with somatostatin analogues and peptide receptor radionuclide therapy. We studied the immunohistochemical expression of SSTR1-5 in 151 primary tumors, including 14 metastasized and 16 SDHB-deficient tumors. SSTR2 and SSTR3 were most abundantly present in these tumors, whereas the tumors were mostly negative for SSTR1, SSTR4, and SSTR5. All metastasized PGLs (9/9), but only one metastasized PHEO (1/5), were strongly SSTR2 positive. SSTR3 expression was lower in metastatic tumors and tumors with a high proliferation rate (MIB1 >= 5%), but tumors had variable individual SSTR profiles. No correlation was found between SDHB status and SSTR expression. Our results suggest that new SSTR analogues with affinity for several SSTRs could be relevant for a subgroup of patients with these tumors. Better knowledge of tumor SSTR profiles could open the door for personalized imaging and treatment in the future. Because SSTR profiles vary in PHEOs and PGLs, individual analysis is required for each tumor. (C) 2018 The Authors. Published by Elsevier Inc.

Background Nasopharyngeal carcinoma (NPC) is related to Epstein-Barr virus (EBV) in endemic areas; however, the role of viruses in nonendemic countries is unclear. Our nationwide study investigated the prevalence and prognostic significance of EBV and human papillomaviruses (HPVs) in Finnish NPC tumors. Methods We analyzed samples from 150 patients diagnosed between 1990 and 2009. Viral status was determined using EBV and HPV RNA in situ hybridizations, and p16 immunohistochemistry. Patient and treatment characteristics were obtained from patient records. Results In our white patient cohort, 93 of 150 (62%) patients were EBV-positive and 21/150 (14%) patients were HPV-positive with no coinfections. Thirty-six (24%) tumors were negative for both viruses. The 5-year disease-specific survival for patients with EBV-positive, HPV-positive, and EBV/HPV-negative tumors was 69%, 63%, and 39%, respectively. In multivariable-adjusted analysis, overall survival was better among patients with EBV-positive (P = .005) and HPV-positive (P = .03) tumors compared to patients with EBV/HPV-negative tumors. Conclusions In our low-incidence population, EBV and HPV are important prognostic factors for NPC.

Current human papillomavirus (HPV) detection methods in oropharyngeal squamous cell carcinoma (OPSCC) have varying sensitivity and specificity. We aimed to compare different HPV-detection methods against the test used in clinical practice, ie, p16 immunohistochemistry (IHC) and to evaluate whether another HPV-detection test additional to p16 IHC would be worthwhile in OPSCC specimens. The study cohort comprised 357 consecutive OPSCC patients during two time periods: 2000-2009 and 2012-2016. From tumor tissue slides, HPV mRNA via in situ hybridization (ISH), HPV DNA via ISH and HPV DNA via polymerase chain reaction (PCR) were detected. The results of these methods were compared with p16 IHC results. Additionally, clinicopathological factors were compared with the methods studied. The sensitivity of HPV mRNA ISH, HPV DNA ISH and HPV DNA PCR were 93.4%, 86.3%, and 83.5%, respectively. The corresponding specificity was 92.4%, 95.3%, and 89.1%, respectively. The negative predictive value for p16 IHC was highest (89.0%) when using mRNA ISH, and followed by DNA ISH (83.5%). ISH for high-risk HPV E6/E7 mRNA was found to be a highly specific and sensitive method for detecting HPV in OPSCC. As p16 protein may be overexpressed due to HPV-independent mechanisms, all p16 IHC positive OPSCCs should be considered for retesting using mRNA ISH in order to verify transcriptionally active HPV. This is especially critical when considering de-escalated treatment approaches for patients with HPV-positive tumors and still maintaining favorable outcomes for this subgroup of patients. (C) 2019 Elsevier Inc. All rights reserved.

Activin A and myostatin, members of the transforming growth factor (TGF)-β superfamily of secreted factors, are potent negative regulators of muscle growth, but their contribution to myocardial ischemia-reperfusion (IR) injury is not known. The aim of this study was to investigate if activin 2B (ACVR2B) receptor ligands contribute to myocardial IR injury. Mice were treated with soluble ACVR2B decoy receptor (ACVR2B-Fc) and subjected to myocardial ischemia followed by reperfusion for 6 or 24 h. Systemic blockade of ACVR2B ligands by ACVR2B-Fc was protective against cardiac IR injury, as evidenced by reduced infarcted area, apoptosis, and autophagy and better preserved LV systolic function following IR. ACVR2B-Fc modified cardiac metabolism, LV mitochondrial respiration, as well as cardiac phenotype toward physiological hypertrophy. Similar to its protective role in IR injury in vivo, ACVR2B-Fc antagonized SMAD2 signaling and cell death in cardiomyocytes that were subjected to hypoxic stress. ACVR2B ligand myostatin was found to exacerbate hypoxic stress. In addition to acute cardioprotection in ischemia, ACVR2B-Fc provided beneficial effects on cardiac function in prolonged cardiac stress in cardiotoxicity model. By blocking myostatin, ACVR2B-Fc potentially reduces cardiomyocyte death and modifies cardiomyocyte metabolism for hypoxic conditions to protect the heart from IR injury., Graphical Abstract, Magga et al. report that systemic blockade of activin receptor type 2 (ACVR2B) ligands protects from cardiac ischemia-reperfusion injury reducing infarct size, apoptosis, and autophagy. Mechanistically, systemic blockade of ACVR2B ligands reduced SMAD2 activation and modified cardiomyocyte metabolism for hypoxic conditions.

Our group originally found and cloned cDNA for a 98-kDa type 1 transmembrane glycoprotein of unknown function. Because of its abundant expression in astrocytes, it was called the protein astroprincin (APCN). Two thirds of the evolutionarily conserved protein is intracytoplasmic, whereas the extracellular domain carries two N-glycosidic side chains. APCN is physiologically expressed in placental trophoblasts, skeletal and hearth muscle, and kidney and pancreas. Overexpression of APCN (cDNA) in various cell lines induced sprouting of slender projections, whereas knockdown of APCN expression by siRNA caused disappearance of actin stress fibers. Immunohistochemical staining of human cancers for endogenous APCN showed elevated expression in invasive tumor cells compared with intratumoral cells. Human melanoma cells (SK-MEL-28) transfected with APCN cDNA acquired the ability of invasive growth in semisolid medium (Matrigel) not seen with control cells. A conserved carboxyterminal stretch of 21 amino acids was found to be essential for APCN to induce cell sprouting and invasive growth. Yeast two-hybrid screening revealed several interactive partners, of which ornithine decarboxylase antizyme-1, NEEP21 (NSG1), and ADAM10 were validated by coimmunoprecipitation. This is the first functional description of APCN. These data show that APCN regulates the dynamics of the actin cytoskeletal and, thereby, the cell shape and invasive growth potential of tumor cells.

International audience; The lymphatic vasculature, which accompanies the blood vasculature in most organs, is indispensable in the maintenance of tissue fluid homeostasis, immune cell trafficking, and nutritional lipid uptake and transport, as well as in reverse cholesterol transport. In this Review, we discuss the physiological role of the lymphatic system in the heart in the maintenance of cardiac health and describe alterations in lymphatic structure and function that occur in cardiovascular pathology, including atherosclerosis and myocardial infarction. We also briefly discuss the role that immune cells might have in the regulation of lymphatic growth (lymphangiogenesis) and function. Finally, we provide examples of how the cardiac lymphatics can be targeted therapeutically to restore lymphatic drainage in the heart to limit myocardial oedema and chronic inflammation.

Matrix metalloproteinases (MMPs), endopeptidases with diverse biochemical functions, can promote cancer cell invasion and metastasis by degrading the extracellular matrix. A high matrix metalloproteinase-14 (MMP-14) expression in gastric cancer tissue has been associated with metastasis and poor prognosis. To further understand this association, we investigated serum MMP-14 as a biomarker in gastric cancer patients. The patient cohort consisted of 240 gastric adenocarcinoma patients who underwent surgery at Helsinki University Hospital, Finland, between 2000 and 2009. We determined the soluble MMP-14 serum levels using an enzyme-linked immunosorbent assay. We then calculated the associations between serum levels and clinicopathologic variables using the Mann-Whitney U-test or the Kruskal-Wallis test. We constructed survival curves using the Kaplan-Meier method and calculating the hazard ratios using the Cox proportional hazard model. We revealed a positive association between a high serum MMP-14 level and stages III-IV (p = 0.029), and between a high serum MMP-14 and distant metastasis (p = 0.022). Patients with a low serum MMP-14 had a 5-year disease-specific survival of 49.2% (95% confidence interval [CI] 45.5-52.9), whereas patients with a high serum MMP-14 had a 5-year survival of 22.1% (95% CI 15.2-29.0; p = 0.001). High serum MMP-14 was a statistically significant prognostic factor among patients with an intestinal type of cancer (hazard ratio [HR] 3.54; 95% CI 1.51-8.33; p = 0.004), but not among patients with a diffuse type. The serum MMP-14 level remained an independent prognostic factor in our multivariate survival analysis (HR 1.55; 95% CI 1.02-2.35; p = 0.040). This study indicates for the first time that high serum soluble MMP-14 levels in gastric cancer serves as a marker for a poor prognosis, possibly indicating the presence of distant metastases.

Background Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment. Methods Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis. Results Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients. Conclusions This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal—not cancer—stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression., The tumour stroma has altered stiffness and matrix architecture compared to normal tissue, which favours proliferation, and invasion. Here, the authors find that the extracellular matrix produced by normal fibroblasts inhibits cancer cell proliferation through mechanosensitive downregulation of JMJD1a.

Vascular endothelial growth factor D (VEGF-D) promotes the lymph node metastasis of cancer by inducing the growth of lymphatic vasculature, but its specific roles in tumorigenesis have not been elucidated. We monitored the effects of VEGF-D in cutaneous squamous cell carcinoma (cSCC) by subjecting transgenic mice overexpressing VEGF-D in the skin (K14-mVEGF-D) and VEGF-D knockout mice to a chemical skin carcinogenesis protocol involving 7,12-dimethylbenz[a] anthracene and 12-O-tetradecanoylphorbol-13-acetate treatments. In K14-mVEGF-Dmice, tumor lymphangiogenesis was significantly increased and the frequency of lymph node metastasis was elevated in comparison with controls. Most notably, the papillomas regressed more often in K14-mVEGF-D mice than in littermate controls, resulting in a delay in tumor incidence and a remarkable reduction in the total tumor number. Skin tumor growth and metastasis were not obviously affected in the absence of VEGF-D; however, the knockout mice showed a trend for reduced lymphangiogenesis in skin tumors and in the untreated skin. Interestingly, K14-mVEGF-D mice showed an altered immune response in skin tumors. This consisted of the reduced accumulation of macrophages, mast cells, and CD4(+) T-cells and an increase of cytotoxic CD8(+) T-cells. Cytokine profiling by flow cytometry and quantitative real time PCR revealed that elevated VEGF-D expression results in an attenuated Th2 response and promotes M1/Th1 and Th17 polarization in the early stage of skin carcinogenesis, leading to an anti-tumoral immune environment and the regression of primary tumors. Our data suggest that VEGF-D may be beneficial in early-stage tumors since it suppresses the pro-tumorigenic inflammation, while at later stages VEGF-D-induced tumor lymphatics provide a route for metastasis.

Summary Background Treatments for small-cell lung cancer (SCLC) after failure of platinum-based chemotherapy are limited. We assessed safety and activity of nivolumab and nivolumab plus ipilimumab in patients with SCLC who progressed after one or more previous regimens. Methods The SCLC cohort of this phase 1/2 multicentre, multi-arm, open-label trial was conducted at 23 sites (academic centres and hospitals) in six countries. Eligible patients were 18 years of age or older, had limited-stage or extensive-stage SCLC, and had disease progression after at least one previous platinum-containing regimen. Patients received nivolumab (3 mg/kg bodyweight intravenously) every 2 weeks (given until disease progression or unacceptable toxicity), or nivolumab plus ipilimumab (1 mg/kg plus 1 mg/kg, 1 mg/kg plus 3 mg/kg, or 3 mg/kg plus 1 mg/kg, intravenously) every 3 weeks for four cycles, followed by nivolumab 3 mg/kg every 2 weeks. Patients were either assigned to nivolumab monotherapy or assessed in a dose-escalating safety phase for the nivolumab/ipilimumab combination beginning at nivolumab 1 mg/kg plus ipilimumab 1 mg/kg. Depending on tolerability, patients were then assigned to nivolumab 1 mg/kg plus ipilimumab 3 mg/kg or nivolumab 3 mg/kg plus ipilimumab 1 mg/kg. The primary endpoint was objective response by investigator assessment. All analyses included patients who were enrolled at least 90 days before database lock. This trial is ongoing; here, we report an interim analysis of the SCLC cohort. This study is registered with ClinicalTrials.gov, number NCT01928394. Findings Between Nov 18, 2013, and July 28, 2015, 216 patients were enrolled and treated (98 with nivolumab 3 mg/kg, three with nivolumab 1 mg/kg plus ipilimumab 1 mg/kg, 61 with nivolumab 1 mg/kg plus ipilimumab 3 mg/kg, and 54 with nivolumab 3 mg/kg plus ipilimumab 1 mg/kg). At database lock on Nov 6, 2015, median follow-up for patients continuing in the study (including those who had died or discontinued treatment) was 198·5 days (IQR 163·0–464·0) for nivolumab 3 mg/kg, 302 days (IQR not calculable) for nivolumab 1 mg/kg plus ipilimumab 1 mg/kg, 361·0 days (273·0–470·0) for nivolumab 1 mg/kg plus ipilimumab 3 mg/kg, and 260·5 days (248·0–288·0) for nivolumab 3 mg/kg plus ipilimumab 1 mg/kg. An objective response was achieved in ten (10%) of 98 patients receiving nivolumab 3 mg/kg, one (33%) of three patients receiving nivolumab 1 mg/kg plus ipilimumab 1 mg/kg, 14 (23%) of 61 receiving nivolumab 1 mg/kg plus ipilimumab 3 mg/kg, and ten (19%) of 54 receiving nivolumab 3 mg/kg plus ipilimumab 1 mg/kg. Grade 3 or 4 treatment-related adverse events occurred in 13 (13%) patients in the nivolumab 3 mg/kg cohort, 18 (30%) in the nivolumab 1 mg/kg plus ipilimumab 3 mg/kg cohort, and ten (19%) in the nivolumab 3 mg/kg plus ipilimumab 1 mg/kg cohort; the most commonly reported grade 3 or 4 treatment-related adverse events were increased lipase (none vs 5 [8%] vs none) and diarrhoea (none vs 3 [5%] vs 1 [2%]). No patients in the nivolumab 1 mg/kg plus ipilimumab 1 mg/kg cohort had a grade 3 or 4 treatment-related adverse event. Six (6%) patients in the nivolumab 3 mg/kg group, seven (11%) in the nivolumab 1 mg/kg plus ipilimumab 3 mg/kg group, and four (7%) in the nivolumab 3 mg/kg plus ipilimumab 1 mg/kg group discontinued treatment due to treatment-related adverse events. Two patients who received nivolumab 1 mg/kg plus ipilimumab 3 mg/kg died from treatment-related adverse events (myasthenia gravis and worsening of renal failure), and one patient who received nivolumab 3 mg/kg plus ipilimumab 1 mg/kg died from treatment-related pneumonitis. Interpretation Nivolumab monotherapy and nivolumab plus ipilimumab showed antitumour activity with durable responses and manageable safety profiles in previously treated patients with SCLC. These data suggest a potential new treatment approach for a population of patients with limited treatment options and support the evaluation of nivolumab and nivolumab plus ipilimumab in phase 3 randomised controlled trials in SCLC. Funding Bristol-Myers Squibb.

Introduction: Burn anemia represents a common complication following a burn injury. Burn anemia etiology carries distinct features occurring at each stage of the post-injury and treatment periods resulting from different causes. We aimed to analyze the use of blood components in Finnish burn victims and to identify patient- and injury-related factors influencing their use. Methods: To study the use of blood products in burn patients, we used data collected from the Optimal Use of Blood registry, developed through co-operation between 10 major hospital districts and the Finnish Red Cross Blood Service. Burn patients ⩾18 years treated at the Helsinki University Hospital between 2005 and 2011 with an in-hospital stay ⩾1 day who received at least one transfusion during their hospital stay were included in this study. Results: Among all 558 burn patients, 192 (34%) received blood products during their hospital stay. The transfused cohort comprised 192 burn patients. The study cohort received a total of 6087 units of blood components, 2422 units of leukoreduced red blood cells, 1728 units of leukoreduced platelets, and 420 units of single-donor fresh frozen plasma or, after 2007, 1517 units of Octaplas® frozen plasma. All three types of blood components were administered to 29% of patients, whereas 45% received only red blood cells and 6% received only Octaplas. Transfused patients were significantly older (p Discussion: We show that Finnish adult burn patients received ample transfusions. The number of blood components transfused varied according to the anatomical location of the injury and patient survival. Whether the additional mortality is related directly to transfusions or is merely a manifestation of the more severe burn injury remains unknown.

The proliferation marker, securin, is involved in the progression of many carcinomas. However, its expression in oral tongue squamous cell carcinoma (OTSCC) has not been previously studied. We examined securin expression by immunohistochemistry in OTSCC. A total of 93 cases treated for OTSCC were included in this study. Expression of securin in OTSCC was studied by immunohistochemistry of tissue microarrays (52 cases) and routine tumor sections (41 cases). Securin overexpression is significantly associated with higher tumor grade (P = 0.03). Overexpression of securin was observed more frequently in advanced stages of OTSCC than in earlier stages but the difference was not statistically significant. These findings suggest that overexpression of securin in OTSCC may be important during progression of this cancer. No significant association was found between securin expression and the prognosis of OTSCC.

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.

Using an in vivo reporter for lymphangiogenesis, a recent study in Nature from Olmeda et al. ( 2017) describes a new subset of melanomas that induce systemic pre-conditioning of distant organs for formation of tumor metastatic niches, and identifies the responsible factor as the pleiotropic cytokine midkine. Non

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer often diagnosed late. Earlier detection is urgently needed. Pancreatic ductal adenocarcinoma is known to associate with increased coagulation activity. We studied whether preoperative coagulation biomarkers are useful in distinguishing PDAC from a benign tumor, intraductal papillary mucinous neoplasm (IPMN) in this observational study. We analyzed standard clinical and coagulation variables in patients operated during 2010 and 2015 at Helsinki University Hospital. Pancreatic ductal adenocarcinoma with preoperative coagulation variables available and no neoadjuvant treatment or other active cancer was observed in 80 patients (stage I-III in 67 and IV in 13) and IPMN in 18 patients. Fibrinogen, factor VIII (FVIII), carbohydrate antigen (CA) 19-9, albumin, alkaline phosphatase, and conjugated bilirubin were higher in both stages I to III and IV PDAC compared to IPMN ( P < .05). Factor VIII was highest in stage IV ( P < .05). Combining these variables in a panel increased sensitivity and specificity for PDAC. In receiver operating characteristic analysis, the area under the curve (95% confidence interval) was 0.95 (0.90-1.00) for the panel, compared to 0.80 (0.71-0.88) for CA 19-9 alone ( P < .01). In conclusion, PDAC was associated with increased fibrinogen and FVIII. Combining these coagulation biomarkers with CA 19-9, albumin, and alkaline phosphatase improves diagnostic accuracy.

A functional protein quality control machinery is crucial to maintain cellular and organismal physiology. Perturbation in the protein homeostasis network can lead to the formation of misfolded and aggregated proteins that are a hallmark of protein conformational disorders and aging. Protein aggregation is counteracted by the action of chaperones that can resolubilize aggregated proteins. An alternative protein aggregation clearance strategy is the elimination by proteolysis employing the ubiquitin proteasome system (UPS) or autophagy. Little is known how these three protein aggregate clearance strategies are regulated and coordinated in an organism with the progression of aging or upon expression of disease-associated proteins. To unravel the crosstalk between the protein aggregate clearance options, we investigated how autophagy and the UPS respond to perturbations in protein disaggregation capacity. We found that autophagy is induced as a potential compensatory mechanism, whereas the UPS exhibits reduced capacity upon depletion of disaggregating chaperones in C. elegans and HEK293 cells. The expression of amyloid proteins A beta(3-42) and Q(40) result in an impairment of autophagy as well as the UPS within the same and even across tissues. Our data indicate a tight coordination between the different nodes of the proteostasis network (PN) with the progression of aging and upon imbalances of the capacity of each clearance mechanism.

Despite gastric cancer being rare nowadays in Western countries, it remains one of the leading causes of cancer death worldwide. The course of the disease varies, so the individual gastric cancer patient’s prognosis is difficult to determine. The need for new biomarkers is crucial. The aim of this study was to evaluate the prognostic value of serum matrix metalloproteinase-8, serum tissue inhibitor of metalloproteinase-1, and tissue matrix metalloproteinase-8 in patients with gastric cancer. Preoperative serum samples from 233 patients with gastric cancer were retrospectively analyzed. Serum levels of matrix metalloproteinase-8 were analyzed with immunofluorometric assay, and tissue inhibitor of metalloproteinase-1 levels were determined by enzyme-linked immunosorbent assay. We also determined the tissue expression of matrix metalloproteinase-8 in 276 gastric cancer samples by immunohistochemistry. Survival data and death causes came from patient records, the Population Register Center of Finland, and Statistics Finland. Patients with a low (131 ng/mL) serum matrix metalloproteinase-8 level had a considerably unfavorable prognosis (p = 0.002). Those patients with a high (≥170 ng/mL) serum tissue inhibitor of metalloproteinase-1 level also had a poor prognosis (p 0.30) molar ratios predicted a worse prognosis (p = 0.020). Tissue matrix metalloproteinase-8 did not influence prognosis. These results suggest that serum matrix metalloproteinase-8, tissue inhibitor of metalloproteinase-1, and the ratio of matrix metalloproteinase-8/ tissue inhibitor of metalloproteinase-1 may prove useful biomarkers for prediction of prognosis in patients with gastric cancer.