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Tables S1, S2 Figures S1-S7

Supplemental Table 4. NK cells were negatively enriched from pooled spleens of 3 WT and 3 Cd47-/- littermate mice and cultured with/out thrombospondin-1 (2nM) for 24 hours in RPMI (without FCS) + IL-15 (2.5 ng/ml). Golgi-stop was added at last 4 hours of culture. FcRs were blocked with rat and rabbit serum and cell surface CD47 and NK1.1 were stained with Aqua Live/Dead. Cells were then fixed, permeabilized and i.c. stained for Ki-67, IL-2, IFNγ, TNFα and GzmB. Values indicate the MFI of indicated proteins.

Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPα. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro. Cd47−/− NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-γ levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47−/− than in wild-type mice. Melanoma-bearing Cd47−/− mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47−/− NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas.

Supplemental Figure S2. CD47 blockade enhances T cell mediated cancer cell killing without affecting target cell proliferation.

Supplemental Figure S1. Suppression of CD47 does not enhance radiation growth delay in and athymic mouse SCC VII model of squamous cell carcinoma.

Supplementary Table 1. Th1 vs. Th2 Cytokine Trend. Supplementary Table 2. Effect of L-NAME on Radiation-induced Cytokine Expression.

LCC9 (A) or ZR-75-1 (B) cells were transfected with control siRNA, GRP78 siRNA, or GRP78 cDNA for 24 hours and then plated in an ACEA E-plate in the presence of vehicle or 100 nM 4-OHT; cell index was measured by electrical impedance every 12 hours for 72 hours. n=3, *p

Supplemental Figure S3. Efficiency of CD47 protein knockdown by CD47 morpholino.

A. Heat map of lipid metabolites in LCC1 and LCC9 cells transfected with control or GRP78 siRNA and treated with or without TAM. n=6. B. Relative levels of oleic ethanolamide and palmitoyl ethanolamide in LCC1 and LCC9 cells treated with vehicle, TAM, GRP78 siRNA or GRP78 siRNA+TAM. n=6. C. LCC9 cells were treated with 10 nM -1 microM oleic ethanolamide (OEA), and/or various doses (vehicle, 10 nM, 100 nM, 1000 nM) TAM for 6 days. Relative cell density was determined by crystal violet assay. n=4, *p

RT-PCR gene expression of SCD1 (A), FASN (B), ACC (C), and CPT1A (D) was measured in LCC1 and LCC9 breast cancer cells transfected with control or GRP78 siRNA for 72 hours and normalized with HRPT housekeeping gene expression. n=3; *p

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657–70. ©2016 AACR.

Supplemental Figure S5. Ratio of Granzyme B gene expression to CD8 alpha gene expression in tumors.

Supplemental Figure S4. Effect of T cell depletion on B16F10 melanoma responses to IR in WT and CD47-null mice.

A. LCC9 breast cancer cells were transfected with control siRNA, GRP78 siRNA, control pcDNA, or GRP78 cDNA for 72 hours and CD47, calreticulin, and HMGB1 protein expression measured by Western blot hybridization. B. LCC9 xenografts from control mice or mice treated with TAM, GRP78M, or GRP78M+TAM were stained for CD47 and visualized at 20X. n=6; *p

Supplemental Figure Legend. Legend for Supplemental Figures S1-S5.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5’-7-methylguanosine (m7G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m7G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m7G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m7G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m7G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex.

CD47 has established roles in the immune system for regulating macrophage phagocytosis and lymphocyte activation, with growing evidence of its cell-intrinsic regulatory roles in natural killer and CD8+ T cells. CD47 limits antigen-dependent cytotoxic activities of human and murine CD8+ T cells, but its role in T cell activation kinetics remains unclear. Using

CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47

In The Longing for Total Revolution, Bernard Yack claims not to account for totalitarianism but simply to unearth a new, specifically modern mindset. Still, the problem of totalitarianism, and what...

Traditionally, Roman temples and shrines in Britain have been contextualised in relation to wider ‘Roman’ religious practices. Until recently, considerations of architectural form and named deities have dominated discussions. The wider turn in archaeological discourse recognising ritual in everyday contexts has highlighted the importance of lived experience and landscape practice in shaping belief. Here we reflect on the implications of such ideas when approaching ritual practice at Roman temples, using a recently excavated example from Wiltshire, southern Britain, as a case study. The exceptional artefactual assemblages from the site demonstrate the importance of local and regional landscape practices and belief in shaping ritual practice in a sacred space. In addition, geophysical survey and analysis of Portable Antiquities Scheme (PAS) finds suggests that those occupying the landscape had long-term access to wealth. Deposition in the temple itself indicates the continuing importance attached to prehistoric objects in the Roman period, but also to the adoption of new votive practices of miniaturisation, mutilation and sacrifice. These rituals, although part of wider grammars of religious behaviour, had their roots in specific local contexts. Our detailed analyses provide a picture of a temple dedicated to a previously unknown local god, Bregneus, framed against that of an active community involved in farming, iron processing, quarrying, hunting and woodland management.

CD47 is a ubiquitously expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and as the counter-receptor for signal regulatory protein-α (SIRPα). Engaging SIRPα on macrophages inhibits phagocytosis, and CD47 thereby serves as a physiological marker of self. However, elevated CD47 expression on some cancer cells also protects tumors from innate immune surveillance and limits adaptive antitumor immunity via inhibitory SIRPα signaling in antigen-presenting cells. CD47 also mediates inhibitory thrombospondin-1 signaling in vascular cells, T cells, and NK cells, and blocking inhibitory CD47 signaling on cytotoxic T cells directly increases tumor cell killing. Therefore, CD47 functions as an innate and adaptive immune checkpoint. These findings have led to the development of antibodies and other therapeutic approaches to block CD47 functions in the tumor microenvironment. Preclinical studies in mice demonstrated that blocking CD47 can limit the growth of hematologic malignancies and solid tumors and enhance the efficacy of conventional chemotherapy, radiation therapy, and some targeted cancer therapies. Humanized CD47 antibodies are showing promise in early clinical trials, but side effects related to enhanced phagocytic clearance of circulating blood cells remain a concern. Approaches to circumvent these include antibody preloading strategies and development of antibodies that recognize tumor-specific epitopes of CD47, SIRPα antibodies, and bivalent antibodies that restrict CD47 blockade to specific tumor cells. Preclinical and clinical development of antibodies and related biologics that inhibit CD47/SIRPα signaling are reviewed, including strategies to combine these agents with various conventional and targeted therapeutics to improve patient outcome for various cancers.

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.

Comprehensive social cognition training programs have been effective to improve social cognition in people with chronic schizophrenia, although there is insufficient quality evidence for recent-onset psychosis. The aim of this study was to assess the effects of Social Cognition and Interaction Training (SCIT) in a sample of recent-onset schizophrenia outpatients. Sixteen participants who had their first psychotic episode for less than 2 years were randomly allocated to the SCIT group during 20 weeks (weekly sessions) or to a psychoeducation group and completed baseline and post-training assessment for cognitive biases, social cognition, clinical symptoms and functioning. Permutation-based analysis revealed improvements in overall functioning (P = 0.036) and blame score (P = 0.070) in the SCIT group compared to the psychoeducation intervention, with large effect sizes (d = 1.438 and d = 1.204, respectively). There were also large effect sizes for hostility, emotion recognition, social perception, positive and total symptoms (d = 0.833-1.158). These results suggest that SCIT may be an effective tool to improve attributional biases and functional outcomes in recent-onset schizophrenia outpatients. Future controlled trials with larger sample size and follow-up assessments should be developed to further understand effective intervention outcomes for this population.

CD47, also known as integrin-associated protein, is a constitutively and ubiquitously expressed transmembrane receptor. CD47 is conserved across amniotes including mammals, reptiles, and birds. Expression is increased in many cancers and, in non-malignant cells, by stress and with aging. The up-regulation of CD47 expression is generally epigenetic, whereas gene amplification occurs with low frequency in some cancers. CD47 is a high affinity signaling receptor for the secreted protein thrombospondin-1 (THBS1) and the counter-receptor for signal regulatory protein-α (SIRPA, SIRPα) and SIRPγ (SIRPG). CD47 interaction with SIRPα serves as a marker of self to innate immune cells and thereby protects cancer cells from phagocytic clearance. Consequently, higher CD47 correlates with a poor prognosis in some cancers, and therapeutic blockade can suppress tumor growth by enhancing innate antitumor immunity. CD47 expressed on cytotoxic T cells, dendritic cells, and NK cells mediates inhibitory THBS1 signaling that further limits antitumor immunity. CD47 laterally associates with several integrins and thereby regulates cell adhesion and migration. CD47 has additional lateral binding partners in specific cell types, and ligation of CD47 in some cases modulates their function. THBS1-CD47 signaling in non-malignant cells inhibits nitric oxide/cGMP, calcium, and VEGF signaling, mitochondrial homeostasis, stem cell maintenance, protective autophagy, and DNA damage response, and promotes NADPH oxidase activity. CD47 signaling is a physiological regulator of platelet activation, angiogenesis and blood flow. THBS1/CD47 signaling is frequently dysregulated in chronic diseases.

Thrombospondin-1 (TSP1) is a matricellular protein with many important roles in mediating carcinogenesis, fibrosis, leukocyte recruitment, and metabolism. We have previously shown a role of diet in the absence of TSP1 in liver metabolism in the context of a colorectal cancer model. However, the metabolic implications of TSP1 regulation by diet in the liver metabolism are currently understudied. Therefore Discrete correlation summation (DCS) was used to re-interrogate data and determine the metabolic alterations of TSP1 deficiency in the liver, providing new insights into the role of TSP1 in liver injury and the progression of liver pathologies such as nonalcoholic fatty liver disease (NAFLD). DCS analysis provides a straightforward approach to rank covariance and data clustering when analyzing complex data sets. Using this approach, our previous liver metabolite data was re-analyzed by comparing wild-type (WT) and Thrombospondin-1 null (Thbs1−/−) mice, identifying changes driven by genotype and diet. Principal component analysis showed clustering of animals by genotype regardless of diet, indicating that TSP1 deficiency alters metabolite handling in the liver. High-fat diet consumption significantly altered over 150 metabolites in the Thbs1−/− livers versus approximately 90 in the wild-type livers, most involved in amino acid metabolism. The absence of Thbs1 differentially regulated tryptophan and tricarboxylic acid cycle metabolites implicated in the progression of NAFLD. Overall, the lack of Thbs1 caused a significant shift in liver metabolism with potential implications for liver injury and the progression of NAFLD.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47

Ischemia reperfusion (IR) injury is a process defined by the temporary loss of blood flow and tissue perfusion followed later by restoration of the same. Brief periods of IR can be tolerated with little permanent deficit, but sensitivity varies for different target cells and tissues. Ischemia reperfusion injuries have multiple causes including peripheral vascular disease and surgical interventions that disrupt soft tissue and organ perfusion as occurs in general and reconstructive surgery. Ischemia reperfusion injury is especially prominent in organ transplantation where substantial effort has been focused on protecting the transplanted organ from the consequences of IR. A number of factors mediate IR injury including the production of reactive oxygen species and inflammatory cell infiltration and activation. In the kidney, IR injury is a major cause of acute injury and secondary loss of renal function. Transplant-initiated renal IR is also a stimulus for innate and adaptive immune-mediated transplant dysfunction. The cell surface molecule CD47 negatively modulates cell and tissue responses to stress through limitation of specific homeostatic pathways and initiation of cell death pathways. Herein, a summary of the maladaptive activities of renal CD47 will be considered as well as the possible therapeutic benefit of interfering with CD47 to limit renal IR.

U-Pb age spectra of detrital zircons are widely used to estimate maximum depositional ages (MDA) for sedimentary successions of various age. Different methods have been proposed for calculating an MDA. The most common are based on calculated ages of either the youngest single grain (YSG), the youngest grain cluster composed of three or more grains that overlap at 2σ (YGC 2σ), or the youngest graphical peak (YPP). Many of these methods produce MDAs consistent with biostratigraphic age or the radiometric age of volcanic horizons within the same unit; however, several studies have shown that MDA estimates based on detrital zircon can be younger than the true depositional age, particularly in active tectonic settings, indicating that the methods should be applied with care for successions where independent depositional age control is lacking.In this contribution we present a compilation of 27 detrital zircon samples from Ordovician to Silurian strata from a part of the Trondheim Nappe Complex of the central Scandinavian Caledonides. The samples belong to six stratigraphically distinct units with independent age control from fossils, dated volcanic horizons or bracketing units of known age. These successions represent various marginal basins filled during the closing stages of the Iapetus Ocean in an overall active tectonic setting with detritus from both continental landmasses and Cambro-Ordovician island arcs. Shortly after deposition, the successions were folded and metamorphosed at up to greenschist facies during Taconian accretionary events and/or the Scandian continent-continent collision.We calculated MDAs by the three methods YSG, YGC 2σ and YPP for all samples based on 206Pb/ 238U ages, applying a rigorous discordance filter of 5% (most studies use 10%), in order to use the most reliable analyses possible. Our analysis shows that the YSG MDA is up to 36 m.y. younger than the known depositional age for 17 of the 27 samples, with up to six individual grains giving too young age estimates in some samples. Hence, YSG MDA obviously does not provide a reliable MDA estimate. Of the YGC 2σ (weighted mean age) estimates, six are still significantly younger than known depositional age; and an additional seven are younger but overlap with the known depositional age when considering the maximum error on the YGC 2σ estimate. The only method which provides an MDA estimate within the age of known deposition or older for all samples is the YPP method.Our results indicate that statistically robust estimates of MDA from detrital zircon data in such an active orogenic setting are provided only by the YPP method; both the YSG and the YGC 2σ methods provided unreliably young estimates even with a discordance filter of 5% (using a filter of only 10% makes the problem considerably worse). The spuriously young ages of up to six near-concordant grains in some samples is probably due to concealed lead loss, possibly caused by (fluid-assisted?) recrystallisation of zircon domains during regional greenschist-facies metamorphism shortly after deposition.

This volume synthesises an extensive programme of geophysical survey of the Roman town at Aldborough, North Yorkshire, and a thorough investigation of all previous excavations of the site. The synt...

Thrombospondins are encoded in vertebrates by a family of 5 THBS genes. THBS1 is infrequently mutated in most cancers, but its expression is positively regulated by several tumor suppressor genes and negatively regulated by activated oncogenes and promoter hypermethylation. Consequently, thrombospondin-1 expression is frequently lost during oncogenesis and is correlated with a poor prognosis for some cancers. Thrombospondin-1 is a secreted protein that acts in the tumor microenvironment to inhibit angiogenesis, regulate antitumor immunity, stimulate tumor cell migration, and regulate the activities of extracellular proteases and growth factors. Differential effects of thrombospondin-1 on the sensitivity of normal versus malignant cells to ischemic and genotoxic stress also regulate the responses to tumors to therapeutic radiation and chemotherapy.

In this essay, David D. Roberts resumes his long and productive exchange of views on fascism with Roger Griffin by revisiting the latter’s conceptual investment in the notion of fascism as ‘palingenetic ultra-nationalism’. Robert’s alternative perspective suggests that a more productive line of enquiry could be based on reconceptualising totalitarianism and probing its relevance as an analytical tool in fascism studies. Roberts reiterates his plea for a recast notion of totalitarianism wound around a new mode of collective action in response to the new sense of possibility, of unprecedented scope for experiment. More attention to the totalitarian trajectory and less reliance on Griffin’s ‘palingenesis’, however, does not mean that we must choose between the two; totalitarianism need not be taken as a completely independent variable, uncoupled from palingenesis altogether. Rather than either/or, this is a matter of degrees, proportions.

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.

Osteobiographies of four individuals whose skeletal remains were recovered in 2015–16 from the Stonehenge World Heritage Site are constructed, drawing upon evidence from funerary taphonomy, radiocarbon dating, osteological study, stable isotope analyses, and microscopic and biomolecular analyses of dental calculus. The burials comprise an adult from the Middle Neolithic period, immediately prior to the building of Stonehenge, and two adults and a perinatal infant dating from the Middle Bronze Age, shortly after the monument ceased to be structurally modified. The two Middle Bronze Age adults were closely contemporary, but differed from one another in ancestry, appearance and geographic origin (key components of ethnicity). They were nevertheless buried in very similar ways. This suggests that aspects they held in common (osteological analysis suggests perhaps a highly mobile lifestyle) were more important in determining the manner of deposition of their bodies than any differences between them in ethnicity. One of these individuals probably came from outside Britain, as perhaps did the Middle Neolithic adult. This would be consistent with the idea that the Stonehenge landscape had begun to draw people to it from beyond Britain before Stonehenge was constructed and that it continued to do so after structural modification to the monument had ceased.

BACKGROUND: A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. METHODS: We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. RESULTS: Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. CONCLUSIONS: Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, absence of CD47 in did not elicit a protective effect but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

In this timely and magisterial distillation of his life’s study of Wessex in the middle centuries of the first millennium AD, Bruce Eagles provides a thorough and carefully thought through contribu...

Tumor cells release extracellular vesicles (EVs) into the tumor microenvironment that may facilitate malignant progression and metastasis. Breast carcinoma EVs express high levels of the thrombospondin-1 and signal regulatory protein-α receptor CD47, which is the target of several experimental therapeutics currently in clinical trials. We analyzed changes in gene expression and function in human umbilical vein endothelial cells (HUVEC) induced by treatment with EVs derived from breast carcinoma cells and the effects of the function-blocking CD47 antibody B6H12 on the resulting intercellular communication. CD47+ EVs exhibited greater uptake by HUVEC compared to CD47− EVs, but the CD47 antibody did not inhibit their uptake. Global and targeted analyses of transcripts demonstrated that treatment of HUVEC with EVs derived from MDA-MB-231 breast carcinomas cells altered pathways associated with tumor necrosis factor-α signaling, angiogenesis, lymphangiogenesis, endothelial-mesenchymal transition, and extracellular matrix. EVs from triple-negative MDA-MB-231 cells were more active than EVs from less metastatic breast carcinoma cell lines. Treatment with MDA-MB-231 EVs down-regulated VEGFR2 mRNA expression and tyrosine phosphorylation while enhancing phosphorylation of the tyrosine phosphatase SHP2. VEGFR2 expression and phosphorylation in HUVEC was further inhibited by the CD47 antibody. Consistent with the observed changes in endothelial-mesenchymal transition genes and SHP2, treatment with MDA-MB-231-derived EVs decreased Zeb1 protein levels in HUVEC, whereas the CD47 antibody increased Zeb1 levels. The induction of E-selectin and other known targets of tumor necrosis factor-α signaling by EVs was also enhanced by the CD47 antibody, and E-selectin was the most up-regulated transcript following CD47 antibody treatment alone. These studies reveal several mechanisms by which therapeutics targeting CD47 could modulate tumor growth by altering the cross talk between cancer-derived EVs and nonmalignant cells in the tumor stroma.

In contrast to structural elements of the extracellular matrix, matricellular proteins appear transiently during development and injury responses, but their sustained expression can contribute to chronic disease. Through interactions with other matrix components and specific cell surface receptors, matricellular proteins regulate multiple signaling pathways, including those mediated by reactive oxygen and nitrogen species and HRedox signaling remains unexplored for many matricellular proteins. Their interactions with multiple cellular receptors remains an obstacle to defining signaling mechanisms, but improved transgenic models could overcome this barrier.Therapeutics targeting the TSP1 receptor CD47 may have beneficial effects for treating cardiovascular disease and cancer and have recently entered clinical trials. Biomarkers are needed to assess their effects on redox signaling in patients and to evaluate how these contribute to their therapeutic efficacy and potential side effects. Antioxid. Redox Signal. 27, 874-911.

Background The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors. Methods and findings This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort. Conclusions Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations., Jeffrey J. Szymanski and colleagues investigate the use of cell-free DNA ultra-low-pass whole genome sequencing to distinguish the malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion in patients with Neurofibromatosis type 1 in United States., Author summary Why was this study done? Neurofibromatosis type 1 (NF1) is the most common inherited cancer predisposition syndrome. The leading cause of mortality in NF1 is malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma that arises from a benign plexiform neurofibroma (PN) precursor lesion. Transformation from PN to MPNST is challenging to detect by imaging (due to difficulty in distinguishing PN from MPNST radiologically) or by biopsy (due to intralesional heterogeneity), which often delays the diagnosis of MPNST and results in a worsened prognosis. What did the researchers do and find? We conducted a multi-institutional study involving 2 large NF1 referral centers, the National Cancer Institute and Washington University in St. Louis, involving 53 patients from whom plasma cell-free DNA (cfDNA) was analyzed using ultra-low-pass whole genome sequencing (ULP-WGS). We found that cfDNA from patients with MPNST harbors a shorter fragmentation profile compared to patients with PN or healthy donors. Using sequencing reads from this fragmentation profile, we quantified genome-wide copy number alterations (CNAs) in cfDNA and used CNAs to estimate the fraction of plasma cfDNA originating from tumor. Tumor fraction in plasma cfDNA distinguished pretreatment MPSNT from PN with 86% accuracy. Plasma cfDNA from MPNST and PN patients harbored focal copy number loss of NF1 not found in healthy donors. Strikingly, MPNST patient cfDNA also had significantly greater tumor genomic instability compared to PN, with CNAs in key genomic loci previously observed in MPNST tissue (i.e., gain of chromosome arm 8q and loss of 9p), which enabled sensitive and specific liquid biopsy discrimination of MPNST from PN. Plasma-derived tumor fraction correlated with tumor size from imaging in MPNST patients, and serial cfDNA analysis demonstrated the potential for noninvasive detection of minimal residual disease, treatment response assessment, and the potential for even greater assay sensitivity. What do these findings mean? Our findings suggest that cfDNA fragment analysis followed by ULP-WGS has the potential to be developed as a biomarker for treatment response and as a screening assay for early detection of MPNST. This study provides, to our knowledge, the first evidence for the ability of liquid biopsy to distinguish between benign and malignant tumors in a heritable cancer predisposition syndrome.