Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

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Abstract

Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies.

Authors

Heike E. Daldrup-Link, Suchismita Mohanty, Celina Ansari, Olga Lenkov, Aubie Shaw, Ken Ito, Su Hyun Hong, Matthias Hoffmann, Laura Pisani, Nancy Boudreau, Sanjiv Sam Gambhir, Lisa M. Coussens

Microvascular resistance of the culprit coronary artery in acute ST-elevation myocardial infarction

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Abstract

BACKGROUND. Failed myocardial reperfusion is common and prognostically important after acute ST-elevation myocardial infarction (STEMI). The purpose of this study was to investigate coronary flow reserve (CFR), a measure of vasodilator capacity, and the index of microvascular resistance (IMR; mmHg × s) in the culprit artery of STEMI survivors.

METHODS. IMR (n = 288) and CFR (n = 283; mean age [SD], 60 [12] years) were measured acutely using guide wire–based thermodilution. Cardiac MRI disclosed left ventricular pathology, function, and volumes at 2 days (n = 281) and 6 months after STEMI (n = 264). All-cause death or first heart failure hospitalization was independently adjudicated (median follow-up 845 days).

RESULTS. Myocardial hemorrhage and microvascular obstruction occurred in 89 (42%) and 114 (54%) patients with evaluable T2*-MRI maps. IMR and CFR were associated with microvascular pathology (none vs. microvascular obstruction only vs. microvascular obstruction and myocardial hemorrhage) (median [interquartile range], IMR: 17 [12.0–33.0] vs. 17 [13.0–39.0] vs. 37 [21.0–63.0], P < 0.001; CFR: 1.7 [1.4–2.5] vs. 1.5 [1.1–1.8] vs. 1.4 [1.0–1.8], P < 0.001), whereas thrombolysis in myocardial infarction blush grade was not. IMR was a multivariable associate of changes in left ventricular end-diastolic volume (regression coefficient [95% CI] 0.13 [0.01, 0.24]; P = 0.036), whereas CFR was not (P = 0.160). IMR (5 units) was a multivariable associate of all-cause death or heart failure hospitalization (n = 30 events; hazard ratio [95% CI], 1.09 [1.04, 1.14]; P < 0.001), whereas CFR (P = 0.124) and thrombolysis in myocardial infarction blush grade (P = 0.613) were not. IMR had similar prognostic value for these outcomes as <50% ST-segment resolution on the ECG.

CONCLUSIONS. IMR is more closely associated with microvascular pathology, left ventricular remodeling, and health outcomes than the angiogram or CFR.

TRIAL REGISTRATION. NCT02072850.

FUNDING. A British Heart Foundation Project Grant (PG/11/2/28474), the National Health Service, the Chief Scientist Office, a Scottish Funding Council Senior Fellowship, a British Heart Foundation Intermediate Fellowship (FS/12/62/29889), and a nonfinancial research agreement with Siemens Healthcare.

Authors

David Carrick, Caroline Haig, Jaclyn Carberry, Vannesa Teng Yue May, Peter McCartney, Paul Welsh, Nadeem Ahmed, Margaret McEntegart, Mark C. Petrie, Hany Eteiba, Mitchell Lindsay, Stuart Hood, Stuart Watkins, Ahmed Mahrous, Samuli M.O. Rauhalammi, Ify Mordi, Ian Ford, Aleksandra Radjenovic, Naveed Sattar, Keith G. Oldroyd, Colin Berry

T cell Bim levels reflect responses to anti–PD-1 cancer therapy

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Abstract

Immune checkpoint therapy with PD-1 blockade has emerged as an effective therapy for many advanced cancers; however, only a small fraction of patients achieve durable responses. To date, there is no validated blood-based means of predicting the response to PD-1 blockade. We report that Bim is a downstream signaling molecule of the PD-1 pathway, and its detection in T cells is significantly associated with expression of PD-1 and effector T cell markers. High levels of Bim in circulating tumor-reactive (PD-1⁺CD11a^hiCD8⁺) T cells were prognostic of poor survival in patients with metastatic melanoma who did not receive anti–PD-1 therapy and were also predictive of clinical benefit in patients with metastatic melanoma who were treated with anti–PD-1 therapy. Moreover, this circulating tumor-reactive T cell population significantly decreased after successful anti–PD-1 therapy. Our study supports a crucial role of Bim in both T cell activation and apoptosis as regulated by PD-1 and PD-L1 interactions in effector CD8⁺ T cells. Measurement of Bim levels in circulating T cells of patients with cancer may provide a less invasive strategy to predict and monitor responses to anti–PD-1 therapy, although future prospective analyses are needed to validate its utility.

Authors

Roxana S. Dronca, Xin Liu, Susan M. Harrington, Lingling Chen, Siyu Cao, Lisa A. Kottschade, Robert R. McWilliams, Matthew S. Block, Wendy K. Nevala, Michael A. Thompson, Aaron S. Mansfield, Sean S. Park, Svetomir N. Markovic, Haidong Dong

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus

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Abstract

To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

Authors

Shereen Oon, Huy Huynh, Tsin Yee Tai, Milica Ng, Katherine Monaghan, Mark Biondo, Gino Vairo, Eugene Maraskovsky, Andrew D. Nash, Ian P. Wicks, Nicholas J. Wilson

Anakinra as a diagnostic challenge and treatment option for systemic autoinflammatory disorders of undefined etiology

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Abstract

BACKGROUND. Some adult patients presenting with unexplained pyrexia, serositis, skin rashes, arthralgia, myalgia, and other symptoms commonly found in autoinflammatory disorders may not fit a specific diagnosis, either because their clinical phenotype is nondiagnostic or genetic tests are negative. We used the term undifferentiated systemic autoinflammatory disorder (uSAID) to describe such cases. Given that well-defined autoinflammatory diseases show responses to IL-1 blockade, we evaluated whether anakinra was useful for both diagnosing and treating uSAID patients.

METHODS. We performed a retrospective analysis of consecutive patients presenting with uSAID between 2012–2015 who were treated with the recombinant IL-1 receptor antagonist anakinra. uSAID was diagnosed after excluding malignancy, infection, and pathogenic mutations in known hereditary fever syndromes (HFS) genes and where clinical criteria for adult onset Still’s disease (AOSD) were not met.

RESULTS. A total of 11 patients presented with uSAID (5 males and 6 females), with a mean time to diagnosis of 3.5 years (1–8 years). Patients were unresponsive or only partially controlled on disease-modifying antirheumatic drug (DMARD)/steroid treatment. Anakinra controlled symptoms within 4–6 weeks of starting treatment in 9 of 11 cases. Two patients discontinued therapy — one due to incomplete response and another due to severe injection-site reactions.

CONCLUSION. This retrospective case series demonstrates that the spectrum of poorly defined autoinflammatory disorders that show responsiveness to anakinra is considerable. Anakinra seems a viable treatment option for these patients, who are unresponsive to standard steroid/DMARD treatments. Moreover, given the mechanisms of action, response to anakinra implicates underlying IL-1 dysregulation in the disease pathogenesis of responding uSAIDs patients.

Authors

Stephanie R. Harrison, Dennis McGonagle, Sharmin Nizam, Stephen Jarrett, Jeroen van der Hilst, Michael F. McDermott, Sinisa Savic

Insulin decreases atherosclerosis by inducing endothelin receptor B expression

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Abstract

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe^–/– mice (Irs1/Apoe^–/–) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE^–/– mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin’s enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca²⁺]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE^–/– mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr^–/– and Irs1/Ldlr^–/– mice decreased NO production and accelerated atherosclerosis, compared with Ldlr^–/– mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production.

Authors

Kyoungmin Park, Akira Mima, Qian Li, Christian Rask-Madsen, Pingnian He, Koji Mizutani, Sayaka Katagiri, Yasutaka Maeda, I-Hsien Wu, Mogher Khamaisi, Simone Rordam Preil, Ernesto Maddaloni, Ditte Sørensen, Lars Melholt Rasmussen, Paul L. Huang, George L. King

Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

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Abstract

Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4⁺CD146⁺CCR5⁺ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4⁺CD146⁺CCR5⁺ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4⁺CD146⁺CCR5⁺ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA–transduced human T cells. Furthermore, the frequency of CD4⁺CD146⁺CCR5⁺ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT.

Authors

Wei Li, Liangyi Liu, Aurelie Gomez, Jilu Zhang, Abdulraouf Ramadan, Qing Zhang, Sung W. Choi, Peng Zhang, Joel K. Greenson, Chen Liu, Di Jiang, Elizabeth Virts, Stephanie L. Kelich, Hong Wei Chu, Ryan Flynn, Bruce R. Blazar, Helmut Hanenberg, Samir Hanash, Sophie Paczesny

Molecular profiling of dilated cardiomyopathy that progresses to heart failure

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Abstract

Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLN^R9C/+). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT, P = 8 × 10^–4) and activation of proinflammatory signaling with notable cardiomyocyte-specific induction of a subset of profibrotic cytokines including TGFβ2 and TGFβ3. These changes progressed through DCM and HF, resulting in substantial fibrosis (17.6% of left ventricle [LV] vs. WT, P = 6 × 10^–33). Cardiomyocytes displayed a marked shift in metabolic gene transcription: downregulation of aerobic respiration and subsequent upregulation of glucose utilization, changes coincident with attenuated expression of PPARα and PPARγ coactivators -1α (PGC1α) and -1β, and increased expression of the metabolic regulator T-box transcription factor 15 (Tbx15). Comparing DCM transcriptional profiles with those in hypertrophic cardiomyopathy (HCM) revealed similar and distinct molecular mechanisms. Our data suggest that cardiomyocyte-specific cytokine expression, early fibroblast activation, and the shift in metabolic gene expression are hallmarks of cardiomyopathy progression. Notably, key components of these profibrotic and metabolic networks were disease specific and distinguish DCM from HCM.

Authors

Michael A. Burke, Stephen Chang, Hiroko Wakimoto, Joshua M. Gorham, David A. Conner, Danos C. Christodoulou, Michael G. Parfenov, Steve R. DePalma, Seda Eminaga, Tetsuo Konno, Jonathan G. Seidman, Christine E. Seidman

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

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Abstract

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes.

Authors

Anup Sood, Alexandra M. Miller, Edi Brogi, Yunxia Sui, Joshua Armenia, Elizabeth McDonough, Alberto Santamaria-Pang, Sean Carlin, Aleksandra Stamper, Carl Campos, Zhengyu Pang, Qing Li, Elisa Port, Thomas G. Graeber, Nikolaus Schultz, Fiona Ginty, Steven M. Larson, Ingo K. Mellinghoff