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Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation,which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model,and near-complete suppression of cyst growth was found by tetrazolo-CFTR(inh)-172,a tetrazolo-derived thiazolidinone,and Ph-GlyH-101,a phenyl-derived glycine hydrazide,without an effect on cell proliferation. These compounds also inhibited cyst number and growth by {\textgreater}80{\%} in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR(inh)-172 and Ph-GlyH-101 to neonatal,kidney-specific PKD1 knockout mice produced stable,therapeutic inhibitor concentrations of {\textgreater}3 microM in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD. View Publication

IDH1 and IDH2 mutations occur frequently in gliomas and acute myeloid leukemia,leading to simultaneous loss and gain of activities in the production of $\alpha$-ketoglutarate ($\alpha$-KG) and 2-hydroxyglutarate (2-HG),respectively. Here we demonstrate that 2-HG is a competitive inhibitor of multiple $\alpha$-KG-dependent dioxygenases,including histone demethylases and the TET family of 5-methlycytosine (5mC) hydroxylases. 2-HG occupies the same space as $\alpha$-KG does in the active site of histone demethylases. Ectopic expression of tumor-derived IDH1 and IDH2 mutants inhibits histone demethylation and 5mC hydroxylation. In glioma,IDH1 mutations are associated with increased histone methylation and decreased 5-hydroxylmethylcytosine (5hmC). Hence,tumor-derived IDH1 and IDH2 mutations reduce $\alpha$-KG and accumulate an $\alpha$-KG antagonist,2-HG,leading to genome-wide histone and DNA methylation alterations. View Publication

Immunotherapy is innovating clinical cancer management. Nevertheless,only a small fraction of patient's benefit from current immunotherapies. To improve clinical management of cancer immunotherapy,it is critical to develop strategies for response monitoring and prediction. In this study,we describe inducible T-cell costimulator (ICOS) as a conserved mediator of immune response across multiple therapy strategies. ICOS expression was evaluated by flow cytometry,89Zr-DFO-ICOS mAb PET/CT imaging was performed on Lewis lung cancer models treated with different immunotherapy strategies,and the change in tumor volume was used as a read-out for therapeutic response. ImmunoPET imaging of ICOS enabled sensitive and specific detection of activated T cells and early benchmarking of immune response. A STING (stimulator of interferon genes) agonist was identified as a promising therapeutic approach in this manner. The STING agonist generated significantly stronger immune responses as measured by ICOS ImmunoPET and delayed tumor growth compared with programmed death-1 checkpoint blockade. More importantly,ICOS ImmunoPET enabled early and robust prediction of therapeutic response across multiple treatment regimens. These data show that ICOS is an indicator of T-cell-mediated immune response and suggests ICOS ImmunoPET as a promising strategy for monitoring,comparing,and predicting immunotherapy success in cancer. SIGNIFICANCE: ICOS ImmunoPET is a promising strategy to noninvasively predict and monitor immunotherapy response.See related commentary by Choyke,p. 2975. View Publication

Expression of BAX,without another death stimulus,proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1 beta-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family),as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However,the fall in mitochondrial membrane potential,production of reactive oxygen species,cytoplasmic vacuolation,and plasma membrane permeability that are downstream of BAX still occurred. Thus,BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases. View Publication

Detection and characterization of rare circulating tumor cells (CTCs) in patients' blood is important for the diagnosis and monitoring of cancer. The traditional way of counting CTCs via fluorescent images requires a series of tedious experimental procedures and often impacts the viability of cells. Here we present a method for label-free detection of CTCs from patient blood samples,by taking advantage of data analysis of bright field microscopy images. The approach uses the convolutional neural network,a powerful image classification and machine learning algorithm to perform label-free classification of cells detected in microscopic images of patient blood samples containing white blood cells and CTCs. It requires minimal data pre-processing and has an easy experimental setup. Through our experiments,we show that our method can achieve high accuracy on the identification of rare CTCs without the need for advanced devices or expert users,thus providing a faster and simpler way for counting and identifying CTCs. With more data becoming available in the future,the machine learning model can be further improved and can serve as an accurate and easy-to-use tool for CTC analysis. View Publication

Mammalian target of rapamycin (mTOR) is frequently upregulated in hepatocellular carcinoma (HCC). Blockage of mTOR was found to induce marked reduction in HCC growth in preclinical models. In the present study,we tested a novel mTOR inhibitor,Torin-2,for its antitumor efficacy in HCC cell lines Hep G2,SNU-182 and Hep 3B2.1-7. The HCC cell lines were cultured in vitro. These cells were treated with Torin-2. Cell apoptosis was evaluated by Annexin V staining. Cell proliferation and cell cycle progression were determined by Ki67 staining and propidium iodide staining,respectively. mTOR signaling,autophagy induction and expression of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) were assessed by western blot analysis. The UHRF1 mRNA level was determined by real-time PCR. We found that Torin-2 effectively suppressed the growth and survival of HCC cell lines,demonstrated by reduced proliferation and a high rate of apoptosis. Further study elucidated that in addition to blocking mTOR complex 1 (mTORC1)-associated cell cycle progression and induction of autophagy,Torin-2 downregulated transcription of UHRF1,an essential regulator of DNA methylation that is highly expressed in HCC cell lines. Consistently,the level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) was higher after treatment of the HCC cell lines with Torin-2. The downregulation of UHRF1 by Torin-1 was partially due to a decrease in the UHRF1 mRNA level. Torin-2 effectively inhibited HCC cell proliferation through induction of autophagy. Torin‑2-induced downregulation of UHRF1 expression may also contribute to its antitumor effect. Our research provides new clues regarding the antitumor effects of Torin-2 and sheds light on a novel therapeutic approach for HCC. View Publication

The function of a T cell depends on its subtype and activation state. Here,we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2,CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99{\%} accuracy,and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97{\%} accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function. View Publication

Pulmonary sarcoidosis has unknown etiology,a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities,but cell sources and mechanisms have not been identified. In the current study,we found that BALF exosomes from sarcoidosis patients,but not from healthy individuals,induced a dose-dependent elevation of intracellular IL-1$\beta$ in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1$\beta$,IL-6 and TNF from both PBMCs and enriched monocytes,suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients,and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast,a cysteinyl leukotriene receptor antagonist. Further,reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation,and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis. View Publication

Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels,but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected,leading to liver-selective pharmacology,when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality,as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441,which reduces glucose levels and improves lipid profiles in an animal model of diabetes. View Publication

Lymphatic endothelial cells (LECs) chemoattract na{\{i}}ve T cells and promote their survival in the lymph nodes and can cross-present antigens to na{\"{i}}ve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However the functional consequence of LEC priming of CD8+ T cells is unknown. Here we show that while many proliferating LEC-educated T cells enter early apoptosis the remainders comprise a long-lived memory subset with transcriptional metabolic and phenotypic features of central memory and stem cell-like memory T cells. In vivo these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.""" View Publication