Serotonin 5-HT(1A) receptors are implicated in the pathophysiology of neuropsychiatric conditions. The goal of this study was to evaluate methods to derive 5-HT(1A) receptor parameters in the human brain with positron emission tomography (PET) and [carbonyl-(11)C]WAY 100635. Five healthy volunteer subjects were studied twice. Three methods of analysis were used to derive the binding potential (BP), and the specific to nonspecific equilibrium partition coefficient (k3/k4). Two methods, kinetic analysis based on a three compartment model and graphical analysis, used the arterial plasma time-activity curves as the input function to derive BP and k3/k4. A third method, the simplified reference tissue model (SRTM), derived the input function from uptake data of a region of reference, the cerebellum, and provided only k3/k4. All methods provided estimates of regional 5-HT(1A) receptor parameters that were highly correlated. Results were consistent with the known distribution of 5-HT(1A) receptors in the human brain. Compared with kinetic BP, graphical analysis slightly underestimated BP, and this phenomenon was mostly apparent in small size-high noise regions. Compared with kinetic k3/k4, the reference tissue method underestimated k3/k4 and the underestimation was apparent primarily in regions with high receptor density. Derivation of BP by both kinetic and graphical analysis was highly reliable, with an intraclass correlation coefficient (ICC) of 0.84 +/- 0.14 (mean +/- SD of 15 regions) and 0.84 +/- 0.19, respectively. In contrast, the reliability of k3/k4 was lower, with ICC of 0.53 +/- 0.28, 0.47 +/- 0.28, and 0.55 +/- 0.29 for kinetic, graphical, and reference tissue methods, respectively. In conclusion, derivation of BP by kinetic analysis using the arterial plasma input function appeared as the method of choice because of its higher test-retest reproducibility, lower vulnerability to experimental noise, and absence of bias.

Inflammation can act as a crucial mediator of epithelial-to-mesenchymal transition (EMT). In this study, we show that oncostatin M (OSM) is expressed in an autocrine/paracrine fashion in invasive breast carcinoma. OSM stimulation promotes spontaneous lung metastasis of MCF-7 xenografts in nude mice. A conspicuous epigenetic transition was induced by OSM stimulation not only in breast cancer cell lines but also in MCF-7 xenografts in nude mice. The expression of miR-200 and let-7 family members in response to OSM stimulation was downregulated in a signal transducer and activator of transcription factor 3 (Stat3)-dependent manner, resulting in comprehensive alterations of the transcription factors and oncoproteins targeted by these microRNAs. Inhibition of Stat3 activation or the ectopic expression of let-7 and miR-200 effectively reversed the mesenchymal phenotype of breast cancer cells. Stat3 promotes the transcription of Lin-28 by directly binding to the Lin-28 promoter, resulting in the repression of let-7 expression and concomitant upregulation of the let-7 target, high-mobility group A protein 2 (HMGA2). Knock down of HMGA2 significantly impairs OSM-driven EMT. Our data indicate that downregulation of let-7 and miR-200 levels initiates and maintains OSM-induced EMT phenotypes, and HMGA2 acts as a master switch of OSM-induced EMT. These findings highlight the importance of Stat3-coordinated Lin-28B-let-7-HMGA2 and miR-200-ZEB1 circuits in the cytokine-mediated phenotypic reprogramming of breast cancer cells.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA-sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging-mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety.

Circulating tumor DNA (ctDNA) in peripheral blood is a “liquid biopsy” that contains representative tumor information including gene mutations. Additionally, repeated ctDNA samples can be easily obtained to monitor response to treatment and disease progression, which may be especially valuable to lung cancer patients with tumors that cannot be easily biopsied or removed. To investigate the changes in ctDNA after surgical tumor resection, tumor and blood samples obtained before and after surgery were collected prospectively from 41 non-small lung cancer (NSCLC) patients. Somatic driver mutations in tumor DNA (tDNA) and pre- and post-op plasma ctDNA sample pairs were identified by targeted sequencing in several genes including EGFR, KRAS, and TP53 with an overall study concordance of 78.1% and sensitivity and specificity of 69.2% and 93.3%, respectively. Importantly, the frequency of 91.7% of ctDNA mutations decreased after surgery and these changes were observed as little as 2 days post-op. Moreover, the presence of ctDNA had a higher positive predictive value than that of six tumor biomarkers in current clinical use. This study demonstrates the use of targeted sequencing to reliably identify ctDNA changes in response to treatment, indicating a potential utility of this approach in the clinical management of NSCLC.

Numerous studies have indicated that primary tumors induce the formation of a pre-metastatic niche in distant organs by secreting tumor-derived factors. The present study shows that pre-exposure to chronic stress enhanced lung colonization efficiency by circulating tumor cells, suggesting that chronic stress critically influences pre-metastatic lungs before the arrival of disseminated tumor cells. Ablation of the sympathetic nerve function by 6-OHDA or blockage of the -adrenergic signaling by propranolol remarkably suppressed stress-induced lung metastasis. Depletion of circulating monocytes or lung macrophages strongly abolished stress-induced lung seeding by tumor cells, whereas treatment of mice with the -adrenergic agonist isoproterenol (ISO) during the pre-metastatic phase promoted the infiltration of macrophages to the lung. Meanwhile, the numbers of monocytes in peripheral blood, spleen, and bone marrow were remarkably increased in response to ISO stimulation. These data indicate that the -adrenergic signaling promotes lung metastatic colonization by tumor cells through increased output of monocytes in the pre-metastatic phase and infiltration of macrophages into the pre-metastatic lung. Mechanistic studies revealed that ISO stimulation upregulated the expression of CCL2 in pulmonary stromal cells and CCR2 in monocytes/macrophages, leading to the recruitment and infiltration of macrophages into the pre-metastatic lung. By inducing a response of monocytes/macrophages driven by the CCL2/CCR2 axis, stress-related catecholamine may act as a crucial factor in regulating the pre-metastatic niche for and lung colonization by tumor cells. Our data demonstrate that disturbance of host macro-environmental homeostasis has an influence on future metastatic organs.

In the present study, we demonstrate that prolonged treatment by trastuzumab induced resistance of NCI-N87 gastric cancer cells to trastuzumab. The resistant cells possessed typical characteristics of epithelial to mesenchymal transition (EMT)/cancer stem cells and acquired more invasive and metastatic potentials both in vitro and in vivo. Long term treatment with trastuzumab dramatically inhibited the phosphorylation of Akt, but triggered the activation of STAT3. The level of IL-6 was remarkably increased, implicating that the release of IL-6 that drives the STAT3 activation initiates the survival signaling transition. Furthermore, the Notch activities were significantly enhanced in the resistant cells, companied by upregulation of the Notch ligand Jagged-1 and the Notch responsive genes Hey1 and Hey2. Inhibiting the endogenous Notch pathway reduced the IL-6 expression and restored the sensitivities of the resistant cells to trastuzumab. Blocking of the STAT3 signaling abrogated IL-6-induced Jagged-1 expression, effectively inhibited the growth of the trastuzumab resistant cells, and enhanced the anti-tumor activities of trastuzumab in the resistant cells. These findings implicate that the IL-6/STAT3/Jagged-1/Notch axis may be a useful target and that combination of the Notch or STAT3 inhibitors with trastuzumab may prevent or delay clinical resistance and improve the efficacy of trastuzumab in gastric cancer.

Circulating tumor cell (CTC) enumeration and analysis has emerged as an important platform for cancer diagnosis and prognosis. A great challenge, however, is to efficiently capture low abundant CTCs with high purity from blood samples in a rapid and high-throughput manner for accurate and sensitive CTC detection. Herein, a new class of DNA-templated magnetic nanoparticle-quantum dot (QD)-aptamer copolymers (MQAPs) is developed for rapid magnetic isolation of CTCs from human blood with high capture efficiency and purity approaching 80%. The phenotype of CTCs is simultaneously profiled with QD photoluminescence (PL) at single cell level. These MQAPs are constructed through hybridization chain reaction to achieve amplified magnetic response, extraordinary binding selectivity for target cells over background cells, and ultra bright ensemble QD PL for single cell detection. MQAPs are free from nonspecific binding that would otherwise compromise the capture purity of target cells. As a result, facile isolation and enumeration of rare CTCs in blood samples could be achieved in 20 min with high sensitivity and accuracy.