Dynamic insulin-stimulated mTOR/GSK3 signaling in peripheral immune cells: Preliminary evidence for an association with lithium response in bipolar disorder.

INTRODUCTION: A key mechanism of lithium is the inhibition of glycogen synthase kinase-3ß (GSK3ß) and activation of mammalian target of rapamycin (mTOR), two contributors to insulin signaling. We explored the relationship between these markers and clinical response to lithium in bipolar disorder (BD). METHODS: Thirty-four subjects with BD who had been taking lithium for ≥2 years and had a maintenance lithium Alda score defined as either high (≥7; n = 20) or low (≤2; n = 14) were included in the study. Baseline protein expression of GSK3ß and mTOR (total and phosphorylated (p)) was obtained from a buffy coat. Peripheral blood mononuclear cells (PBMCs) from a subset of each group (n = 11) were stimulated with insulin (10 µg) and change in protein expression was determined using Western blot. RESULTS: In buffy coat samples, significantly higher levels of pmTOR were present in subjects with an Alda score ≤2 (lithium non-responsive), relative to those with scores ≥7 (lithium-responsive). No differences were observed for pGSK3ß. In contrast, functional PBMC responses to 5 min of insulin stimulation demonstrated robust increases in pGSK3ß (87.05 ± 43.41%) and pmTOR (105.7 ± 66.48%) in the lithium responsive group only. This contrasted observed decreases in pGSK3ß (34.08 ± 16.12%) and pmTOR (37.84 ± 14.39%) 5 mins post-insulin in non-responders. CONCLUSIONS: Dynamic increases in pmTOR and pGSK3ß post-insulin stimulation may reflect an immunometabolic state that facilitates lithium response. Further prospective analyses are needed to replicate and extend these preliminary findings and further investigate the role of insulin signaling in lithium response in BD.

Maternal Monocytes Respond to Cell-Free Fetal DNA and Initiate Key Processes of Human Parturition.

Throughout gestation, the maternal immune system is tightly modulated to allow growth of a semiallogeneic fetus. During the third trimester, the maternal immune system shifts to a proinflammatory phenotype in preparation for labor. What induces this shift remains unclear. Cell-free fetal DNA (cffDNA) is shed by the placenta and enters maternal circulation throughout pregnancy. Levels of cffDNA are increased as gestation progresses and peak before labor, coinciding with a shift to proinflammatory maternal immunity. Furthermore, cffDNA is abnormally elevated in plasma from women with complications of pregnancy, including preterm labor. Given the changes in maternal immunity at the end of pregnancy and the role of sterile inflammation in the pathophysiology of spontaneous preterm birth, we hypothesized that cffDNA can act as a damage-associated molecular pattern inducing an inflammatory cytokine response that promotes hallmarks of parturition. To test this hypothesis, we stimulated human maternal leukocytes with cffDNA from primary term cytotrophoblasts or maternal plasma and observed significant IL-1ß and CXCL10 secretion, which coincides with phosphorylation of IFN regulatory factor 3 and caspase-1 cleavage. We then show that human maternal monocytes are crucial for the immune response to cffDNA and can activate bystander T cells to secrete proinflammatory IFN-γ and granzyme B. Lastly, we find that the monocyte response to cffDNA leads to vascular endothelium activation, induction of myometrial contractility, and PGE2 release in vitro. Our results suggest that the immune response to cffDNA can promote key features of the parturition cascade, which has physiologic consequences relevant to the timing of labor.

Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation.

Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(-) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Identification of novel, immune-mediating extracellular vesicles in human lymphatic effluent draining primary cutaneous melanoma.

Epithelial tumors including melanoma often first metastasize to regional, sentinel lymph nodes (SLN). Thus, the presence of SLN metastases is a critical prognostic factor of survival. Prior to metastasis, accumulating evidence suggests the SLN is immunologically compromised; however, the process by which pre-metastatic niche formation occurs remains unknown. In this prospective study, freshly dissected, afferent lymphatic fluid was obtained during SLN biopsy in three patients with primary cutaneous melanoma. Lymphatic extracellular vesicles (L-EV) were visualized by transmission electron microscopy and proteomic cargo profiled by mass spectrometry. Flow cytometry assessed L-EV effects on autologous dendritic cell maturation in vitro. Immunogold electron microscopy and immunohistochemistry visualized expression of EV cargo within the primary tumor and SLN. Lymphatic extracellular vesicles from each afferent lymphatic channel demonstrated inhibition of autologous dendritic cell maturation. Proteomic profiling identified 81 peptides shared among the L-EV preparations including a signature of 18 immune-modulating proteins including previously established inhibitor of dendritic cell maturation, S100A9. Immunohistochemistry and immunogold electron microscopy confirmed S100A9 tracking along the lymphatic path, from keratinocytes in the primary tumor to sub-capsular macrophages in the SLN. Our findings suggest L-EV cargo may serve as early mediators of tumor-induced immune subversion in regional lymph nodes, by preceding malignant cells and trafficking within the lymphatic vasculature to harbor the first pre-metastatic niche.

Insulin-stimulated mTOR activation in peripheral blood mononuclear cells associated with early treatment response to lithium augmentation in rodent model of antidepressant-resistance.

Lithium has been shown to have some therapeutic efficacy as an adjunctive treatment for intractable forms of major depression. Activation of mammalian target of rapamycin (mTOR) and inhibition of glycogen synthase kinase-3ß (GSK3ß) have been implicated in its putative mechanisms of action. These proteins are integral components of the insulin signaling pathway, which may serve as a critical co-regulator of drug action. Utilizing an animal model of tricyclic antidepressant resistance, we investigated the relationship between insulin signaling and antidepressant response to lithium augmentation. Pre-treatment with adrenocorticotropic hormone (ACTH 100 µg/day i.p.) for 14 days effectively blocked the immobility-reducing effects of an acute dose of imipramine (10 mg/kg i.p.) in the forced swim test (FST). Lithium augmentation (100 mg/kg i.p.) rescued the antidepressant-like effects of imipramine in this model. Total and phosphorylated (p) levels of protein kinase B (Akt), mTOR, and GSK3ß protein were quantified in the infralimbic cortex (ILPFC) following FST stress via Western blot. Levels of mTOR and pmTOR were further quantified in isolated peripheral blood mononuclear cells (PBMCs) following insulin stimulation (10 mg/mL for 5 min) via ELISA. Elevated levels of phosphorylated insulin signaling proteins were present in the ILPFC of ACTH-pretreated animals that received both imipramine and lithium, together with a concurrent increase in mTOR activation in PBMCs. Large correlations were observed between immobility time and insulin-stimulated mTOR levels in PBMCs. We propose that PBMC insulin challenge may be a useful probe for predicting antidepressant response to lithium administration, and potentially other therapies acting via similar mechanisms of action.

CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment.

In patients with metastatic melanoma, high blood levels of galectin-9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin-9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin-9 with macrophages. We aimed to determine whether galectin-9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor-supportive. It was determined that incubation of CD68+ macrophages with galectin-9 or anti-CD206 blocked target binding and that both CD206 and galectin-9 were detected by immunoprecipitation of cell lysates. CD206 and galectin-9 had a binding affinity of 2.8 × 10-7  m. Galectin-9 causes CD206+ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP-1), but less macrophage-derived chemokine (MDC). Galectin-9 had no effect on classical monocyte subsets, but caused expansion of the non-classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin-9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin-9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti-tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Contraction of T cell richness in lung cancer brain metastases.

Very little is known about how the adaptive immune system responds to clonal evolution and tumor heterogeneity in non-small cell lung cancer. We profiled the T-cell receptor ß complementarity determining region 3 in 20 patients with fully resected non-small cell lung cancer primary lesions and paired brain metastases. We characterized the richness, abundance and overlap of T cell clones between pairs, in addition to the tumor mutation burden and predicted neoantigens. We found a significant contraction in the number of unique T cell clones in brain metastases compared to paired primary cancers. The vast majority of T cell clones were specific to a single lesion, and there was minimal overlap in T cell clones between paired lesions. Despite the contraction in the number of T cell clones, brain metastases had higher non-synonymous mutation burdens than primary lesions. Our results suggest that there is greater richness of T cell clones in primary lung cancers than their paired metastases despite the higher mutation burden observed in metastatic lesions. These results may have implications for immunotherapy.

Antibody-targeted paclitaxel loaded nanoparticles for the treatment of CD20+ B-cell lymphoma.

We developed a nano-antibody targeted chemotherapy (nATC) delivery strategy in which tumor specific and clinically relevant antibodies (rituximab, anti-CD20) are non-covalently bound to the albumin scaffold of nab-paclitaxel (ABX). We define the nanoparticle formed when the 2 drugs are bound (AR160). The newly created nATC retains the cytotoxicity of ABX and CD20 affinity of rituximab in vitro. We describe the binding characteristics of the ABX and rituximab in AR160 using peptide mapping/Biacore approach. Flow-based methods, including ImageStream and nanoparticle tracking, were used to characterize the AR160 particles in vitro. A mouse model of human B-cell lymphoma was utilized to test in vivo efficacy of AR160 therapy, which suggested improved tumor targeting (biodistribution) as the most likely mechanism of AR160 therapeutic superiority over ABX or rituximab alone. These data suggest a novel platform for nATC delivery using a slight modification of existing cancer drugs with significantly improved treatment efficacy.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Antidepressant actions of lateral habenula deep brain stimulation differentially correlate with CaMKII/GSK3/AMPK signaling locally and in the infralimbic cortex.

High frequency deep brain stimulation (DBS) of the lateral habenula (LHb) reduces symptoms of depression in severely treatment-resistant individuals. Despite the observed therapeutic effects, the molecular underpinnings of DBS are poorly understood. This study investigated the efficacy of high frequency LHb DBS (130Hz; 200µA; 90µs) in an animal model of tricyclic antidepressant resistance. Further, we reported DBS mediated changes in Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα/ß), glycogen synthase kinase 3 (GSK3α/ß) and AMP-activated protein kinase (AMPK) both locally and in the infralimbic cortex (IL). Protein expressions were then correlated to immobility time during the forced swim test (FST). Antidepressant actions were quantified via FST. Treatment groups comprised of animals treated with adrenocorticotropic hormone alone (ACTH; 100µg/day, 14days, n=7), ACTH with active DBS (n=7), sham DBS (n=8), surgery only (n=8) or control (n=8). Active DBS significantly reduced immobility in ACTH-treated animals (p<0.05). For this group, western blot results demonstrated phosphorylation status of LHb CaMKIIα/ß and GSK3α/ß significantly correlated to immobility time in the FST. Concurrently, we observed phosphorylation status of CaMKIIα/ß, GSK3α/ß, and AMPK in the IL to be negatively correlated with antidepressant actions of DBS. These findings suggest that activity dependent phosphorylation of CaMKIIα/ß, and GSK3α/ß in the LHb together with the downregulation of CaMKIIα/ß, GSK3α/ß, and AMPK in the IL, contribute to the antidepressant actions of DBS.

Peripheral proinflammatory markers associated with ketamine response in a preclinical model of antidepressant-resistance.

Ketamine, N-methyl-d-aspartate (NMDA) receptor antagonist and anti-inflammatory agent, has rapid therapeutic effects in a subset of patients with more intractable forms of depression. Irregular proinflammatory cytokine and acute-reactive protein levels have been reported in clinical and preclinical depression research. We explored the association between the rapid antidepressant-like effects of ketamine and peripheral proinflammatory profile in a model of antidepressant-resistance. Male Wistar rats were pre-treated with ACTH-(1-24) 100µg/d or saline (0.9%) for 14d. Antidepressant-like effects were assessed with the forced swim test (FST). Ketamine (10mg/kg) significantly reduced immobility duration in saline-pretreated control animals. In contrast, a divergent response was observed in ACTH-pretreated antidepressant resistant animals, with 50% responders and 50% non-responders. Plasma samples were analyzed via enzyme-linked immunosorbent assay (ELISA) for interleukin 6 (IL-6), tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP). Levels of CRP and TNFα differentiated ketamine responders and non-responders.

Generation and sequencing of pulmonary carcinoid tumor cell lines.

INTRODUCTION: Pulmonary carcinoid tumors account for approximately 5% of all lung malignancies in adults, and comprise 30% of all carcinoid tumors. There are limited reagents available to study these rare tumors, and consequently no major advances have been made for patient treatment. We report the generation and characterization of human pulmonary carcinoid tumor cell lines to study underlying biology, and to provide models for testing novel chemotherapeutic agents. METHODS: Tissue was harvested from three patients with primary pulmonary typical carcinoid tumors undergoing surgical resection. The tumor was dissociated and plated onto dishes in culture media. The established cell lines were characterized by immunohistochemistry, Western blotting, and cell proliferation assays. Tumorigenicity was confirmed by soft agar growth and the ability to form tumors in a mouse xenograft model. Exome and RNA sequencing of patient tumor samples and cell lines was performed using standard protocols. RESULTS: Three typical carcinoid tumor lines grew as adherent monolayers in vitro, expressed neuroendocrine markers consistent with the primary tumor, and formed colonies in soft agar. A single cell line produced lung tumors in nude mice after intravenous injection. Exome and RNA sequencing of this cell line showed lineage relationship with the primary tumor, and demonstrated mutations in a number of genes related to neuronal differentiation. CONCLUSION: Three human pulmonary typical carcinoid tumor cell lines have been generated and characterized as a tool for studying the biology and novel treatment approaches for these rare tumors.

Ovarian cancer-associated mutations disable catalytic activity of CDK12, a kinase that promotes homologous recombination repair and resistance to cisplatin and poly(ADP-ribose) polymerase inhibitors.

Mutations in the tumor suppressors BRCA1 and BRCA2, which encode proteins that are key participants in homologous recombination (HR) repair, occur in ∼20% of high grade serous ovarian cancers. Although only 20% of these tumors have mutations in BRCA1 and BRCA2, nearly 50% of these tumors have defects in HR. Notably, however, the underlying genetic defects that give rise to HR defects in the absence of BRCA1 and BRCA2 mutations have not been fully elucidated. Here we show that the recurrent somatic CDK12 mutations identified in ovarian cancers impair the catalytic activity of this kinase, which is involved in the transcription of a subset of genes, including BRCA1 and other DNA repair genes. Furthermore, we show that disabling CDK12 function in ovarian cancer cells reduces BRCA1 levels, disrupts HR repair, and sensitizes these cells to the cross-linking agents melphalan and cisplatin and to the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888). Taken together, these findings suggest that many CDK12 mutations are an unrecognized cause of HR defects in ovarian cancers.

Role for putative hepatocellular carcinoma stem cell subpopulations in biological response to incomplete thermal ablation: in vitro and in vivo pilot study.

PURPOSE: To investigate the potential role for CD44(+) and CD90(+) hepatocellular carcinoma (HCC) cellular subpopulations in biological response to thermal ablation-induced heat stress. METHODS: This study was approved by the institutional animal care committee. The N1S1 rat HCC cell line was subjected to sublethal heat stress (45 °C) or control (37 °C) for 10 min, costained with fluorescent-labeled antibodies against CD44, CD90, and 7-AAD after a 48-h recovery and analyzed by flow cytometry to assess the percentage of live CD44(+) and CD90(+) HCC cells (n = 4). Experiments were repeated with pretreatment of N1S1 cells with a dose titration of the dual PI3K-mTOR inhibitor BEZ235 or vehicle control (n = 3). Rats bearing orthotopic N1S1 tumors were subjected to ultrasound-guided partial laser ablation (n = 5) or sham ablation (n = 3), euthanized 24 h after ablation, and liver/tumor analyzed for immunohistochemical staining of CD44 and CD90. Differences between groups were compared with an unpaired t test. RESULTS: Sublethal heat stress induced a significant increase in the relative proportion of live CD44(+) and CD90(+) HCC cells compared to the control group: CD44(+)CD90(-) (5.3-fold; p = 0.0001), CD44(-)CD90(+) (2.4-fold; p = 0.003), and CD44(+)CD90(+) (22.0-fold; p < 0.03). Inhibition of PI3K-mTOR prevented heat stress-induced enrichment of the population of live CD44(+) HCC cells (p < 0.01), but not CD90(+) cells (p > 0.10). Immunohistochemical analysis demonstrated preferential localization of clusters of CD44(+) cells at both the tumor margin and ablation margin. CONCLUSION: These studies provide experimental evidence supporting a role for HCC cells expressing the putative stem cell marker CD44 in HCC response to heat stress.

A comparative genomic approach for identifying synthetic lethal interactions in human cancer.

Synthetic lethal interactions enable a novel approach for discovering specific genetic vulnerabilities in cancer cells that can be exploited for the development of therapeutics. Despite successes in model organisms such as yeast, discovering synthetic lethal interactions on a large scale in human cells remains a significant challenge. We describe a comparative genomic strategy for identifying cancer-relevant synthetic lethal interactions whereby candidate interactions are prioritized on the basis of genetic interaction data available in yeast, followed by targeted testing of candidate interactions in human cell lines. As a proof of principle, we describe two novel synthetic lethal interactions in human cells discovered by this approach, one between the tumor suppressor gene SMARCB1 and PSMA4, and another between alveolar soft-part sarcoma-associated ASPSCR1 and PSMC2. These results suggest therapeutic targets for cancers harboring mutations in SMARCB1 or ASPSCR1 and highlight the potential of a targeted, cross-species strategy for identifying synthetic lethal interactions relevant to human cancer.

ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status.

Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

Molecular bioluminescence imaging as a noninvasive tool for monitoring tumor growth and therapeutic response to MRI-guided laser ablation in a rat model of hepatocellular carcinoma.

OBJECTIVES: The objective of this study was to quantitatively compare tumor imaging by magnetic resonance imaging (MRI) and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2-dimensional BLI and 3-dimensional diffuse luminescence tomography (3D DLIT) in an orthotopic rat model of hepatocellular carcinoma. MATERIALS AND METHODS: This study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (n = 3) or a heat shock element luciferase reporter (HSE-luc; n = 4) into the liver. All rats underwent MRI to assess tumor establishment and volume and 2-dimensional BLI to assess tumor luminescence at day 7 with subsequent MRI and 2D BLI and 3D DLIT in select animals at days 14 and 21. Magnetic resonance imaging-guided laser ablation of the tumor was performed with preablation and postablation 2D BLI and/or 3D DLIT (n = 2). The tumors underwent histopathologic analysis to assess tumor viability. RESULTS: The MRI scans demonstrated hyperintense T2-weighted lesions at 3 of 3 and 4 of 4 sites in the empty vector and HSE-luc rats, respectively. Two-dimensional BLI quantitation demonstrated 23.0-fold higher radiance in the HSE-luc group compared with the empty vector group at day 7 (P < 0.01) and a significant correlation with tumor volume by MRI (r = 0.86; P < 0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. Three-dimensional DLIT quantitation demonstrated better agreement with the percentage of nonviable tumor by histopathology than did 2D BLI quantitation after the MRI-guided laser ablation. CONCLUSIONS: Bioluminescence imaging is feasible as a noninvasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of hepatocellular carcinoma.

MicroRNA-mRNA interactions in a murine model of hyperoxia-induced bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary dysplasia is a chronic lung disease of premature neonates characterized by arrested pulmonary alveolar development. There is increasing evidence that microRNAs (miRNAs) regulate translation of messenger RNAs (mRNAs) during lung organogenesis. The potential role of miRNAs in the pathogenesis of BPD is unclear. RESULTS: Following exposure of neonatal mice to 80% O2 or room air (RA) for either 14 or 29 days, lungs of hyperoxic mice displayed histological changes consistent with BPD. Comprehensive miRNA and mRNA profiling was performed using lung tissue from both O2 and RA treated mice, identifying a number of dynamically regulated miRNAs and associated mRNA target genes. Gene ontology enrichment and pathway analysis revealed that hyperoxia modulated genes involved in a variety of lung developmental processes, including cell cycle, cell adhesion, mobility and taxis, inflammation, and angiogenesis. MiR-29 was prominently increased in the lungs of hyperoxic mice, and several predicted mRNA targets of miR-29 were validated with real-time PCR, western blotting and immunohistochemistry. Direct miR-29 targets were further validated in vitro using bronchoalveolar stem cells. CONCLUSION: In newborn mice, prolonged hyperoxia induces an arrest of alveolar development similar to that seen in human neonates with BPD. This abnormal lung development is accompanied by significant increases in the levels of multiple miRNAs and corresponding decreases in the levels of predicted mRNA targets, many of which have known or suspected roles in pathways altered in BPD. These data support the hypothesis that dynamic regulation of miRNAs plays a prominent role in the pathophysiology of BPD.

c-Myc regulates self-renewal in bronchoalveolar stem cells.

BACKGROUND: Bronchoalveolar stem cells (BASCs) located in the bronchoalveolar duct junction are thought to regenerate both bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury. The mechanisms directing self-renewal in BASCs are poorly understood. METHODS: BASCs (Sca-1(+), CD34(+), CD31(-) and, CD45(-)) were isolated from adult mouse lung using FACS, and their capacity for self-renewal and differentiation were demonstrated by immunostaining. A transcription factor network of 53 genes required for pluripotency in embryonic stem cells was assessed in BASCs, Kras-initiated lung tumor tissue, and lung organogenesis by real-time PCR. c-Myc was knocked down in BASCs by infection with c-Myc shRNA lentivirus. Comprehensive miRNA and mRNA profiling for BASCs was performed, and significant miRNAs and mRNAs potentially regulated by c-Myc were identified. We explored a c-Myc regulatory network in BASCs using a number of statistical and computational approaches through two different strategies; 1) c-Myc/Max binding sites within individual gene promoters, and 2) miRNA-regulated target genes. RESULTS: c-Myc expression was upregulated in BASCs and downregulated over the time course of lung organogenesis in vivo. The depletion of c-Myc in BASCs resulted in decreased proliferation and cell death. Multiple mRNAs and miRNAs were dynamically regulated in c-Myc depleted BASCs. Among a total of 250 dynamically regulated genes in c-Myc depleted BASCs, 57 genes were identified as potential targets of miRNAs through miRBase and TargetScan-based computational mapping. A further 88 genes were identified as potential downstream targets through their c-Myc binding motif. CONCLUSION: c-Myc plays a critical role in maintaining the self-renewal capacity of lung bronchoalveolar stem cells through a combination of miRNA and transcription factor regulatory networks.

Severe osteogenesis imperfecta in cyclophilin B-deficient mice.

Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB-deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB-deficient cells and tissues from CypB-knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone.