Influenza-Specific T Cell Memory: Influenza of Obesity, Weight Loss, Weight Gain

Obesity is a global epidemic, with 10% of men and 14% of women obese worldwide. Obesity is a known risk factor for increased complications and death from infection with influenza virus, and impairs the T cell response to both influenza infection and vaccination. As obesity is primarily a metabolic disorder, and immune cell function is dictated by metabolism of the immune cell, the effect of obesity on memory T cell metabolism following a secondary influenza infection was investigated. This dissertation addressed whether the metabolic environment at the time of memory T cell generation or at the time of re-challenge would influence T cell metabolism and function. C57BL/6J high fat diet-induced obese mice were infected with X-31 influenza virus to generate memory T cells, then switched to a low-fat diet to induce weight loss. Following weight loss and normalized fasting glucose levels, mice were re-infected with influenza Puerto Rico/8/34 (PR8) to activate the memory T cells in a newly generated lean state. Conversely, lean mice were infected with X-31 to generate memory T cells followed by a diet switch to a high fat diet to induce obesity. Following weight gain and elevated fasting glucose levels, mice were re-exposed to PR8. Compared with mice that were always lean, mice that were obese for both primary and secondary influenza infections had impaired T cell metabolism and function. Mice that lost weight maintained a metabolic phenotype that paralleled the always obese metabolic phenotype along with dysregulated frequencies of central memory, effector memory, and tissue resident memory T cell populations and decreased function of influenza-specific memory T cell subsets. Mice that had gained weight, and were previously lean, maintained a metabolic profile similar to the mice that were always lean, although also had T cell subset alterations and diminished function. Altogether, this data demonstrates that metabolic environment present at the time of memory T cell generation and at time of secondary immune challenge both impact T cell function. For the first time, obesity has been shown to alter T cell metabolism, and we demonstrate that weight loss will not restore T cell metabolism or function.Doctor of Philosoph

Genomic loss of tumor suppressor miRNA-204 promotes cancer cell migration and invasion by activating AKT/mTOR/Rac1 signaling and actin reorganization

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis

Lineage of origin in rhabdomyosarcoma informs pharmacological response

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.Stem Cell and Regenerative Biolog

Detection of early-stage lung cancer in sputum using automated flow cytometry and machine learning

Abstract Background Low-dose spiral computed tomography (LDCT) may not lead to a clear treatment path when small to intermediate-sized lung nodules are identified. We have combined flow cytometry and machine learning to develop a sputum-based test (CyPath Lung) that can assist physicians in decision-making in such cases. Methods Single cell suspensions prepared from induced sputum samples collected over three consecutive days were labeled with a viability dye to exclude dead cells, antibodies to distinguish cell types, and a porphyrin to label cancer-associated cells. The labeled cell suspension was run on a flow cytometer and the data collected. An analysis pipeline combining automated flow cytometry data processing with machine learning was developed to distinguish cancer from non-cancer samples from 150 patients at high risk of whom 28 had lung cancer. Flow data and patient features were evaluated to identify predictors of lung cancer. Random training and test sets were chosen to evaluate predictive variables iteratively until a robust model was identified. The final model was tested on a second, independent group of 32 samples, including six samples from patients diagnosed with lung cancer. Results Automated analysis combined with machine learning resulted in a predictive model that achieved an area under the ROC curve (AUC) of 0.89 (95% CI 0.83–0.89). The sensitivity and specificity were 82% and 88%, respectively, and the negative and positive predictive values 96% and 61%, respectively. Importantly, the test was 92% sensitive and 87% specific in cases when nodules were < 20 mm (AUC of 0.94; 95% CI 0.89–0.99). Testing of the model on an independent second set of samples showed an AUC of 0.85 (95% CI 0.71–0.98) with an 83% sensitivity, 77% specificity, 95% negative predictive value and 45% positive predictive value. The model is robust to differences in sample processing and disease state. Conclusion CyPath Lung correctly classifies samples as cancer or non-cancer with high accuracy, including from participants at different disease stages and with nodules < 20 mm in diameter. This test is intended for use after lung cancer screening to improve early-stage lung cancer diagnosis. Trial registration ClinicalTrials.gov ID: NCT03457415; March 7, 201

Genomic Loss of Tumor Suppressor miRNA-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization

<div><p>Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including <em>brain-derived neurotrophic factor</em> (<em>BDNF</em>), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.</p> </div

MiR-204 regulates expression of BDNF in cancers.

<p>A–C, increased BDNF expression correlates strongly with lower miR-204 expression in multiple cancers. Graphical representation of qRT-PCR analysis showing the inverse correlation between miR-204 and <i>BDNF</i> in pediatric renal tumors (<i>n</i> = 38; A), advanced stage ovarian cancers (<i>n</i> = 11; B) and breast cancers (<i>n</i> = 10; C), compared to normal matched control kidney (<i>n</i> = 38), normal ovarian tissues (<i>n</i> = 5) and normal matched breast tissues (<i>n</i> = 10). D–H, <i>BDNF</i> is a bonafide target of miR-204. D, schematic of the putative miR-204 binding sequence in the <i>BDNF</i> 3′ UTR. E, HEK-293 cells were co-transfected with Renilla luciferase expression construct pRL-TK and firefly luciferase constructs containing either pMIR-<i>BDNF</i> 3′ UTR in the absence and presence of miR-204 mimic or pMIR-<i>BDNF</i> 3′ UTR mutant. Firefly luciferase activity of each sample was normalized to Renilla luciferase activity. Mean±SEM of three independent experiments (performed in duplicate for each experiment). (**) <i>P</i><0.01; (***) <i>P</i><0.001. F, qRT-PCR analysis of miR-204 overexpressing cells and cells transfected with miR-204 inhibitors using <i>BDNF</i>-specific primers. G, western blot analysis of HEK-293 cells transfected with miR-204 mimic using anti-BDNF antibody (1∶1000). β-actin was used as a loading control. Gel photographs are representative of three independent experiments. H, graphical representation of band intensities quantified using the Total Labs TL100 1D gel analysis software (<i>n</i> = 3; Nonlinear). BDNF protein level for the control was set to 100.</p

Systemic delivery of miR-204 suppresses tumor metastasis.

<p>A, injection of miR-204 oligonucleotide into tail vein suppressed lung metastasis. Live bioluminescence images of mice injected with miR-204 (<i>n</i> = 6) or miR-204 mutant (neg. control; <i>n</i> = 6) oligonucleotides using the Xenogen In Vivo Imaging System (IVIS) (Xenogen). Images were taken after subcutaneously injecting 150 mg/kg D-luciferin substrate in PBS to anesthetized mice. B, tumor metastasis volume was assessed starting from day 10 until animals were sacrificed at day 60. Using ROI analysis, tumor light intensity was calculated in photon/s, which corresponds with the number of live cells in vivo. C, representative lung images showing GFP^+ve foci (red circle) in neg. control groups. D, representative lung sections showing metastatic foci in neg. control groups. E, no hepatotoxicity in miR-204 injected mice. Sections of liver from miR-204 injected mice show no signs of hepatotoxicity. The presence of multifocal periportal lymphocytes is not unusual and is a common finding in young animals.</p

MiR-204 inhibits tumor growth and metastasis.

<p>A, miR-204 inhibits anchorage-independent growth. Nutrient consumption (left) and graph (right) depict the number of colonies formed in soft agar wells by HEK-293 cells stably overexpressing either scramble or miR-204, further transfected with miR-204 inhibitor. (*) <i>P</i><0.05; (**) <i>P</i><0.01. Results are the average of three independent experiments. B, miR-204 overexpression inhibits tumor growth. Photograph shows representative features of tumor growth in <i>RAG2</i>^−/−, γc^−/− SCID mice injected (in the kidney capsule) with HEK-293 cells stably overexpressing either scramble control or miR-204. Bar graph shows mean tumor volume for miR-204 (<i>n</i> = 9) and scramble (<i>n</i> = 9) transfectants. (*) <i>P</i><0.001. C, histological analysis of sections from tumor xenografts overexpressing either scramble (control) or miR-204. Images shown in the right panel represent magnified view of boxed region indicated in the left panel. Tumor invasion in control transfectants is reflected by the invasion of tumor into renal tissue. D, basement membrane matrix invasion assay of MDA-MB-231 cells transfected with 75 nM scrambled sequence (control) or miR-204 mimic (miR-204) or miR-204 mimic transfected cells further transfected with miR-204 inhibitor (miR-204+inhibitor). Bar graph shows the average number of invaded cells counted microscopically in five different fields per filter. (***) <i>P</i><0.001.</p