Browsing by Author "Garlotte, Isabelle"
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Item EXOSOME PROFILING OF BRONCHIAL LAVAGE FLUID IN A MOUSE MODEL OF SURGERY RESECTION OF BREAST CANCER WITH LUNG METASTASIS(2024-03-21) Marikh, Morad; Brown, Ainsley; Hall, Courtney; Donkor, Michael; Garlotte, Isabelle; Subasinghe, Kumudu; Elkassih, Omar; Jones, Harlan; Phillips, NicoleThe lung serves as a primary site for breast cancer metastasis, carrying profound implications for patient prognoses. About 60% of people diagnosed with metastatic breast cancer have lesions in either the lungs or the bones, with triple-negative breast cancer (TNBC) more likely than other types of breast cancers to metastasize to the lungs. Although current targeted chemo-radiotherapy and surgery result in higher survivorship, studies have documented that such curative treatments may also increase risk of lung metastasis. To date, the causal factors that mediate metastasis in the context of cancer treatments remain elusive. Our long-term goal is that a deeper understanding of the mechanisms that mediate relocation of breast tumor cells from its primary origin to its distal site (e.g., lung) will reveal novel complementary diagnostic and preventative treatments to improve TNBC survivorship. Exosomes, serving as tiny extracellular vesicles within tumor cells and other cells (e.g., immune cells) release diverse biomolecules have been implicated in tumor pathogenesis. Specifically, miRNAs as cargo within exosomes are known to regulate cellular function. miRNAs are small RNA molecules that can bind to messenger RNA (mRNA) and inhibit protein synthesis or promote mRNA degradation. This regulatory function allows miRNAs to modulate the expression of multiple genes involved in various cellular processes and their dysregulation has been implicated in various diseases, including cancer. The objective of this study was to determine the expression of miRNA-200b-3p and miRNA-141-5p as known regulators of lung cancer are influenced by surgical removal of a primary breast cancer. We hypothesized that miRNA-200b-3p and miRNA-141-5p mRNA expression is increased in response to surgery. Using an established model of breast cancer metastasis, exosomes were isolated from the bronchiole alveolar lavage fluid (BALF) of tumor bearing mice and mice in which primary tumors were resected compared to tumor-free mice. Results demonstrated that miRNA-200b-3p was present in both tumor-bearing and non-tumor-bearing mice. In contrast, miRNA-141-5p was not expressed in tumor-bearing, non-tumor-bearing mice, and naïve mice determined by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). In conclusion, as we navigate the intricacies of miRNA dynamics in the lung microenvironment, future studies will involve broadening the miRNA panel and refining exosome recovery techniques. This strategic evolution aims to enhance sensitivity, facilitating the detection of elusive, tumor-derived exosome miRNAs. All studies have been approved by UNTHSC IACUC, approval number #2018-0031. Acknowledgement: This research is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) to Dr. Jamboor K. Vishwanatha and National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01 (Vishwanatha, Jones-Project 1).Item Imputation Accuracy of Apolipoprotein E ε Alleles in Genome-wide arrays and real-time SNP Genotyping assays(2022) Subasinghe, Kumudu; Garlotte, Isabelle; O'Bryant, Sid E.; Barber, Robert C.; Phillips, NicolePurpose: The vast majority of the established genetic-based risk for late-onset Alzheimer's disease (AD) is attributable to variation within the apolipoprotein E (APOE) gene. This gene, which encodes a protein implicated in various aspects of AD pathology, is characterized by two single nucleotide polymorphisms (SNPs; rs429358 and rs7412) that result in three distinct isoforms (ε4, ε3 and ε2). Most population-based genome-wide association studies to date have identified the APOE ε4 and ε2 alleles as the strongest genetic-based risk and protective factors for AD, respectively. APOE genotype is not only critical for determining disease risk and diagnosis, but also for developing individualized therapeutic strategies. Genotyping via real-time quantitative PCR (qPCR) is the gold standard for APOE isoform determination; however, if genome wide SNP data is available, imputation of APOE (i.e., probabilistic genotyping through inference) may eliminate the need for qPCR genotyping. In this project, we evaluate the concordance of APOE genotypes obtained via qPCR and a genome-wide SNP chip in non-Hispanic White and Mexican American individuals from the Health & Aging Brain among Latino Elders (HABLE) cohort. Method: DNA was extracted from buffy coat samples (n = 1650) on the Hamilton robotic system with the Mag-Bind Blood & Tissue DNA HDQ 96 Kit. qPCR was then performed using the TaqMan Genotyping Kit as per manufacturer's protocol. Results produced via qPCR were then compared to those imputed for rs429358 and directly typed for rs7412 on the Illumina Infinium Global Screening Arrays (GSA) and analyzed with Genome Studio 2.0. Samples with call rates less than 98% were repeated or excluded. Results: Concordance between the APOE genotypes obtained from qPCR and Infinium GSA was 99.32%. Discordance was likely due to poor sample quality and low-frequency imputation errors of rs429358, which may be corrected with more conservative thresholding of the imputed genotype confidence statistics. Conclusion: Genotype imputation from SNPs commonly typed in the APOE region is an effective method for APOE isoform determination, even in Mexican Americans who are more genetically heterogenous due to ancestral admixture; this method may be effectively implemented in large population-based studies of aging and AD.Item Oxidative Stress and Release of Cell-free Mitochondrial DNA from Trophoblast Cells(2022) Gardner, Jennifer; Cushen, Spencer; Bradshaw, Jessica L.; Garlotte, Isabelle; Phillips, Nicole; Cunningham, Rebecca; Goulopoulou, StylianiCell free mitochondrial DNA (mtDNA) is an indicator of cellular stress and systemic inflammation. These properties are accentuated when mtDNA undergoes oxidative damage. In addition, toll-like receptor 9 (TLR9), a receptor of the innate immune system, is activated by mtDNA. Inflammation, oxidative stress, and cell death are characteristics of placental ischemia, a common feature of preeclampsia. Recent work from our lab has shown dysregulation of circulating cell-free mtDNA in pregnancies with preeclampsia and association of this dysregulation with preeclampsia diagnosis. However, mechanisms underlying the release of mtDNA remain unclear. We hypothesized that human trophoblast cells exposed to oxidative stress via antimycin A, an inhibitor of complex III of the electron transport chain, would induce release of mtDNA via cell death-dependent mechanisms, leading to increased TLR9 activation. BeWo cells (ATCC? CCL-98) were treated with increasing concentrations of antimycin A (10, 50, 100, 320 µM) and vehicle (ethanol, 0.16% v/v) for 4 hours. Supernatants were collected and snap frozen in liquid nitrogen. Absolute real-time qPCR quantification with TaqMan™ probes and chemistry was used to quantify cell-free mtDNA (amplification target: MT-ND5 gene) and nuclear DNA (nDNA). Flow cytometry was used to assess the activation of cell death mechanisms in response to oxidative stress. To determine TLR-9-associated immunostimulatory potency of cell culture supernatants, we used an engineered cell line of human embryonic kidney 293 cells transfected with a human TLR-9 gene (HEK-BlueTM hTLR9). Exposure of trophoblast cells to antimycin A did not induce the release of mtDNA (p>0.05) or nDNA (p>0.05). Similarly, there were no differences in TLR9 activation between groups (p>0.28). Antimycin A (320 µM) reduced cell viability (Vehicle: 64.44 ± 5.46% vs Antimycin A: 18.14 ± 5.78%, p< 0.05) and increased necrosis (Vehicle: 10.39 ± 3.11% vs Antimycin A (100, 320 µM): 30.51 ± 4.43%, 40.16 ± 5.08%, P< 0.05), while apoptosis levels remained unchanged (P>0.1). Activation of oxidative stress pathways, via inhibition of complex III of the electron transport chain, leads to cell death, but does not affect release of mtDNA. These data suggest other cellular mechanisms, such as mitophagy or activation of antioxidant pathways, may serve a cytoprotective role against oxidative stressors in trophoblast cells. This study extends our pre-clinical knowledge about the links between placental oxidative stress and immunogenic factors in trophoblast cells. These findings may contribute to development of novel therapeutic targets for treatment of maternal cardiovascular dysfunction in preeclampsia.