Distinct Phenotypic Changes Research Articles

TMIC-01. TARGETABLE GENE EXPRESSION CHANGES MEDIATE EXTRAVASATION OF BREAST CANCER CELLS FROM THE CIRCULATION AND LOCAL PROLIFERATION IN THE BRAIN

Abstract As breast cancer migrates into the brain, it acquires distinct genomic, immunologic and phenotypic changes through interactions with the local microenvironment. We sought to determine how targetable gene expression changes mediate breast cancer extravasation into and proliferation within the brain. We created a brain metastatic cell line (“brain-seeking”) via serial intracardiac injection of mCherry+ 4T1 breast cancer cells in Balb/c mice and flow cytometry isolation of micrometastases. We conducted in vitro and in-vivo studies to uncover immunological and genetic differences between primary breast cancer and brain metastases. In culture, we observed significant upregulation in adhesion (p=0.01) and invasion (p=0.0004) along with upregulation of adhesion-promoting genes such as Sdc3, Serpine2 or ECM1 in brain-seeking cells. We observed significant differences in survival of Balb/c mice injected intracranially with brain-seeking cells vs. breast cancer cells (p=0.04). In the mammary fat pad (MFP), parental 4T1 cells caused increased mortality vs. brain-seeking cells (p=0.003). This rejection of brain-seeking 4T1 cells in the MFP could be due to prior adaptation to the brain microenvironment, while rejection of parental 4T1 cells in the brain could be due to lacking said adaptations. Consistent with this finding, of 20,382 analyzed genes in both cell lines, 108 related to immune regulation were significantly upregulated in brain-seeking cells including Cxcl1, Dusp1, C3. Flow cytometry showed increased cytotoxic and helper T cells in intracranially injected parental vs. brain-seeking cells (p <0.001). In the MFP, however, 4T1 parental cells displayed CD3+ T cell depletion and an increased number of neutrophils (p <0.05) while the “brain-seeking cells” showed increased numbers of CD3+ and CD4+ cells (p <0.01) and macrophages (p<0.05). This suggests that brain metastases evolve from their primary tumor through adhesive and invasive adaptations promoting extravasation, along with unique immunological adaptations that allow their proliferation free of immune surveillance in the brain microenvironment.

A Stem-like Patient-Derived Ovarian Cancer Model of Platinum Resistance Reveals Dissociation of Stemness and Resistance.

To understand chemoresistance in the context of cancer stem cells (CSC), a cisplatin resistance model was developed using a high-grade serous ovarian cancer patient-derived, cisplatin-sensitive sample, PDX4. As a molecular subtype-specific stem-like cell line, PDX4 was selected for its representative features, including its histopathological and BRCA2 mutation status, and exposed to cisplatin in vitro. In the cisplatin-resistant cells, transcriptomics were carried out, and cell morphology, protein expression, and functional status were characterized. Additionally, potential signaling pathways involved in cisplatin resistance were explored. Our findings reveal the presence of distinct molecular signatures and phenotypic changes in cisplatin-resistant PDX4 compared to their sensitive counterparts. Surprisingly, we observed that chemoresistance was not inherently linked with increased stemness. In fact, although resistant cells expressed a combination of EMT and stemness markers, functional assays revealed that they were less proliferative, migratory, and clonogenic-features indicative of an underlying complex mechanism for cell survival. Furthermore, DNA damage tolerance and cellular stress management pathways were enriched. This novel, syngeneic model provides a valuable platform for investigating the underlying mechanisms of cisplatin resistance in a clinically relevant context, contributing to the development of targeted therapies tailored to combat resistance in stem-like ovarian cancer.

Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) system to the newly synthesized megabase Sc2.0 chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by (1) removing most of the chromosome content or (2) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and specific chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic chromosome resource opens new paradigms in studying aneuploidy.

Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release

Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca2+ and reactive oxygen species, cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing, PS exposure and Cyto.C release into the cytosol. Removing EtOH at predetermined time points revealed that all phenomena except Cyto.C release occurred in a phase of neuronal cell death in which full recovery to a neurite-bearing cell was still possible. Our findings underscore a strategy of treating chronic neurodegenerative diseases by removing stressors from neurons and harnessing intracellular targets that delay or prevent trespassing the point of no return.

Abstract 5507: A potential combinatorial treatment strategy to overcome olaparib resistance in high-grade ovarian cancer

Abstract PARP1 inhibitors have evolved the treatment of BRCA1/2-mutated high-grade serous ovarian cancer. However, its resistance is frequently observed. In our initial combinatorial drug screening, OSU-03012 (AR-12) appeared as a promising drug to be combined with PARP inhibitor Olaparib in multiple ovarian cancer cell lines, regardless of BRCA mutation status. OSU-03012 is designed as an orphan drug with anti-microbial activity. It has also shown cell-context-dependent anti-cancer activity. Using imaging-based high-throughput drug sensitivity analysis, we found that OSU-03012 and BX-912, while both classified as 3-Phosphoinositide Dependent Protein Kinase 1 (PDK-1) inhibitors, not only differed in their synergistic effect together with Olaparib, also caused distinct phenotypic changes in ovarian cancer cells. Cell senescence was observed in BX-912 but not OSU-03012 treatment, alone or together with Olaparib. Recently, dihydroorotate dehydrogenase (DHODH) has been identified as a secondary target of OSU-03012, which explains its anti-cancer activity by regulating nucleotide metabolism and thus affecting DNA repair. However, in our study, OSU-03012 did not similarly affect cell viability and phenotype on ovarian cancer cell lines as a DHODH inhibitor BAY2402234. Interestingly, compared to BX-912 and BAY2402234, OSU-03012 showed higher selectivity on ovarian cancer cell lines over normal epithelial carrying BRCA and TP53 mutations; it also exhibited reduced toxicity on a human fibroblast cell line. We are further clarifying the putative targets of OSU-03012 using thermal proteome profiling. To investigate the molecular evidence of OSU-03012 for treating ovarian cancer, we performed a differential gene expression analysis using Genomics of Drug Sensitivity in Cancer (GDSC) database. All ovarian cancer cell lines included in GDSC drug screening are classified as Olaparib- or OSU-03012-sensitive/insensitive, based on the respective IC50 threshold. We found that OSU-03012 sensitivity is positively associated with several known genes regulating ovarian cancer progression, such as LUM and COL3A1, as well as genes (e.g., PTPRZ1) that are associated with Olaparib insensitivity in this analysis. Interestingly, angiogenesis signatures were found to be enriched in both Olaparib and OSU-03012-sensitive cancer cell lines. We are currently establishing a cancer-vascular cell system in a microfluidic setting to study how such combinatorial drug treatment would connect to tumor microenvironment remodeling. Altogether, we identified a potential non-cancer drug for ovarian cancer treatment and to overcome Olaparib resistance. A multidisciplinary approach highlights the importance of drug target profiling and sensitivity signatures scoring to improve treatment stratification. Citation Format: Jie Bao, Eva Daniela Mendoza Ortiz, Jessica Agudelo, Roman Filimonov, Anniina Färkkilä, Jing Tang. A potential combinatorial treatment strategy to overcome olaparib resistance in high-grade ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5507.

Abstract 3262: Single-cell spatial dynamics of the tumor-immune microenvironment of high-grade serous ovarian cancer during neoadjuvant chemotherapy

Abstract Neoadjuvant Platinum-based combination chemotherapy (NACT) is commonly offered to patients with high-grade serous ovarian cancer (HGSC) harboring unresectable disease in the primary setting. In order to develop effective immunotherapeutic and combinatorial treatment strategies for HGSC, a deeper knowledge on the effects of NACT on the spatial tumor-immune interactions is required. We performed genetic and single-cell spatial characterization of the tumor-immune microenvironment of 22 HGSC samples collected before and after NACT using Whole Genome Sequencing and tissue cyclic immunofluorescence (t-CycIF) highly-multiplexed imaging platform. We used image analysis and bioinformatics to analyze 30 antibodies in over 6 500 000 single-cells in the context of their spatial tissue architecture. The results were integrated with genomic features and clinical data. We identified four distinct tumor cell metaclusters, eight immune cell subtypes and six stromal cell subpopulations. Chemotherapy induced distinct phenotypic changes in the tumor and immune cells including glutamine metabolism, cell proliferation and antigen presentation. Using single-cell spatial analyses, we identified 18 unique tumor-immune-stromal communities, characterized by different tumor, stromal and immune cell proportions, as well as the tumor-stromal interface. Interestingly, we observed increased CD8+T-cell and decreased CD163+ macrophage infiltration specifically at the tumor-stroma interface induced by chemotherapy. Furthermore, NACT induced spatial CD8+T-cell and macrophage cell-cell interaction patterns specifically in the tumor-stromal interface. Further dissection of the functional properties of the spatial cell-cell interactions using spatial transcriptomics revealed distinct tumor-cell clonal dynamics and gene expression programs on the tumor-stroma interface during NACT. In conclusion, we identified distinct spatial features in the tumor-immune microenvironment indicating the tumor-stromal interface as the underpinning of spatial dynamics in HGSC during NACT. Using a spatial single-cell multi-omics approach has the potential to improve patient stratification and immunotherapeutic strategies in HGSC. Citation Format: Inga-Maria Pauliina Launonen, Angela Szabo, Fernando Perez, Julia Casado, Zoltan Maliga, Sampsa Hautaniemi, Jaana Oikkonen, Johanna Hynninen, Tuulia Vallius, Ajit Johnson Nirmal, Peter Sorger, Anna Vähärautio, Anniina Färkkilä. Single-cell spatial dynamics of the tumor-immune microenvironment of high-grade serous ovarian cancer during neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3262.

Abstract 5362: AI-enabled novel workflow to evaluate T-cell activity in vitro using 3D spheroid models

Abstract T-cell therapies are designed to help our immune system eliminate cancer cells. Those include CAR T-cells (Chimeric Antigen Receptor engineered T-cells), tumor infiltrating lymphocytes (TIL), and other genetically modified T-cells. In recent years, the field of cell therapy has started to expand, including the launch of the first CAR T-cell therapies to treat blood cancer in 2017, which was a critical milestone in this field. Despite its boom, the discovery of novel immunotherapies that specifically enhance T-cell response against cancer cells remains a challenging task, limited by the absence of robust in vitro models to evaluate these immunotherapies throughout their development. The use of CAR T-cells on solid tumors has been lagging due to challenges that include tumor heterogeneity, immunosuppressive microenvironments, and the lack of unique tumor antigens that can be recognized by the CAR-T cells. As such, the ability to screen for CAR T-cells (e.g. with CRISPR) that effectively target and kill tumors is an area of active research. Here we describe a method for assessment of T-cell effect and penetration into the multi-cellular 3D tumor spheroids as a proof-of-concept model for in vitro CAR T assays. Spheroids were formed from Hela cell lines in 96-well round bottom plates. We activated the human peripheral blood mononuclear (PBMC) cells with PMA/ionomycin for 6 hours, then treated the spheroids with stimulated PBMC for 72 hours. Samples were imaged with a confocal imager every 2 hours for a 72 hour period. High-content imaging and analysis allowed us to observe and measure phenotypic changes in cancer spheroids and the process of T-cell penetration over a period of 72 hours. To optimize the workflow, we developed an image analysis approach that uses a deep learning-based segmentation model and a machine learning-based classification model to quantify the T-cell induced phenotypic changes in the spheroids using brightfield images. Our results show clear distinct phenotypic changes among different treatment groups and the feasibility of using AI-based analysis workflows to accurately predict the efficacy of T-cells in an in vitro assay. Moreover, the information associated with the penetration of T-cell into the 3D spheroids was explored by calculating the coordinates of the penetrated T-cells and T-cell movements from the nearest edge of the spheroids where the T-cell penetration speed was estimated. Our results show that the stimulated T-cells have much greater penetration speed compared to non-stimulated T-cells. Image analysis also allowed us to measure and quantitate the deterioration of cancer spheroids and changes in their morphology. Overall, our results show that the 3D spheroid models and the high-content analysis workflow may potentially be used as a metric to evaluate the efficacy of cell therapy in vitro. Citation Format: Zhisong Tong, Oksana Sirenko, Misha Bashkurov, Angeline Lim. AI-enabled novel workflow to evaluate T-cell activity in vitro using 3D spheroid models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5362.

Identification and functional validation of super-enhancers in Arabidopsis thaliana

Super-enhancers (SEs) are exceptionally large enhancers and are recognized to play prominent roles in cell identity in mammalian species. We surveyed the genomic regions containing large clusters of accessible chromatin regions (ACRs) marked by deoxyribonuclease (DNase) I hypersensitivity in Arabidopsis thaliana. We identified a set of 749 putative SEs, which have a minimum length of 1.5 kilobases and represent the top 2.5% of the largest ACR clusters. We demonstrate that the genomic regions associating with these SEs were more sensitive to DNase I than other nonpromoter ACRs. The SEs were preferentially associated with topologically associating domains. Furthermore, the SEs and their predicted cognate genes were frequently associated with organ development and tissue identity in A. thaliana. Therefore, the A. thaliana SEs and their cognate genes mirror the functional characteristics of those reported in mammalian species. We developed CRISPR/Cas-mediated deletion lines of a 3,578-bp SE associated with the thalianol biosynthetic gene cluster(BGC). Small deletions (131-157 bp) within the SE resulted in distinct phenotypic changes and transcriptional repression of all five thalianol genes. In addition, T-DNA insertions in the SE region resulted in transcriptional alteration of all five thalianol genes. Thus, this SE appears to play a central role in coordinating the operon-like expression pattern of the thalianol BGC.

TRAF3 enhances type I interferon (IFN I) receptor signaling in T cells through modulation of the phosphatase PTPN22

Abstract Type I interferons (IFN I) are among the most powerful tools host cells deploy against intracellular pathogens. Their effectiveness is due to both the rapid, directly anti-viral effects of IFN-stimulated gene products, and to the effects of IFN I on responding immune cells. IFN I signaling through its receptor, IFNAR, is tightly regulated at multiple steps in the signaling cascade. Despite the distinct phenotypic changes induced by IFN I in different immune cell types, few studies have addressed the possibility of cell type-specific differences in regulation. In this study, we show that TNFR-associated factor 3 (TRAF3) promotes robust IFNAR signaling specifically in CD4+ T cells by preventing recruitment of the negative regulatory phosphatase PTPN22 to the IFNAR complex. The balance between signals through IFNAR and other differentiating cytokine receptors strongly influences CD4+ T cell differentiation and function during infections. Our work introduces TRAF3 and PTPN22 as newly appreciated regulators key to influencing CD4+ T cell activation by IFN I. Supported by R01 AI123107, T32 AI007260, T32 HL007344, Department of Veterans Affairs

Integrin Beta 4E Promotes Endothelial Phenotypic Changes and Attenuates Lung Endothelial Cell Inflammatory Responses.

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 μM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI).

PACAP and VIP Modulate LPS-Induced Microglial Activation and Trigger Distinct Phenotypic Changes in Murine BV2 Microglial Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1β, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.

Metabolic Changes in Aging Bone Marrow

Introduction: Aging is associated with complex molecular changes at the cellular level. Bone marrow undergoes distinct phenotypic changes with aging, the mechanisms of which are not completely understood. Current metabolomic approaches provide a comprehensive analysis of tissue-related changes that occur due to exposure to external agents, environmental shifts, or the passage of time. A limited number of studies have investigated the metabolome in aging models and have shown significant alterations in lipid and amino acid metabolism. However, no study has investigated the metabolome of aging bone marrow. In this study, we comprehensively characterized the metabolome and transcriptome of aging murine bone marrow and compared it with bone marrow from young healthy mice and chemotherapy treated mice; chemotherapy treatment is known to induce age-related changes in hematopoiesis.Methods: Mice experiment: Twenty 8-week old and eleven 18-week old BALB/c mice were used in this experiment. 8-week old mice were injected with 0.15mg/g of cyclophosphamide (chemo group, N=10) or saline (young group, N=10) intraperitoneally every 28 days, for a total of three cycles. During the same period, the 18-month old mice (N=11) were observed in similar vivarium conditions. 28 days after the last chemotherapy/saline treatment, all mice were sacrificed (after 16 hours of fasting) and bone marrow was extracted from tibia, femur, and spine. One chemotherapy-treated mouse died before bone-marrow extraction. Subsequently, red blood cells were depleted using red blood cell lysis buffer. Metabolon assay: A 200 ul aliquot of the cells in suspension were used for RNA extraction and the remaining cells were used for the metabolomic assay (Metabolon, Durham, NC, USA). For the metabolomic analysis, the bone marrow cells were pelleted (25 ul), snap frozen and stored at -80°C until the assay was performed. Statistical analysis of the metabolon assay: A one-way ANOVA followed by Tukey's test was used to test the null hypothesis of no difference in the mean values of each metabolite between experimental-groups. A P < 0.05 was used as the criteria to reject the null hypothesis in each analysis. IBM SPSS StatisticsTM ver. 24 was used to perform ANOVA and Tukey analyses. Outlier detection was determined using a median-based technique in R-package Rallfun-v27 23 and RStudio 1.0.44. RNA sequencing: Three samples from each experimental-group were used for RNA-sequencing. RNA was isolated from frozen bone marrow and 250 nanograms was used to generate RNA-sequencing libraries using TruSeq stranded mRNA library prep kit. Sequencing was performed on an Illumina NextSeq500 system that produced paired-end 75bp reads¾ an estimated 33 million reads per sample. Statistical analysis of RNA sequencing: Pair-wise-differential expression between treatment-groups (aged and chemotherapy-treated) and control (young) was determined with a limma-based general linear model using Edge-R software. Genes with a P <0.05 and FDR <0.05 were considered differentially expressed. Genes with a P <0.05, regardless of FDR score, were considered marginally differentially expressed. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA). Statistically significant pathways were identified using a Fisher's exact test. Pathways with a P < 0.05 were considered significantly activated. IPA was also used to predict regulator genes and their downstream target molecules that were differentially expressed in our dataset.Results: Our results revealed broad changes in lipids, amino acids, and nucleotides in aging marrow tissue. In particular, the metabolome of the aging bone marrow exhibited a signature of suppressed fatty-acid oxidation: accumulation of free fatty acids, reduced acyl-carnitines and low β-hydroxy butyric acid. The aged bone marrow also exhibited a significant reduction in amino acid and nucleic acid pool. The transcriptome of the aging bone marrow revealed a signature of oxidative stress, known to be associated with mitochondrial dysfunction. Lastly, the metabolic and transcriptomic profiles of the bone marrow of chemotherapy treated mice did not show broad age-related changes but rather mostly resembled young healthy mice, suggestive of a lack of ‘metabolic aging’ with chemotherapy exposure. Together, these data provide significant insights into metabolic alterations associated with aging in bone marrow. DisclosuresNo relevant conflicts of interest to declare.

Hyperactivation of monocytes and macrophages in MCI patients contributes to the progression of Alzheimer's disease

BackgroundAlzheimer’s disease (AD) is the most common neurodegenerative disease ultimately manifesting as clinical dementia. Despite considerable effort and ample experimental data, the role of neuroinflammation related to systemic inflammation is still unsettled. While the implication of microglia is well recognized, the exact contribution of peripheral monocytes/macrophages is still largely unknown, especially concerning their role in the various stages of AD.ObjectivesAD develops over decades and its clinical manifestation is preceded by subjective memory complaints (SMC) and mild cognitive impairment (MCI); thus, the question arises how the peripheral innate immune response changes with the progression of the disease. Therefore, to further investigate the roles of monocytes/macrophages in the progression of AD we assessed their phenotypes and functions in patients at SMC, MCI and AD stages and compared them with cognitively healthy controls. We also conceptualised an idealised mathematical model to explain the functionality of monocytes/macrophages along the progression of the disease.ResultsWe show that there are distinct phenotypic and functional changes in monocyte and macrophage populations as the disease progresses. Higher free radical production upon stimulation could already be observed for the monocytes of SMC patients. The most striking results show that activation of peripheral monocytes (hyperactivation) is the strongest in the MCI group, at the prodromal stage of the disease. Monocytes exhibit significantly increased chemotaxis, free radical production, and cytokine production in response to TLR2 and TLR4 stimulation.ConclusionOur data suggest that the peripheral innate immune system is activated during the progression from SMC through MCI to AD, with the highest levels of activation being in MCI subjects and the lowest in AD patients. Some of these parameters may be used as biomarkers, but more holistic immune studies are needed to find the best period of the disease for clinical intervention.

Microglia Phenotypes Converge in Aging and Neurodegenerative Disease.

Microglia, the primary immune cells of the central nervous system, hold a multitude of tasks in order to ensure brain homeostasis and are one of the best predictors of biological age on a cellular level. We and others have shown that these long-lived cells undergo an aging process that impedes their ability to perform some of the most vital homeostatic functions such as immune surveillance, acute injury response, and clearance of debris. Microglia have been described as gradually transitioning from a homeostatic state to an activated state in response to various insults, as well as aging. However, microglia show diverse responses to presented stimuli in the form of acute injury or chronic disease. This complexity is potentially further compounded by the distinct alterations that globally occur in the aging process. In this review, we discuss factors that may contribute to microglial aging, as well as transcriptional microglia alterations that occur in old age. We then compare these distinct phenotypic changes with microglial phenotype in neurodegenerative disease.

Microglial Polarization: Novel Therapeutic Strategy against Ischemic Stroke.

Ischemic stroke, which is the second highest cause of death and the leading cause of disability, represents ~71% of all strokes globally. Some studies have found that the key elements of the pathobiology of stroke is immunity and inflammation. Microglia are the first line of defense in the nervous system. After stroke, the activated microglia become a double-edged sword, with distinct phenotypic changes to the deleterious M1 types and neuroprotective M2 types. Therefore, ways to promote microglial polarization toward M2 phenotype after stroke have become the focus of attention in recent years. In this review, we discuss the process of microglial polarization, summarize the alternation of signaling pathways and epigenetic regulation that control microglial polarization in ischemic stroke, aiming to find the potential mechanisms by which microglia can be transformed into the M2 polarized phenotype.

Automated segmentation and analysis of retinal microglia within ImageJ

Microglia are immune cells of the central nervous system capable of distinct phenotypic changes and migration in response to injury. These changes most notably include the retraction of fine dendritic structures and adoption of a globular, phagocytic morphology. Due to their characteristic responses, microglia frequently act as histological indicators of injury progression. While algorithms seeking to automate microglia counts and morphological analysis are becoming increasingly popular, few exist that are adequate for use within the retina and manual analysis remains prevalent. To address this, we propose a novel segmentation routine, implemented within FIJI-ImageJ, to perform automated segmentation and cell counting of retinal microglia. We show that our routine could perform cell counts with accuracy similar to manual observers using the I307N Rho model. Tracking cell position relative to retinal vasculature, we observed population migration towards the photoreceptor layer beginning 12 h post light damage. Using feature selection with Chi2 and principal component analysis, we resolved cells along a morphological gradient, demonstrating that extracted features were sufficiently descriptive to capture subtle morphological changes within cell populations in I307N Rho and Balb/c TLR2−/− retinal degeneration models. Taken together, we introduce a novel automated routine capable of efficient image processing and segmentation. Using data retrieved following segmentation, we perform morphological analysis simultaneously on whole populations of cells, rather than individually. Our algorithm was built entirely with open-source software, for use on retinal microglia.

Uncovering the Molecular Underpinnings of Oxidative Stress-Induced Senescence

The aging process is sexually dimorphic, with males having higher occurrence rates of cancer and facing a greater risk of mortality. Sexual dimorphism in the response to cellular damage may account for distinct phenotypic changes with age as they relate to the accumulation of cellular damage leading to cancer. Cellular senescence triggers permanent cell cycle arrest in order to protect against malignant growth. However, organismal senescence increases with age and is associated with the release of pro-inflammatory signals (cytokines, chemokines, and proteases) known as the ‘senescence-associated-secretory-phenotype’ (SASP) that, if unchecked, accelerates tissue damage and creates a microenvironment ripe for cancer development. In this study, we hypothesized that sexual disparities in mortality and cancer prevalence stems from differences in the rate of accumulation of senescent cells in mice. Male and female C57BL/6J mice were fed ad libitum or subjected to 30% calorie restriction, a nutritional intervention known to delay the onset of various cancers and prevent senescent cell accumulation. Primary skin fibroblasts were collected longitudinally to allow measurement of cell proliferation, wound healing and the release of SASP factors. The results indicate that when compared to males, fibroblasts of CR-fed females showed significant improvements in cell growth rate, wound healing and SASP markers vs. AL controls. Work is underway to determine how sex influences cellular protective pathways. Thus, like other cell processes, cellular senescence is unequal between males and females and CR delays the emergence of the senescence phenotype.

Maneb alters central carbon metabolism and thiol redox status in a toxicant model of Parkinson's disease

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson's disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.

Immune Senescence and Exhaustion Correlate with Response to Flotetuzumab, an Investigational CD123×CD3 Bispecific Dart® Molecule, in Acute Myeloid Leukemia

Immune Senescence and Exhaustion Correlate with Response to Flotetuzumab, an Investigational CD123×CD3 Bispecific Dart® Molecule, in Acute Myeloid Leukemia