Vol. 2, 2019 — Featured publications for the IncuCyte® Live-Cell Analysis System

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Neuroinflammation

Glutamine antagonism attenuates physical and cognitive deficits in a model of MS

Advancing the quest for better multiple sclerosis treatments

Effective, well-tolerated treatments to help curb the immune-mediated damage to the central nervous system during multiple sclerosis (MS) have remained elusive. The dysregulation of glutamate and activation of T-cells are known to be involved in disease pathogenesis, spurring a number of pharmacological attempts to treat disease effects with limited success.

In this study by Hollinger and colleagues at Johns Hopkins, the drug JHU-083 (a liphophilic glutamine antagonist) was tested in an attempt to overcome peripheral toxicity encountered in the use of DON (6-diazo-5-oxo-L-norleucine) drug candidates and their limited penetration into the brain. JHU-083, a liphophilic prodrug candidate of DON, was designed to be inert in the circulation to reduce toxicity, but upon penetration into the brain, becomes cleaved and is activated. The antiproliferative effects on T-cell proliferation of the drug were examined in vitro, along with additional in vivo physical and cognitive assessment in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE).

Key findings include:

• JHU-083 inhibited the in vivo proliferation of T cells and decreased their activation, as determined by flow cytometry. There was no effect on bone marrow derived dendritic cells.

• To confirm these findings, and to determine if the anti-proliferative effects of JHU-083 were due to drug cytotoxicity, IncuCyte® Live-Cell Imaging and Analysis was used to measure proliferation and viability of CD4+ T cells isolated from the naive mouse spleens. The activation of CD4+ T cells was followed using the IncuCyte reagents Cytotox Green (for cell death) and NucLight Rapid Red (for cell number). IncuCyte analysis revealed that all doses of the drug caused reduced CD4+ proliferation and reduced cell number, even the lower doses, which was important for reduced toxicity. Further, IncuCyte analysis also revealed that the drug was not cytotoxic as compared to DMSO control.

• In vivo administration of JH-083 reduced the severity of EAE symptoms in two testing conditions: prevention and treatment. Most interestingly, the cognitive impairment associated with this EAE murine model was reversed, as assessed by Barnes maze testing.

• JHU-083 shows promise as a novel potential therapeutic for the treatment of physical and cognitive symptoms of MS, which may also have important implications for other neuroinflammatory-related conditions.

Read the full paper in Neurology, Neuroimmunology & Neuroinflammation, August 2019.

Cell Motility and Migration

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility

A new discovery leads to dynamic insights in Nt-acetylation

Actin is a critical component of the cytoskeleton, the organization and dynamic assembly of which are involved in changes in cellular shape, motility, and processes such as gene transcription.  Post-translational modifications of the actin protein, such and N-terminal (Nt)-acetylation, can affect cellular functions but this process has thus far been uncharacterized.

In this study by Drazic, Aksnes, and Marie, et al. of the Arnesen lab at the University of Bergen, the authors report on the exciting discovery of actin’s N-terminal acetyltransferase: NAA80. Digging deeper, this work investigates the various activities of Nt-acetylation that affect cytoskeletal morphology and cell hypermotility, emphasizing how this important actin modification impacts both functional and cellular responses, opening an avenue to manipulate this process through gene targeting of NAA80. 

Key findings include:

• The authors discovered that NAA80, N-terminal acetyltransferase (NAT) enzyme, is responsible for the Nt-acetylation of actin.
• To study the effect of Nt-acetylation on actin on the control of cellular motility, IncuCyte® Live-Cell Imaging and Analysis of wound healing assays were employed along with other microscopy methods to capture this dynamic process in two NAA80 knockouts (KO1 and KO2). IncuCyte image analysis was used to for average gap size. Using additional analysis methods, the authors were able to calculate cell-front velocity of NAA80 knockout cells, which closed the wound faster than in control cells, validating the important role for this enzyme.
• IncuCyte® chemotaxis assays were then used to measure chemotaxis (directed migration) as well as random migration. Knockout of NAA80 resulted in enhanced motility in both of these processes. Thus cells with reduced actin Nt-acetylation are hypermotile.
• Actin Nt-acetylation was also found to affect the morphology of the cytoskeleton, with assessment of the formation of filopodia and lamellipodia by phalloidin staining.
• Nt-acetylation also influenced actin polymerization and stability, as assessed by studies of cytoskeletal structure recovery rates in cells treated with an actin depolymerizing drug latrunculin A (LatA), pyrene-actin polymerization assays, and actin assembly properties in response to the formins, mDia1 and mDia2.

Read the full paper in PNAS, April 2018.

Watch the supplemental video of wound closure from this paper.

Development

Defective endothelial cell migration in the absence of Cdc42 leads to capillary-venous malformations

The loss of a GTPase leads to vascular abnormalities

Cdc42 is a GTPase that regulates signaling for a variety of cell functions in vitro, including changes in cellular morphology, cell polarity, the migration of cells, and cell cycle.  This is accomplished via activation of some families of kinases, which may influence the actin organization and cellular migration.  Though these effects have been studied in vitro, the in vivo effects have not been well characterized.

In this international, collaborative study by Lavina et al., the authors examined the regulatory role of small GTPase cell division cycle 42 (Cdc42) on endothelial cell functions, angiogenesis, and vascular morphogenesis in a mouse retinal model.  The authors combined the power of conditional, mosaic gene ablation and computational modeling to identify the cellular functions that were dependent on Cdc42, and which were related to vascular morphogenesis.

Key findings include:

• Deletion of Cdc42 resulted in capillary-venous malformations.
• In an assessment of EC (endothelial cell) axial polarization and migration, the authors used the IncuCyte® WoundMaker to create homogenous scratch wounds of GFP-labeled primary brain endothelial cells isolated from control and Cdc42i^ΔEC animals that were grown in confluent monolayers. Cell migration was recorded using IncuCyte® for 72 h and assessed with IncuCyte® Live-Cell Analysis. Study of EC polarization revealed that Cdc42 controlled the axil polarity of the endothelial cells, and this was specific to the type of vessel.
• CDc42 knockout resulted in impaired migration, and it is required for the motility of EC and helps to regulate the actin cytoskeleton. It is also required for endothelial tip cell selection, directed cell migration, and the formation of filopodia.
• The absence of Cdc42 results in defective endothelial axial polarization (previously unknown) as well as malformations in capillaries and veins due to altered cell migration of endothelial cells. The authors proposed that EC, lacking CdC42, remain sequestered in the capillaries and veins, making then unable to redistribute within the greater vascular network that is developing.

Read the full paper in Development, July 2018.

Neurology

Antagonism between the RNA-binding protein Musashi1 and miR-137 and its potential impact on neurogenesis and glioblastoma development

Key RNA binding proteins impact on cell fate decisions

The importance of the interactions between RNA-binding proteins and miRNAs in the control of gene expression is becoming more apparent, and such interactions may be crucial for determining cell fate, neurogenesis, brain development, and neurological disease pathology.

In this multi-institutional study by Velasco et al., the authors built upon their previous finding that tumor suppressor miRNAs regulate Musashi1 (Msi1), an RNA binding protein and stem cell marker, which also regulates cell fate either towards self-renewal or cellular differentiation. In particular, the miRNA miR-137 can upregulate Msi1 to promote differentiation, thereby preventing the development of glioblastoma. In this follow-up investigation, the authors attempted to further characterize this relationship, proposing an antagonistic model, whereby by Msi1 and miR-137 act to regulate via activation or repression, influencing the determination of cell fate.

Key findings include:

• The binding site of miR-137 to Msi1 was highly conserved in both vertebrates and Drosophila, indicating a key regulatory role.
• U251 glioblastoma cells were transfected with a lentivirus expressing Msi1 or control vector. Following transfection with miRNA-137 or mimics, cells were cultured in IncuCyte® Image Lock plates. The cells were monitored for confluence in response to knockdown of a gene set, for which the impact on neurogenesis and glioblastoma was unknown. The results from these experiments supporting the hypothesis that Msi1 and miR-137 share targets that are activated in cancer cells.
• Neurite outgrowth of the neuroblastoma cell line BE-(2)-C was assessed in response to miR-137 and Msi1 to determine the effects of these miRNA on neuronal differentiation. Cells were transfected with either miR-137 or control mimics, as well as Msi1 lentivirus expression vectors or control. IncuCyte® Live-Cell Imaging and Analysis and IncuCyte® NeuroTrack Analysis Software Module were used to assess proliferation, as well as neurite outgrowth to monitor differentiation. Neurite formation was enhanced with miR-137, but this effect was reduced when Msi1 was used.
• Msi1 and Mir-137 were found to act as regulators between the cell fates of self-renewal and differentiation with opposite expression patterns and associated functions, which may be important for studying the development of glioblastoma neurodegenerative diseases.

Read the full paper in RNA, April 2019.

Oncology Therapeutics

Alveolar macrophage secretion of vesicular SOCS3 represents a platform for lung cancer therapeutics

Leveraging an endogenous antitumor mechanism for therapeutic development

Lung cancer continues to be the leading cause of cancer deaths, partially due to late–stage diagnosis and resulting delay in treatment. This necessitates the continued, urgent search for more effective therapeutics. Of particular interest for drug targeting are the endogenous signal transducer and activator of transcription (STAT) proteins, which are involved in various aspects of the immune response and may be downregulated during tumor development.

In this multidisciplinary study from the University of Michigan, Speth et al. creatively approached this challenge by developing synthetic liposomes for the delivery of an endogenous STAT inhibitor suppressor of cytokine signaling (SOCS3).  This agent was utilized both in vitro, and in an in vivo in a lung cancer xenograft mouse model. The authors attempted to harness the power of an endogenous antitumor mechanism, performing a therapeutic rescue investigation based on their previous discovery that alveolar macrophages (AM) curb inflammatory responses in lung epithelial cells (ECs).

Key findings include:

• SOCS3, secreted in AM, can provide an additional source of SOCS3, resulting in the inhibition of STAT3 in the adenocarcinoma cell line A549, thereby reducing activation, proliferation, and survival of these cancer cells. This provides a new mechanism for the progression of lung tumors through the loss of SOCS3 secreted by AM.
• During tumor development, this mechanism becomes dysregulated.
• SOCS3 liposomes were generated and their functional activity against the human lung cancer cell line A549 were assessed in rescue experiments. IncuCyte® Live-Cell Imaging and Analysis was used to assess cell confluence and apoptosis, via the inclusion of IncuCyte® Caspase-3/7 Green Apoptosis Reagent. Confluence which was reduced in response to the SOCS3 containing liposome treatment with greater caspase-3/7 activation, indicating increased apoptosis.
• Treatment with liposomes containing SOCS3 inhibited EC transformation and expansion in vitro , as well as in vivo and tumor growth in an A549 xenograft athymic nude mouse model.

Read the full paper in JCI Insight, October 2019.

Oncology

Drug sensitivity prediction models reveal a link between DNA repair defects and poor prognosis in HNSCC

The hunt for DNA repair defects and their clinical impact

Many cancer treatments, such as chemotherapy and radiation, inflict DNA damage on tumors.  The inability of a tumor to repair this DNA damage could theoretically increase treatment efficacy, but this requires further investigation and characterization.

In this study by Essers et al. from the Netherlands Cancer Institute and VUmc Cancer Center, the authors utilized machine learning for the development of gene expression models. They identified functional defects in gene repair pathways in head and neck squamous cell carcinoma (HNSCC), with subsequent validation and impact on patient prognosis.  This paper shows the power of an efficient screening mechanism for DNA repair defect to gain important insights for the HNSCC patient response to chemo-radiotherapy and outcome assessment.

Key findings include:

• Novel DNA repair defect markers were identified in HNCC cell lines representing cross link repair, HR/FA pathway defects, as well as compensatory repair mechanisms and shifts in replication repair.
• Machine learning was applied to 25 HNSCC cell lines for the identification of functional DNA repair defects as determined by hypersensitivity to mitomycin C and olaparib. Expression profiles were created, pointing to mutations in crosslink repair genes, as well as the downregulation of the DNA damage response and repair genes.
• To assess the functional output of migration, the IncuCyte® Live-Cell Imaging and Analysis System, along with the IncuCyte® WoundMaker, were utilized for scratch wound assays of crosslink repair impaired HNSCCs, with measurement of relative wound density. Invasion assays were also performed in vitro using both IncuCyte® and transwell assays, with the addition of extracellular matrix proteins to assess invasive capacity in response to Rad51 inhibition.
• HNSCC cell lines with defective crosslinking were found to be more migratory and invasive. Repair competent cell lines were treated with the RAD51 inhibitor BO2, as well as inhibitors of DNA-PK (NU7026, ATM Ku-55933 and WNT XAV-939t). The cell lines displayed similar migratory capacity, which could be reduced through the inhibition of DNA-PK.
• A validation study of this profiling method was assessed using datasets from the Cancer Genome Project (CGP), as well as analysis of patient samples from HNSCC patients receiving chemo-radiotherapy (cisplatin-based), DNA repair that were associated with reduced survival and poor prognosis.

Read the full paper in Cancer Research, September 2019.