Vol. 3, 2018 — Featured publications for the IncuCyte® Live-Cell Analysis System

Subscribe to Live Cell Insights eNewsletter

Don't miss out on new and notable IncuCyte® publications! Be the first to see IncuCyte Live-Cell Analysis System in peer-reviewed journals. 

SUBSCRIBE NOW

Ask a Scientist!

Let us know how we can help you get your research published using the IncuCyte!

Let us know what you need

NEUROSCIENCE

An in vitro paradigm to assess potential anti-Aβ antibodies for Alzheimer’s disease

Development of a bioassay to screen and identify anti-amyloid β-protein therapeutics

Synthetic amyloid β-protein (Aβ) exists in vitro in an array of assemblies, but it remains unclear if these assemblies are found in the human brain. Studies on Aβ in aqueous extracts from brains of Alzheimer’s disease patients suggest that Aβ forms variously-sized assemblies, only some of which have disease-relevant bioactivity. At least nine anti-Aβ monoclonal antibodies (mAbs) are currently in various stages of clinical development, but anti-Aβ immunotherapy so far has had limited success in humans. The disappointing success is partly due to potential therapies targeting a broad range of Aβ species, including inactive forms, so that the necessary therapeutic concentration against active forms cannot be reached.

Jin et al. of the Laboratory for Neurodegenerative Research at Brigham and Women’s Hospital describe a process for identifying anti-Aβ antibodies in vitro based on their ability to target human neurotoxic Aβ. The researchers developed a bioassay to quantify the protective effects of potential anti-Aβ antibodies. Their report shows:

• The potential mAb therapies 1C22 and 3D6 bind to protofibrils better than monomers. Jin et al. used a constant concentration of plate-immobilized Aβ monomers and potofibrils (PFs) with three potential mAb therapies, 1C22, 3D6, and 266, diluted in solution across plates to show that 1C22 and 3D6 bound Aβ PFs better than monomers. In contrast, 266 bound PFs and monomers about equally. In a reciprocal experiment with immobilized mAbs and Aβ in solution, Jin et al. confirmed that 1C22 and 3D6 bound Aβ PFs better than monomers and 266 bound PFs and monomers equally.
• The preference of 1C22 for soluble polymers is likely due to its requirement for an extended or conformational epitope. Using the surface plasmon resonance (SPR) technique comparing binding of a monovalent form of the antibody to the intact bivalent form, Jin et al. explored the mechanism by which 1C22 binds PF polymers more tightly than monomers. Aggregates have multiple potential binding sites in close proximity, so bivalent IgGs will be bound at 2 adjacent antigen sites.
• Anti-Aβ antibodies attenuate the effects of neuritic dystrophy on human induced neurons exposed to Aβ derived from brain extracts of patients with Alzheimer’s disease. Using the The IncuCyte® Live-Cell Analysis System, Jin et al. assessed the maturation and effects of Aβ from brain extracts of patients with Alzheimer’s disease on human induced neurons in the absence and presence of potential mAb therapies. The neurons showed neuritic dystrophy (decreased neurite length and branch points, a feature of Alzheimer’s disease) upon exposure to soluble Aβ from brain extracts of patients with Alzheimer’s disease. Potential mAb therapies attenuated the effects of neuritic dystrophy on these neurons in a time-dependent way.

Read the full paper in Nature Communications, July 2018.

INFECTIOUS DISEASE

Marek’s disease virus oncoprotein Meq physically interacts with the chicken infectious anemia virus-encoded apoptotic protein Apoptin

Molecular mechanism involved in increased pathogenesis of MDV-CAV co-infection

The Marek’s disease virus (MDV) oncoprotein Meq is one of the few viral proteins expressed in the cell line MSB-1, a cell model used to study virus-induced T-cell lymphomas. Meq is expressed primarily in the nucleus and induces neoplastic transformation of T cells, in part, through inhibition of apoptosis. Chickens infected with Marek’s disease (MD) are often coinfected with chicken anemia virus (CAV), which expresses a protein, Apoptin (or VP3), that uniquely induces cell cycle arrest and apoptosis in transformed cells. CAV, thus, replicates in MDV-transformed T-cells, but the mechanism remains poorly understood.

Brown et al. of the Nair lab group at the Pirbright Institute undertook to determine whether the proteins Meq and Apoptin directly interact in MDV-transformed cell lines. Their research demonstrates that Meq interacts with and inhibits Apoptin, suggesting mechanisms for coinfection of MDV and CAV. Brown et al. found that:

• Meq and Apoptin colocalize to the nucleus of transfected cell lines and interact biochemically. Immunohistochemistry showed colocalization of Meq and Apoptin in the nucleus of cotransfected CEF and DF-1 cell lines. Likewise, Meq and Apoptin also colocalize in the nucleus of Apoptin-FLAG transfected MSB-1 cells. Reciprocal GST pull-down and immunoprecipitation experiments showed that Meq and Apoptin co-precipitated with one another.
• The Meq-Apoptin interaction occurs between amino acid residues 130 and 140 of the Meq protein. Meq is known to interact with a number of proteins through a CtBP-binding motif or leucine zipper motif. However, mutational analysis with C-terminal truncated Meq constructs indicated that the interaction between Meq and Apoptin occurs between the amino acid residues 130 and 140.
• Meq inhibits Apoptin in the DF-1 cell line. Brown et al. used the IncuCyte® Live-Cell Analysis System and Caspase 3/7 Apoptosis Assay Reagent to compare apoptosis rates in Meq-Apoptin cotransfected and Meq- or Apoptin-transfected DF-1 cells. Apoptosis was significantly lower in the Meq-Apoptin cotransfected DF-1 cells than in cells transfected with only Meq or Apoptin.

Read the full paper in Oncotarget, March 2018.

ONCOLOGY

Pharmacological and genomic profiling of neurofibromatosis type 1 plexiform neurofibroma-derived schwann cells

Characterization NF Schwann cell lines as pharmacological models of NF1 mutant derived tumors

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic syndrome wherein patients develop multiple peripheral nervous system tumors, including plexiform neurofibromas (pNFs). pNFs are benign, slow-growing, yet heterogeneous nerve sheath tumors that are thought to develop from NF1-/- Schwann cells and that can transform into aggressive sarcomas. People with NF1 are generally heterozygous for NF1 (+/-), but may develop pNF when there is a second, somatic mutation at NF1. Therapeutic research for pNFs has been hindered by clinical and cellular heterogeneity, as well as lack of preclinical model systems. Recently, viable NF1 cell lines have been developed to test potential therapies.

Ferrer et al. of the National Center for Advancing Translational Sciences (NCATS) division of the NIH report on dose response quantitative high-throughput screen (qHTS)-compatible cell proliferation assays and their use screening a collection of oncology drugs of the NCATS Mechanism Interrogation PlatE (MIPE 4.0) library. These data represent a new and validated scientific resource and toolset to facilitate therapeutic discovery for NF1 mutant-derived tumors.

• Ferrer et al. gathered and compared high-throughput genomic and transcriptomic data on immortalized and primary cell lines from pNF patient samples, including data from SNP Arrays, RNA-Seq, and ExomeSeq. Then, they optimized culture conditions to use six immortalized cell lines for the high-throughput drug-screening assays.
• The IncuCyte® Live-Cell Analysis System was used to characterize growth rates for the cell lines for comparisons during the high-throughput drug screening.
• All data was validated and made publically available online at http://www.synapse.orp/pnfCellCulture.

Read the full paper in Sci Data, June 2018.

PHARMACOLOGY

Changes in cell morphology guide identification of tubulin as the off-target for protein kinase inhibitors

Development of a cell-morphology assay to identify kinase inhibitors that directly target tubulin

Determining the correct mechanism of action by which kinase inhibitors kill malignant cells is an important part of the drug discovery process. Some kinase inhibitors have been found to target non-kinase proteins, including microtubules. Microtubules, composed of α/β-tubulin polymers, are a main component of the cytoskeleton, and are essential for chromosomal segregation during cell division and help determine cell shape and morphology.

Hoque et al. test the hypothesis that a kinase inhibitor that actually targets tubulin causes unique changes in cell morphology. In these studies, the researchers found that early changes in cell morphology after drug treatment may indicate tubulin targeting rather than direct kinase targeting. Key finds include:

• Tivantinib inhibited tubulin polymerization, whereas (R)-crizotinib, centrinone, and ponatinib had no effect on the rate of polymerization. Hoque et al. tested five well-known drugs for tubulin polymerization and cell viability: tivantinib, (R)-crizotinib, paclitaxel, centrinone, and ponatinib. Vinblastine (a known tubulin inhibitor) and paclitaxel (a known accelerator of tubulin polymerization) were used as positive controls. Hoque et al. also determined half maximal effective concentrations (EC₅₀) for the drugs.
• Glioblastoma cells treated with trivantinib caused cell morphology changes within 30 minutes. Hoque et al. treated glioblastoma cells with 5-fold higher concentration than EC₅₀ of trivantinib, vinblastine, (R)-crizotinib, centrinone, and ponatinib and monitored cell morphology changes using the IncuCyte® Live-Cell Analysis System. Cells became rounder and smaller after trivatinib and vinblastine treatment, whereas (R)-crizotinib, centrinone, and ponatinib had no effect on cell morphology.
• Shrinking and rounding of cells is not due to mitosis or onset of apoptosis. Rounding may indicate cells entering mitosis. So, to establish cell cycle progression, Hoque et al. tested the cells using the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) system in the IncuCyte® Live-Cell Analysis System. Distribution of cells within cell cycle phases was not statistically significantly different compared to control within the first eight hours of treatment. Changes in cell cycle distribution became significant after 24 hours of drug treatment. Likewise, analysis of apoptosis by Annexin V staining showed that increased levels of apoptosis did not begin until at least 24 hours after drug treatment.

Read the full paper in Pharmacol Res, June 2018.

TRANSCRIPTOMICS

A transcriptome-wide association study of 229,000 women identifies new candidate susceptibility genes for breast cancer

Novel insights into breast cancer genetics and biology

Genetic factors play an important role in the risk of developing breast cancer, one of the most common cancers in women. Genome-wide association studies (GWAS) have identified about 170 genetic loci associated with risk of breast cancer, but these loci still account for less than 20% of familial risk for breast cancer. Transcriptome-wide association studies (TWAS) are used to investigate the association between GWAS-identified gene expression and disease risk. Wu et al. report on a TWAS study using data from 122,977 cases and 105,974 controls of European descent from the Breast Cancer Association Consortium (BCAC) database.

These studies describe the identification of new risk loci and candidate genes using a TWAS study to evaluate associations between predicted genetic loci and breast cancer risk in a large cohort of women of European ancestry. Key findings include:

• Significant associations between expression and risk were found for 48 loci of the 8,597 genetic loci evaluated, 14 of which were previously unidentified. Wu et al. used transcriptome and genotyping data from the Gentoype-Tissue Expression (GTEx) project or data about SNPs located in promoter or enhancer regions of genes to build models of RNA expression in normal breast tissue. They compared this data with The Cancer Genome Atlas (TCGA) and identified 8,597 genes for association analyses, which they performed using meta-analyses from the 122,977 cases and 105,974 controls of European ancestry in the BCAC database. From this analysis, they identified 48 loci with significant association for risk, 14 of these genes were 500 kb away from any risk variant previously identified using GWAS.
• Thirteen genes were selected for in vitro assays of gene function. The authors used integrated expression quantitative trait and in silico prediction of GWAS target (INQUISIT) scores and Ingenuity Pathway Analysis (IPA) to further assess the 48 loci they had identified by knockdown.
• Knockdown of 11 of the 13 genes identified showed an effect on cell proliferation and colony-forming in normal breast and breast cancer cell lines. Using the The IncuCyte® Live-Cell Analysis System, Wu et al. assessed effects of knockdown of 19 genes (13 identified above plus negative and positive control genes) on cell-proliferation of a normal mammary cell line and two luminal cancer cell lines and colony-forming of a luminal cancer cell line.

Read the full paper in Nature Genetics, June 2018.

IMMUNOLOGY

Neutrophil extracellular traps prevent HIV infection in the female genital tract

Role of genital neutrophil NETs in preventing HIV infection

Neutrophils found in the female genital tract are phenotypically and functionally different from neutrophils found in blood. Female genital tract neutrophils, present in the stratified epithelium of the vagina and on mucosal surfaces, are rapidly recruited to sites of trauma, where viruses may enter the bloodstream. Blood neutrophils are known to inactivate microbes, in part, through the extrusion of neutrophil extracellular traps (NETs), DNA fragments associated with antimicrobial activity. Barr et al., in collaboration with the Rodriguez-Garcia lab at Dartmouth, sought to understand the role of genital neutrophil NETs in preventing HIV infection.

NETs produced by neutrophils in the genital tract inactivate HIV; this could represent a previously unrecognized mucosal protection mechanism against HIV infection. To establish their conclusion, Barr et al. found that:

• Genital neutrophils trap HIV particles in NETs. Previous experiments had shown that dendritic cells capture HIV particles. Using purified neutrophils from the ectocervix (ECX), endocervix (CX), and/or endometrium (EM) of 6 women who were HIV-negative and received hysterectomies for benign conditions, Barr et al. first showed that genital neutrophils, like blood neutrophils, could release NETs. By stimulating the neutrophils with HIV-virus-like particles (HIV-VLPs) or calcium ionophore (a known NET inducer), and staining with the nucleic acid dye Sytox they showed that genital neutrophils released DNA in response to HIV-VLP stimulation. Next, they showed that, like those released from dendritic cells, NETs released from genital neutrophils colocalize with HIV-VLP-GFP, suggesting that NETs released from genital neutrophils capture HIV-VLPs.
• Genital neutrophil release of NETs upon HIV-VLP stimulation is time-dependent and dose-dependent. Using the IncuCyte® Live-Cell Analysis System for real-time, time-lapse imaging, Barr et al. characterized the dynamics of HIV-VLP capture by genital neutrophil NETs. They showed, after stimulation with HIV-VLPs, genital neutrophils released NETs from ECX, CX, and EM tissues quickly for about 10 minutes and continued release for about 30 minutes before tapering. Increasing levels of HIV-VLP per cell also increased the NET total area.
• NETs released from genital neutrophils inactivate HIV and prevent infection of CD4⁺ T cells. Barr et al. isolated NETs from genital neutrophils and incubated with HIV before adding CD4⁺ cells and measuring the rate of infection with fluorescent microscopy and ELISA detection of p24. The presence of isolated genital neutrophil NETs incubated for 7 days with HIV prevented HIV infection of CD4⁺ cells, as indicated by lack of GFP fluorescence and decreased levels of p24. Near complete inhibition of HIV virions occurred after 7 days.
• Different strains of HIV, HIV lacking envelope, and Toll-like receptor (TLR) 7/8 induced genital neutrophils to release NETs, giving insight into the mechanisms by which NETs inactivate HIV particles.

Read the full paper in Mucosal Immunology, June 2018.

ONCOLOGY

P53/PUMA are potential targets that mediate the protection of brain-derived neurotrophic factor (BDNF)/TrkB from etoposide-induced cell death in neuroblastoma (NB)

Role of P53 and PUMA in escaping chemotherapeutic drug-induced apoptosis

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children. Patients with high-risk disease have a poor prognosis, with a long-term survival rate of < 40%. About 50%-60% of high-risk patients have elevated expression of brain-derived neurotrophic factor (BDNF) and/or its kinase receptor TrkB in their tumors. Elevated expression of BDNF/TrkB is known to induce chemoresistance of tumors, one of the main challenges of treating patients with high-risk NB. However, the mechanism by which BDNF/TrkB elevated expression protects tumors from chemotherapy drugs have yet to be fully explored.

Hua et al. of the Medical Research Center in Shenyang, China explored the mechanism by which BDNF/TrkB may protect NB cells from the drug etoposide. They found BDNF partly blocks etoposide-induced increases of p53 and PUMA in TrkB-expressing NB cells. Their report shows:

• BDNF/TrkB protects NB cells from etoposide-induced death by down-regulating p53. Hua et al. used two tetracycline-regulated TrkB-expressing cell lines, TB3 and TB8, and showed a significantly decreased cell survival rate in cells treated with etoposide alone compared to those also pretreated with BDNF. They showed reduced p53 expression in cells pretreated with BDNF followed by treatment with etoposide. Cell survival was significantly reduced in cells with siRNA-induced knockdown of p53 expression, despite etoposide treatment, suggesting that reduced expression of p53 in cells pretreated with BDNF is the mechanism by which BDNF protects cells treated with etoposide from cell death.
• BDNF and etoposide treatment have opposite effects on the expression of some BCL-2 proteins, and BDNF pretreatment of cells attenuates the increased expression of pro-apoptotic protein PUMA caused by etoposide. The BCL-2 family of proteins are downstream targets of p53 and play varying roles in apoptosis. Hua et al. explored if BCL-2 family of proteins are involved in the protection of BDNF/TrkB from etoposide. They treated TB3 and TB8 cells with BDNF or etoposide and examined expression of several BCL-2 family proteins. In etoposide-treated cells, the expression of proteins known to promote apoptosis increased (including PUMA), and one protein known to inhibit apoptosis decreased. In contrast, after treatment with BDNF, PUMA expression decreased and the expression of two proteins know to inhibit apoptosis increased. Using BDNF-pretreated cells, they also show that PUMA expression is decreased in etoposide-treated cells relative to etoposide-only treated cells, suggesting that BDNF attenuates etoposide-induced expression of PUMA.
• Knockdown and overexpression of PUMA transcripts in TB3 and TB8 cell lines shows that PUMA expression is both necessary and sufficient for the increased cell survival observed in etoposide-treated cells. The researchers used siRNAs to knockdown expression of PUMA and measured cell survival and confluence rate in the IncuCyte® Live-Cell Analysis System after etoposide treatment. Knockdown of PUMA expression in etoposide-treated cells showed increased cell survival and decreased percent confluence. Overexpression of PUMA by lentivirus decreased cell survival, but the survival could be increased by treatment with BDNF.

Read the full paper in Apoptosis, June 2018.