Vol. 2, 2018 — Featured publications for the IncuCyte® Live-Cell Analysis System

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Developmental Biology

Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus

Role of GSK3 in cell migration during neural crest development

Neural crest migration is vital to the function of neural crest cells, and its misregulation is associated with cancer. However, many questions remain about the mechanisms that control its vital migratory activity. Scientists from the Centre for Craniofacial and Regenerative Biology, King’s College London, studied the effect of the serine/threonine kinase GSK3 — previously shown to play an important role in craniofacial development — in neural crest migration in mouse and Xenopus.

Their studies show that GSK3 is tyrosine phosphorylated in migratory neural crest (mNC) cells, and its activity is under the control of a protein associated with neuroblastoma. Their findings include:

• mRNA and reporter gene expression analysis in frog Xenopus and mouse showed that both known GSK3α/β isoforms are expressed in mNC cells. Antibodies recognizing active-site tyrosine revealed GSK3α/β phosphorylation in the migratory cells.
• Cranial neural crest cells did not migrate in mouse embryos with a conditional knockout of both GSK3 isoforms α/β. Time-controlled pharmacological inhibition of GSK3 in Xenopus embryos confirmed these findings. Both treatments affected the cytoskeletal dynamics via control of key regulators FAK, Rac1, cdc42 and lamellipodin.
• Anaplastic lymphoma kinase (ALK), a protein associated with neuroblastoma, was expressed in mouse neural crest cells as verified by mRNA in situ hybridization. Pharmacological inhibition of ALK led to a loss of GSK3 phosphorylation, and mimicked the phenotypic effects of direct GSK3 inhibition.
• Western blot analysis of Neuroblastoma cell lines with high ALK expression (Kelly cells) showed coexpression of phosphorylated GSK3, which decreased when treated with ALK inhibitors.
• The IncuCyte® Scratch Wound Cell Migration Kit and IncuCyte® system were used to show that ALK inhibitors blocked Kelly cell migration, suggesting ALK activity is closely linked to GSK3 activity in neuroblastoma cells.

Read the full paper in Nature Communications, March 2018.

Infectious Disease

Safety implication of Salmonella based Brucella vaccine candidate in mice and in vitro human cell culture

Safely profile of anti-Brucella vaccine candidate

Current efforts to develop an anti-Brucella vaccine focus on safer approaches that utilize inactive, subunit vaccines and vectors that avoid introducing the complete Brucella organism. Scientists led by Dr. Lalsiamthara at the Chonbuk National University, Iksan Campus, in the Republic of Korea, previously developed an anti-Brucella vaccine candidate based on a Salmonella Typhimurium vector cocktail delivering immunogenic Brucella proteins superoxide dismutase (SOD), Brucella lumazine synthase (BLS), outer membrane protein-19 (OMP19) and proline racemase protein A (prpA). This study assesses the lethal dose in mice and toxicity to cultured human cells.

The data show high safety in mouse models and good safety profile in cultured human cells. They found that:

• Oral and subcutaneous administration of vaccine did not cause mortality in BALB/c mice, even when administered at 100 times the doses generally used in mice.
• The IncuCyte® Live-Cell Analysis System was used to measure apoptosis (Annexin V binding to phosphatidylserine) and nitric oxide (NO) production in human cells. The IncuCyte® Cytotox Green Reagent was used to measure cytotoxicity based on green fluorescence on dead cells. Both Caco-2 and THP-1 derived macrophages showed apoptosis and cytotoxicity. NOS production, as measured by a standard fluorescence assay, peaked at 24 h post infection, in both vaccine and control groups.
• ELISA assays showed lower level of TNF-α and IL-1β cytokine induction in vaccine-treated cells compared to the control group.

Read the full paper in Vaccine, March 2018.

ONCOLOGY

Small molecule Neuropilin‑1 antagonists combine antiangiogenic and antitumor activity with immune modulation through reduction of Transforming Growth Factor Beta (TGFβ) production in regulatory T‑cells

Development and evaluation of small molecule neuropilin-1 antagonists

The cell-surface coreceptor Neuropilin-1 (NRP1) binds a number of different growths factors, including isoforms of vascular endothelial growth factor (VEGF). Given the role of the immune system in cancer progression, NRP1 antagonists are desirable candidates for development as anticancer drugs.

In a joint study between the Institute of Structural and Molecular Biology, University College London, and the Department of Pharmacology, Stony Brook University, in New York, scientists used a previously published NRP1/VEGF-A antagonist (S)-2-(3-(benzo[c][1,2,5]thiadiazole-4-sulfonamido)-thiophene-2-carboxamido)-5-((diaminomethylene)amino)-pentanoic acid (EG00229) as a starting point to develop better candidates. They identified a new lead 1 (EG013777) and demonstrated antimigratory and antitumor activity in preliminary studies.

• Structure-based design was used to develop a series of optimized small molecule antagonists of NRP1/VEGF-A resulting in lead compound 1, with good affinity to NRP1 and favorable pharmacokinetic profile.
• Western blot analysis showed a decrease in VEGF-A-induced tyrosine phosphorylation of VEGF-R2/KDR in HUVEC cells treated with 1. Transwell assays demonstrated a 60% drop in HUVEC cell migration when treated with 30 μM 1, which is more potent than EG00229. Wound healing scratch assays performed on the IncuCyte® Live-Cell Analysis System showed that 1 delayed VEGF-induced wound closure. These data suggest that NRP1 activity is important for HUVEC cell migration.
• An organotypic endothelial−fibroblast coculture assay that mimics angiogenic behavior in endothelial cells resulted in reduced VEGF-induced branch points in tubular networks, overall network area and length when treated with 1. Similar effects on angiogenesis were seen with an ex vivo mouse aortic rings assay. These data potentiate NPR1 as a drug candidate for metastatic cancers.
• Treatment with 1 blocked VEGF-A-induced invasion in A375P (malignant melanoma) spheroid, which better represent in vivo cell growth, highlighting the role of VEGF-A signaling in aggressive melanoma.
• NRP1 is involved in the function and survival of regulatory T-cells (Tregs), which limit antitumor response in mouse models. Purified Treg populations of Tregs from mice treated with 1 showed a block in Treg activation, as shown by glioma-conditioned medium-induced increase in TGFβ production.

Read the full paper in J Med Chem, May 2018.

Pharmacology

Chondroitin sulfate protects vascular endothelial cells from toxicities of extracellular histones

The therapeutic mechanisms of chondroitin sulfate (CS) in lethal thrombosis

Extracellular histone release in patients with disseminated intravascular coagulation (DIC) causes platelet aggregation, neutrophil migration, and lethal thrombosis. Linear polysaccharide heparin binds negatively to charged histones, but it also blocks the coagulation pathway. Chondroitin sulfate (CS) has been used as a nutraceutical and a drug for osteoarthritis and is less inhibitory to the coagulation pathway. A study led by Dr. Nagano at the Department of Analytical Pharmacology, Meijo University, in Japan, investigated the interaction between CS and extracellular histones as a new strategy to treat lethal thrombosis induced by histones.

The data supports using CS as a novel agent to treat lethal thrombosis in DIC patients. Key finds are:

• Vascular endothelial cells were incubated with calf thymus histones with either heparin or CS. Heparin and CS protected endothelial cells in a dose dependent manner, with heparin eliciting better protection in lower concentrations. The IncuCyte® Live-Cell Analysis System was used to monitor cells and automatically calculate caspase-3/7 expression as a readout for cell death. Direct binding of CS to histones was confirmed via surface plasmon resonance.
• Heparin- and CS-treated mice injected with calf thymus histones showed reduced levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and serum creatinine (SCr), which are elevated in liver and renal injuries.
• Histone-induced platelet aggregation, white blood cell (WBC) activation and increased PLA levels are associated with embolism and inflammation. Heparin- and CS-treated mice injected with histones showed normal WBC and platelet counts with reduced PLA formation.
• Prothrombin time (PT) and activated partial thromboplastin time (APTT) tests revealed that CS has a lesser effect on coagulation compared to heparin, qualifying CS as a better candidate for treating lethal thrombosis with the risk of hemorrhage.

Read the full paper in Eur J Pharmacol, May 2018.

ONCOLOGY

Proteomic analysis of cancer-associated fibroblasts reveals a paracrine role for MFAP5 in human oral tongue squamous cell carcinoma

The role of cancer-associated fibroblasts (CAFs) within the tumor stroma

Cancer-associated fibroblasts (CAFs) secrete a variety of factors and extracellular vehicles (EVs) that promote tumor growth. Dr. Principe and colleagues at the Princess Margaret Cancer Centre in Canada embarked on a proteomics approach to better understand the role of CAFs in oral tongue squamous cell carcinoma (OTSCC) and to identify novel CAF associated biomarkers.

They identified 4247 proteins, including microfibrillar-associated protein 5 (MFAP5) as a novel CAF-enriched protein. Their report shows:

• Patient-derived fibroblasts were extensively characterized using an LC−MS-based proteomics approach. The researchers identified 4247 protein groups, the largest of its kind to date, and proceeded on with detailed analysis. Enrichment analysis revealed that distinct proteins were present in the exosome and conditioned media secretomes.
• The IncuCyte® Live-Cell Analysis System was used to monitor cell proliferation and migration. OTSCC cells treated with CAF-derived exosomal-enriched fraction (EXO) showed increased cell proliferation and migration compared to exosomal-free media (EFM). Heat denaturation negated the effects observed with the EXO fraction, highlighting the importance of intact exosomes in cell proliferation and migration. 
• Comparative proteomics revealed new proteins that are associated with a CAF-like state. Of note is MFAP5, a protein component of extracellular microfibrils, which was enriched in CAF secretomes. Protein expression analysis in OTSCC tissue microarray showed that MFAP5 expression in cancer-associated stroma was associated with patient survival.
• In vitro assays in OTCC cells showed that human recombinant MFAP5 induced cell growth and migration via the Mitogen-activated protein kinase (MAPK) and AKT pathways, consistent with the roles of these pathways in cancer malignancy.

Read the full paper in J Proteome Res, June 2018.