A ring barrier-based migration assay to assess cell migration in vitro

Cell migration is a key feature of virtually every biological process, and it can be studied in a variety of ways. Here we outline a protocol for the in vitro study of cell migration using a ring barrier-based assay. A 'barrier' is inserted in the culture chamber, which prevents cells from entering a defined area. Cells of interest are seeded around this barrier, and after the formation of a peripheral monolayer the barrier is removed and migration into the cell-free area is monitored. This assay is highly reproducible and convenient to perform, and it allows the deduction of several parameters of migration, including total and effective migration, velocity and cell polarization. An advantage of this assay over the conventional scratch assay is that the cells move over an unaltered and virgin surface, and thus the effect of matrix components on cell migration can be studied. In addition, the cells are not harmed at the onset of the assay. Through computer automation, four individual barrier assays can be monitored at the same time. The procedure can be used in a 12-well standard plate allowing higher throughput, or it can be modified to perform invasion assays. The basic procedure takes 2-3 and to complete

Wnt5a promotes human colon cancer cell migration and invasion but does not augment intestinal tumorigenesis in Apc1638N mice

Whereas aberrant activation of canonical Wnt/-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression

Applicability and Reproducibility of Biomarkers for the Evaluation of Anti. Inflammatory Therapy in Allergic Rhinitis

Background: We aimed to study the reproducibility of several biomarkers of allergic rhinitis to investigate their potential as outcome measures in clinical intervention trials. Furthermore, we investigated the kinetics of the biomarkers studied in nasal lavage and brush material following a placebo-controlled nasal allergen challenge. Methods: We performed a skin prick test and measured serum specific immunoglobulin (Ig) E levels and inflammatory biomarkers in nasal lavage and brush material in 20 patients with allergic rhinitis on 2 separate days (washout, 14-21 days). The patients were then randomly assigned to undergo an intranasal challenge with a relevant allergen (n = 10) or diluent (n =10) in order to assess the kinetics of several biomarkers of allergic airway inflammation in nasal lavage and brush samples. Results: Baseline serum IgE levels and skin wheal sizes were highly reproducible measurements, with a coefficient of variation (CV) of 13.4% and 18.2%, respectively. This was not the case with the majority of inflammatory biomarkers, whose CV varied considerably (range, 6.1%-224.1%). The nasal allergen challenge induced an increase in composite symptom scores in all patients. Compared to placebo, tryptase (P=.004), eosinophilic cationic protein (ECP) (P=.03) and (alpha 2-macroglobulin (P=.002) were increased in nasal lavage at 20 minutes post allergen. Nasal lavage ECP levels and nasal brush eosinophils were still significantly increased at 7 hours (P=.03 and P=.04), but all statistical significance had been lost at 24 hours post challenge. Conclusion: Serum specific IgE assays and skin prick tests exhibited good reproducibility in patients with clinically stable allergic rhinitis. M were also able to investigate the kinetics of allergen-incluced upper airway inflammatory markers in nasal lavage and brush material. Hence, nasal allergen challenge, when used in combination with nasal lavage and brush sampling, is a suitable research tool for early drug development

Tissue inhibitor of metalloproteinase-3 (TIMP3) expression decreases during melanoma progression and inhibits melanoma cell migration

Aims: Malignant melanoma is the most aggressive form of skin cancer, and metastatic dissemination to regional and visceral sites is responsible for the majority of melanoma-related mortalities. In a recent study by our group, we observed reduced expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in the majority of stage III melanoma samples studied. TIMP3 has been reported as a tumour suppressor in several human malignancies, with reduced expression correlating with poor clinical outcome. In this study, we investigated the changes in TIMP3 expression during melanoma progression. Patients and methods: TIMP3 expression was analysed by immunohistochemistry in sequential archived tumour material from stage I/II, stage III and stage IV samples from melanoma patients (n = 33). Protein expression was investigated for associations with disease-free survival and overall survival. Methylation status of the gene promoter was determined using methylation-specific PCR. In vitro assays were used to investigate the functional consequences of TIMP3 expression on behavioural aspects of melanoma cells. Results: We show that TIMP3 expression decreases with melanoma progression although no significant clinical associations were obtained. Analysis of the status of promoter methylation using methylation-specific PCR revealed it to be a low-frequency event in melanoma. Additionally, through gene modulation experiments in melanoma cell lines, we show that TIMP3 negatively regulates cell migration, invasion and anoikis resistance. Conclusions: Collectively, our data suggests that TIMP3 functions as a tumour suppressor in melanoma and negatively regulates several aspects of the metastatic cascade. (C) 2016 Elsevier Ltd. All rights reserved