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A sensitive screening assay for secreted motility-stimulating factors
S W Nam1, T Clair, E Schiffmann
1Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. swnam@box-s.nih.gov
Cell Motility and the Cytoskeleton
|August 30, 2000
Summary
Researchers developed a new motility assay to detect low concentrations of secreted factors like Autotaxin (ATX). This rapid method identifies cells secreting tumor-promoting motility factors, aiding cancer research.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Secreted motility-stimulating factors are challenging to detect at low concentrations using traditional methods like Northern or Western blots.
- Autotaxin (ATX) is a secreted factor linked to tumor invasion and metastasis, but its detection is difficult.
- ATX's enzymatic activities are less sensitive indicators than its ability to induce chemotaxis.
Purpose of the Study:
- To develop a sensitive and adaptable motility-based assay for screening cells that secrete active Autotaxin (ATX).
- To overcome the limitations of detecting low concentrations of motility-stimulating factors in cancer research.
Main Methods:
- Transfected NIH3T3 fibroblasts with ATX cDNA to create cells expressing the factor.
- Utilized a modified Boyden chamber assay to assess the chemotactic response of A2058 human melanoma cells to secreted factors.
- Employed densitometry measurements of stained membranes to quantify the motility response and identify positive clones.
Main Results:
- Successfully screened 36 clones and identified 4 positive cell lines secreting active ATX.
- The modified Boyden chamber assay demonstrated sufficient accuracy and sensitivity for detecting secreted motility-stimulating factors.
- The assay provides a rapid and adaptable means to identify ATX-secreting cells.
Conclusions:
- The developed motility-based assay is a rapid and highly adaptable tool for identifying cells that secrete motility-stimulating factors like ATX.
- This method offers a sensitive alternative to conventional techniques for detecting low-concentration secreted proteins.
- The assay has significant potential for advancing research in tumor invasion and metastasis.
