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A simple and efficient "cell in situ collagen zymography" technique to evaluate cellular collagenase activities in
Ege Gokce Savas1,2, Seniz Inanc Surer3, Murat Sipahi4
1Department of Chemistry, Faculty of Science, Dokuz Eylul University, Izmir, 35390, Türkiye.
Background:
Collagenases, a subgroup of matrix metalloproteinases (MMPs), play crucial roles in local invasion and metastasis in cancer. While substrate zymography and in situ zymography are commonly used to analyze the collagenases, traditional techniques have limitations in determining their local activities in vitro.
Objectives:
We aimed to develop a new "cell in situ collagen zymography" technique to enhance the efficiency of studying local collagenase activities in vitro.
Methods:
We utilized human thyroid cancer cell lines (8505 C, B-CPAP, FTC-133) and normal follicular thyroid cell line (Nhty-ori-3-1). We compared collagenase levels across these cell lines and selected 8505 C as a model due to its highest collagenase activity. We optimized factors including (i) fixation method (methanol, ethanol and zinc), (ii) dye-quenched (DQ) collagen concentration and (iii) collagen gel configuration. For gel configuration, cells were seeded under, on the top of, or between (sandwich) collagen gel layers. As controls, enzymatic activity was suppressed in the presence of EDTA, piroxicam and matrix metalloproteinase 8 inhibitor I. The optimized method was also applied to BCPAP, FTC-133, and Nthy-ori-3-1.
Results:
Our optimization process revealed that that the best visualization of collagenase activity in 8505 C was provided by the "sandwich model" of gel, containing 25 µg/mL of DQ-collagen with 100% cold methanol fixation. We confirmed the optimized method's applicability in other thyroid cell lines. The use of inhibitors validated the specificity of the fluorescent signal to MMP activity.
Conclusion:
The innovative "cell in situ collagen zymography" technique offers an efficient, cost-effective, and rapid method for analyzing local collagenase activities in vitro.
