A simple and efficient “cell in situ collagen zymography” technique to evaluate cellular collagenase activities in thyroid cancer cell lines

Savas E. G., Surer S. I., Sipahi M., Keles D., Oktay G.

MOLECULAR BIOLOGY REPORTS, cilt.52, sa.52, ss.1-17, 2025 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 52 Sayı: 52
  • Basım Tarihi: 2025
  • Doi Numarası: 10.1007/s11033-024-10158-8
  • Dergi Adı: MOLECULAR BIOLOGY REPORTS
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, CAB Abstracts, Chemical Abstracts Core, Veterinary Science Database
  • Sayfa Sayıları: ss.1-17
  • Anahtar Kelimeler: In situ zymography, Matrix metalloproteinases, Collagenases, Gelatinases, DQ-collagen, Thyroid cancer
  • Dokuz Eylül Üniversitesi Adresli: Evet

Özet

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. 

Keywords In situ zymography · Matrix metalloproteinases · Collagenases · Gelatinases · DQ-collagen · Thyroid cancer