Journal des biomarqueurs moléculaires et du diagnostic

Application of Proteases as Molecular Imaging Tools for Early Gastric Cancer Detection: In Vitro and Ex Vivo Investigation

Abstract

Wajana Lako Labisso and Daniel Seifu

Background: Substantial miss-rate of gastric cancer by conventianl endoscope was reported, indicating the importance of novel biomarkers for early detection of precancerous and cancerous lesions.

Objective: To identify potential biomarker molecules for early gastric cancer detection and evaluation of their application for molecular in vivo imaging.

Method: Besides the CEA-TAG+/- mouse model, 3 human and 2 murine gastric cancer cell lines were applied in this study. Cells lines were cultured at standard conditions. To determine the expression pattern of the targets, total RNA was extracted from cultured cells; cDNA was synthesised and qPCR was performed using spcefic primers. The murine stomach was removed and washed with PBS. The stomach was macroscopically categorized into tumor region and normal tissues. Histopathology and imunohistochemitry was performed in one half of the tumor tissues using specific antibodies. In the other half of the tumour tissue, RNA was extracted, reverse transcribed into cDNA and qPCR was performed. Histopathology, immhunohistochemistry and qPCR was performed for tumor-adjescent normal tissues to compare the difference in expression pattern of the target. Finally, a cathepsin activatable probe was injected into mice intravenously. Ex vivo fluorescent imaging was performed after 24 h of intravenous injection of a cathepsin activatable probe. Gene expression was quantified by standard method with normalizing CT values to the housekeeping gene (Cyclophilin A).

Results: Significantly elevated expression of cathepsins B, H and MMP2 in human and murine gastric cancer cell lines was detected. In addition, increased expression of cathepsins and MMPs was observed in murine primary gastric tumour specimens compared to the corresponding adjacent normal gastric mucosa (p<0.05, tumor versus normal mucosa). Accordingly, the NIRF probe was specifically activated in stomach tumor of CEA-TAG+/- mice and allowed ex vivo detection of the stomach tumor by Odyssey planar near-infrared scanner. Furthermore, immunohistochemical stainings with antibodies specific for cathepsins and MMPs indicated a stronger signal towards the tumor tissues compared to the adjacent normal mucosa.

Conclusion: Cathepsins and MMPs are potential biomarkers for detection of high grade dysplasia and early gastric cancer in mouse models, revealing the potential applicability of the biomarkers in tumor imaging and therapeutic monitoring.