gms | German Medical Science

123. Kongress der Deutschen Gesellschaft für Chirurgie

Deutsche Gesellschaft für Chirurgie

02. bis 05.05.2006, Berlin

In vivo detection and quantification of apoptosis in colorectal cancer metastases

Meeting Abstract

  • corresponding author V. Yagublu - Chirurgische Klinik, Universitätsklinikum Mannheim, Mannheim, Germany
  • M. Keese - Chirurgische Klinik, Universitätsklinikum Mannheim, Mannheim, Germany
  • T. Neufang - Chirurgische Klinik, Universitätsklinikum Mannheim, Mannheim, Germany
  • S. Post - Chirurgische Klinik, Universitätsklinikum Mannheim, Mannheim, Germany
  • P.I. Bastiaens - European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Deutsche Gesellschaft für Chirurgie. 123. Kongress der Deutschen Gesellschaft für Chirurgie. Berlin, 02.-05.05.2006. Düsseldorf, Köln: German Medical Science; 2006. Doc06dgch5118

The electronic version of this article is the complete one and can be found online at:

Published: May 2, 2006

© 2006 Yagublu et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.



Einleitung: The development an optical functional in vivo apoptosis assay, which is capable to localize and quantify apoptotic changes in a living organism, is an important target of cancer research. Such an assay should allow accurate monitoring of tumour growth/inhibition in vivo. Therefore, we developed an assay which allows visual quantification of apoptosis commitment in metastasized colorectal tumor cells in response to anticancer drug treatment at different time points in living mice.

Material und Methoden: A novel caspase-3 sensor based on EGFP and tHcred1 and a 14 amino acid linker containing the caspase-3 cleavage site for readout of apoptotic activity in cancer cells by Fluorescence Lifetime Imaging (FLIM) has been introduced by our research group recently. Fluorescence resonance energy transfer (FRET) between EGFP and tHcred1 in intact sensor results in reduction of fluorescence lifetime of the donor – EGFP. This reduction can be correctly and reproducibly detected by fluorescence lifetime imaging microscopy - FLIM. Activation of Caspase-3 in the process of apoptosis disrupts the covalent linkage between EGFP and tHcred1 and effectively eliminates FRET, thus conveying that a cell is apoptotic. This EGFP-DEVD-tHcred1 construct was stably transfected into C26 murine colon carcinoma cells, which are syngeneic to BALB/c mice. To validate in vivo functionality of the assay, the models of peritoneal and liver metastases were generated in 10-12 weeks old female BALB/c mice by intraperitoneal and intraportal injection of EGFP-DEVD-tHcred1 expressing C26 cells. Animals were treated with intraperitoneal injection of 30 mg/kg 5 FU for 0, 36, 72 and 120 hours. By the end of the tenth day after tumor inoculation, the animals were sacrificed under narcosis and exposed for FLIM.

Ergebnisse: The maximal apoptosis rate was achieved after 36 hours of 5-FU treatment in peritoneal metastasis (50,01%). In non-treated group 25,23% apoptotic cells were identified. The rate of apoptosis decreased during further 5-FU treatment. After 72 hours treatment, only 27,44% of cells demonstrated apoptotic lifetimes and at 120 hour point of 5 FU treatment no apoptotic cells could be detected. In the liver metastasis, the maximal proportion of apoptotic cells was found again at 36 hours of 5-FU treatment (45%), however the decrease in the rate of apoptotic cells with ongoing 5-FU treatment was slower than in peritoneal metastases. In the 72 hours 5-FU treatment group 44,71% of the cell were apoptotic and at 120 hours only 13,59% of the cells underwent apoptosis.

Schlussfolgerung: In summary, we show that an optical sensor can be successfully applied to monitor apoptosis as determined by caspase-3-induced proteolysis in living cells in the context of a living organism. This assay allows to monitor the changes in the level of apoptotic and healthy tumor cell populations throughout chemotherapy, thus facilitating further therapeutic interventions at the right time to push for a further regression in tumor growth.