Repetitive bioluminescence imaging of Fluc+ tumor bearing mice: comparison of the variability of intraperitoneal and intravenous administration of D-luciferin.

Student thesis: Master-after-master


Background: Animal models are of great importance in cancer research for the development and evaluation of effective anti-cancer therapies. This research has been complicated by inaccurate means of in vivo tumour burden assessment. Various techniques have been developed to compensate for this shortcoming. In vivo reporter gene imaging based on firefly luciferase (Fluc) and bioluminescent detection (BLI) allows sensitive and non invasive assessment of tumour burden. This method is used for testing the effects of new therapeutics. However, measurements can show substantial variability.
Aim: The aim of this thesis is to correlate photon emission and cell count in vitro and in vivo of a luciferase transduced R1M rhabdomyosarcoma cell line. Secondly the intensity and kinetic profile and repeatability will be compared after IV and IP administration of D-Luciferin. The ultimate goal is to use the characterized in vivo model for the evaluation of experimental anti-cancer therapies.
Materials and methods: Photon emission of increasing numbers of Fluc-positive R1M rhabdomyosarcoma cells was measured in vitro using an ICCD-camera. For in vivo measurements Fluc-positive R1M cells (1x105) were subcutaneously inoculated in nu/nu-mice. Dynamic-BLI was performed after IV or IP administration of D-Luciferin. A time profile of the photon emission was generated and the Peak Photon Emission (PPE) was calculated based on the 95th percentile of the curve. For the assessment of repeatability every D-BLI acquisition was repeated after 4h. Repeatability was assessed using Bland-Altman analysis. A separate group of animals were serially imaged from day 3 to day 21 post tumour inoculation. Administration of D-Luciferin via IV or IP route was alternated on subsequent days to allow for comparison within the same group of mice. In a selected group of animals photon emission after IV administration of D-Luciferin was correlated with the number of cells counted on histological samples.
Results: Photon emission correlates with cell number in vitro (r2= 0.99). The mean difference between repeated measurements was 29.0% for IV and 30.5% for IP which is explained by tumour growth during the 4h-interval. The Bland-Altman curve for IV shows that there is a trend towards an improved repeatability with increasing peak photon emission. This trend is not present for IP. The 95% confidence interval was smaller for IV (-25.6% to 83.3%) as compared to IP (-41.5% to 102.5%). The coefficient of repeatability was 80.2% for IV versus 95.0% for IP. Time to peak is significantly shorter for IV compared to IP (pConclusion: In vitro bioluminescence shows an excellent linear correlation between cell number and photon emission. Photon emission in vivo increases with increasing cell count, however attenuation and limited substrate penetration decrease the measured photon emission per cell with increasing tumour size. Photon emission after IV injection of D-Luciferin is more intense when compared to IP injection. Time to peak photon emission for IV measurements is significantly shorter and shows less variability than IP. Repeatability of bioluminescence measurements is moderate, but better for IV compared to IP. These results indicate that for the evaluation of experimental therapies IV administration of D-Luciferin combined with dynamic BLI offers the best sensitivity for the quantification of therapeutic efficacy.
Date of Award7 Jun 2007
Original languageEnglish
SupervisorTony Lahoutte (Promotor)


  • molecular imaging
  • Biioluminescence

Cite this