Modeling and quantifying the detection thresholds of fluorochromes pairedwith a molecular probe of integrins regarding the development of an imaging strategy for the lymph node involvement

The concept of sentinel lymph nodes in oncological surgery is based on the fact that the spread of solid tumors in an orderly manner passes first through the lymphatic system. Thus, the sentinel lymph node, meaning the first lymph node encountered by lymphatic drainage from the site of the tumor, is likely to be the first one affected by metastasis. Therefore, a negative sentinel lymph node makes it very unlikely that other lymph nodes along the same lymphatic pathway will be affected. Our interest in the development of an alternative molecular imaging technique for the localization and the detection of the sentinel lymph node micro-invasion is highly triggered by the limits of the current techniques. In this context, RGD mimetic molecular probes are prime targets for fluorescence invivo imaging. The work we report here consist in validating the detection of a fluorochrome paired with a mime RGD from the lymph nodes collected ex vivo. In a translational research approach, we have chosen as animal model some healthy sheep lymph nodes. For the various intra-ganglionic fluorochromes tested, AF750-RGD could be used to give good fluorescence signal from the 1/100 dilution, i.e. 10 nmol, and this fluorescence increases with the concentration of AF750-RGD. For the Cy5.5 the net fluorescence signal appears only from the dilution 1/20 i.e. 5nmol and intensifies with dilution 1/10. Finally, for the ICG (Indo Cyanine Green), a good fluorescence signal is obtained with respect to the sample at the dilution 1/500, i.e. 7.75 mmol. However the unmixing spectral, better than the subtraction of background noise, improves the contrast. Validation of these detection thresholds from these lymph nodes directs us on the choice of fluorochromes and their concentration in a targeting strategy of integrins by fluorescence imaging.