Abstract
In this study we implemented an axillary SLN invasion model to develop highly sensitive imaging strategies enabling detection of a very small amount of tumor cells. A highly diffusible molecular probe targeting αvβ3 and αvβ5 integrins was investigated either via IV or locoregional injections. We additionally documented the potential interferences of this Near Infrared Fluorescence Probe with Blue Patente V and ICG dyes routinely used to facilitate lymph node detection during surgery. The human mammary adenocarcinoma MDA-MB-231-luc model was injected into the forepaw of nude female rats to obtain a controlled invasion of the axillary LN. Thanks to its high sensitivity, BLI was selected to achieve in vivo quantitation of tumor cells in SLNs and determine eligible animals for the study. NIRF of integrins was performed at 680 nm both in vivo and ex vivo using spectral unmixing to suppress auto-fluorescence signal and preserve sensitivity. In vivo BLI was quite reliable in estimating discrete invasion by cancer cells in the LN with thresholds of detection and quantitation of about 500 and 1500 cells respectively. For fluorescence at 680 nm, in vivo imaging is not suitable to detect micro-invasion, but ex vivo fluorescence with spectral unmixing of SLNs confirmed the presence of a tumor burden as low as 1500 cells expressing αvβ3/αvβ5 integrins. Targeting few tumor cells inside a micro-invaded sentinel lymph node by molecular probes is not sensitive enough to provide direct in vivo or peroperative imaging. At the time NIRF is performed on the excised specimen, high sensitivity imaging associated with spectral unmixing allowed such detection within less than 1 minute of examination.
Highlights
From the primary tumor, at the time tumor cells disseminate via lymph ducts, they accumulate in the first drainage site which is named the Sentinel Lymph Node (SLN)
We performed preclinical investigations about assessment of tumor micro-invasion in a rat model of SLN. For such a purpose we considered Bioluminescence Imaging (BLI) thank to its very high sensitivity detecting a tiny number of tumor cells in order to select animals with a predefined SLN micro-invasion status
1) Assessments of the fluorescence quenching, and wash-out kinetics in lymph nodes from healthy rats: There was no statistical difference in the fluorescence intensities quantified on ex vivo resected LNs after forepaw injection of IntegriSense680 versus Blue Patente V (BPV) + IntegriSense680 (Figure 1(A))
Summary
At the time tumor cells disseminate via lymph ducts, they accumulate in the first drainage site which is named the Sentinel Lymph Node (SLN). For the imaging of invasion, labeled dedicated Monoclonal antibodies (MAbs) and RGD mimetic agents for targeting αVβ3 and αVβ5 integrins have been identified as efficient probes for in vivo imaging (PET, SPECT, MRI, NIRF) Targeting integrins from both tumor cells and tumor-related angiogenesis thank to low molecular weight highly diffusible probes should theoretically contribute to significantly increase the detection’s sensitivity [13]. We performed preclinical investigations about assessment of tumor micro-invasion in a rat model of SLN For such a purpose we considered Bioluminescence Imaging (BLI) thank to its very high sensitivity detecting a tiny number of tumor cells in order to select animals with a predefined SLN micro-invasion status. Considering the routinely used dyes such as Blue Patente V (BPV) by surgeons to evidence the SLNs, we investigated its potential quenching effect on fluorescence intensities from both an integrin targeting probe and ICG
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