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 αvβ 3 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.
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). The interest in exploring the sentinel lymph node is to obtain information on the tumor invasion stage [
1) Ethics Statement
All procedures on animals were performed in accordance with European ethical guidelines (European directives 2010/63/EU) and were approved by the Regional Committee for Animal Care and Ethics in Animal Experiments (CECCO n˚ 3).
2) Cell Culture
The MDA-MB-231-luc-D3H2LN human breast adenocarcinoma cancer cell line was obtained from Perkin Elmer (France). The cancer cell line was maintained according to the supplier’s instructions.
3) Animals
Pathogen-free 8 to 12 week-old female nude rats were purchased from Taconic (Germany). Rats were acclimated for 7 days in the laboratory before experimentation and were maintained in sterilized filter-stopped cages inside a controlled ventilated rack with access to food and water ad libitum. They were examined daily for clinical signs, distress, decreased physical activity and weighed 3 times a week.
4) Cell xenografts
Human breast cancer xenografts from MDA-MB-231-luc cells were established in NIH nude rats. We performed forepaw injection, so that 15 rats were anaesthetized by inhalation of 1.5% isoflurane with air (IsofloÒ, AXIENCE S.A.S, France) and inoculated by 2 × 106 tumor cells in 10 μL PBS into the forepaw. The rats received pre-treatment with 10 mg/kg of busulfan 2 days before cell injection, so that immunodeficiency of rats was improved.
5) Bioluminescence Imaging
BLI was performed twice a week until the end of the study (Day 28) using an IVIS-Lumina II (Perkin Elmer, France). Each rat was IP injected with 20 mg luciferin potassium salt (Promega, France). Rats anesthetized by 1.5% isoflurane were placed on a thermostatically controlled heating pad (37˚C) during imaging. Acquisition binning and duration were set depending on tumor activity. Signal intensity was quantified as the total flux (photons/seconds) within ROIs drawn manually around the tumor area using Living Image 4.4 software (Perkin Elmer, France).
6) Estimation of the number of tumor cells present in the SLN
A calibration curve was determined from known amounts of cells (500, 1000, 5000, 10,000 & 50,000 cells) either deposited on the skin or subcutaneously injected. The injected volume was 100 µL of culture medium supplemented with luciferin potassium salt (300 µg/mL). Immediately after luciferin addition, the cell suspension was deposited or injected, and then BLI was performed 5 minutes later.
7) Near Infrared Fluorescence Imaging
NIRF was performed in vivo and ex vivo on excised specimens using an IVIS-Lumina II (Perkin Elmer, France) operated according to the spectral unmixing mode. Rats selected from BLI were IV injected with 20 nmol of IntegriSense680 (Perkin Elmer, France). Animals anesthetized by 1.5% isoflurane were placed on a thermostatically controlled heating pad in supine or lateral position. Signal intensity was quantified as the total radiant efficiency ([photons/seconds]/[µW∙cm−1]) within ROIs drawn manually around LN area using Living Image 4.4 software (Perkin Elmer, France).
8) Assessment of fluorescence quenching
For these experiments, 50 µL of Blue Patente V (0.5 nmol, Guerbet, France), IntegriSense680 (0.5 nmol, Perkin Elmer, France), ICG (129 nmol, SERB France), or 50 µL of a mix of the two dyes (BPV + IntegriSense680 or BPV + ICG) were injected in the forepaw of the rat, and then axillary LNs were resected 15 min after injection, so that NIRF was performed on excised LNs.
9) Sacrifice and organ removal
Rats under anesthesia were sacrificed by lethal IV injection of pentobarbital (Ceva Santé Animale, Libourne, France) and LNs were collected from each animal for immediate ex vivo assessments.
10) Statistical analysis
Statistical analysis was performed using Graph Pad Prism software 5.0 (Graph Pad, USA). Statistical analysis was performed with the Student’s unpaired t test.
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 BPV + IntegriSense680 (
After forepaw injection of IntegriSense680, the dye diffuses mainly through the lymphatic ducts, then reaches LNs where it accumulates momentarily before being eliminated (
2) Validation of BLI quantitation:
The minimal invasion status was determined thanks to in vivo calibration experiments by the quantification of known amounts of cancer cells suspended in a biological medium in the presence of luciferin. The bioluminescence signal was stable during at least 9 minutes following injection and attenuation of photons
by skin was about 77% (
3) Sentinel Lymph Node invasion’s model:
Thanks to BLI, it was possible to confirm the minimal invasion of tumor cells in both brachial and axillary LNs in vivo (
4) αVβ3 and αVβ5 integrins targeting:
Since locoregional injection of Integrin targeting probe IntegriSense680 results in a non-complete clearance in healthy LN, this route cannot be used to detect minimal invasion. So, only IV injections of the fluorescent probe were considered. In animals with SLNs containing almost 2000 cells, based on quantitative BLI, NIRF did not allow detection of any significant fluorescent signals in vivo. However, after excision of the SLN, ex vivo NIRF associated with spectral unmixing to suppress auto-fluorescence, enables confirmation of the minimal invasion of left axillary LN by cancer cells (
In clinical practice, blue dyes injected in the vicinity of primary tumors stain SLNs, so that clinicians can easily identify SLNs with the naked eyes during surgery [
detection of SLN location through the skin. Considering the technical and regulatory constraints associated with the use of radioisotopes, direct imaging of the SLNs via a fluorescent probe (i.e. ICG) should be a convenient and powerful alternative. To validate the use of infrared fluorescent probes at the same time as blue dyes, we assessed the potential quenching effect when either co-injected or injected alone. Our results demonstrate the absence of quenching by BPV towards fluorescence of ICG and integrin targeting agents. So, fluorescence imaging is compatible with standard BPV-based surgery procedures.
When considering the simultaneous use of the two fluorescence probes (ICGexcitation = 800 nm; ICGemission = 843 nm and IntegriSense680excitation = 675; IntegriSense680emission = 693 nm), optimized settings of excitation and emission filters allow for in vivo imaging of the SLN without reciprocal interferences. Due to the very lowIntegriSense680 fluorescence intensity present in micro-invaded SLNs as well as significant auto-fluorescence background, it is not possible to perform in vivo direct detection of IntegriSense680. So, ex vivo examinations on excised SLNs were performed using the spectral unmixing mode to suppress auto-fluorescence and contribution of ICG. In these conditions, the high quantum yield of fluorescence from IntegriSense680 is favorable for the level of detection requested and allows for evidencing of micro-invasion in SLNs as low as 1500 cells.
Fluorescent dyes with an emission of around 800 nm are usually more suitable for intraoperative imaging due to decreased auto-fluorescence of tissues and compatibility with lighting in the operating room [
BLI cannot be considered in humans but it is the most sensitive imaging modality to assess minimal tumor invasion (less than 1500 cells) for oncology research in animals. It was a valuable resource to acquire a relevant model with minimal invasion. NIRF of integrins targeted dyes in an invaded LN is possible in vivo or during surgery, but its sensitivity is not sufficient enough to demonstrate micro-invasion by a direct in vivo examination. When performed on an excised LN, ex vivo high sensitivity NIRF with spectral unmixing allows for detection of the micro-invasion.
The authors thank CNRS for financial support of this study.
None reported.
Moussa, B.S., Florian, R., Sobilo, J., Sharuja, N., Stéphanie, L. and Alain, L.P. (2018) Minimally Invaded Sentinel Lymph Node Model for the Development of Intraoperative Infrared Fluorescence Imaging. Advances in Molecular Imaging, 8, 48-58. https://doi.org/10.4236/ami.2018.84005