Murine Model Study Of A New Receptor-targeted Tracer For Sentinel Lymph Node In Breast Cancer

Purpose: Sentinel lymph node biopsy(SLNB)has replaced axillary lymph node dissection(ALND)as the standard staging procedure for early breast cancer patients with clinically negative axillary lymph nodes.To investigate indocyanine green(ICG)-Rituximab as a new receptor-targeted tracer for breast cancer sentinel lymph node(SLN)biopsy with a newly-built murine SLN model.Methods: After establishment of the murine SLN model,(1)different doses(containing 0.48 ?g,0.24 ?g,0.12 ?g,or 0.06 ?g of ICG respectively)of ICG-Rituximab were subcutaneously injected into the hindlimb instep of each mouse of each subgroup.After the injections,an MDM-I fluorescence vascular imager was used to continuously monitor the SLN in each mouse and to produce images at 5-min intervals over 3 h.The times required for SLN fluorescence to initiate and for obtaining the optimal images were recorded.The mice were sacrificed via cervical dislocation after 3 h,prior to the dissection of the popliteal space and lymph node drainage.Five minutes prior to the dissections,methylene blue was injected into the mice using the same procedure as described above.The stained SLNs,secondary lymph nodes,and tertiary lymph nodes were collected,and the MDM-I fluorescence vascular imager was then used to detect the presence or absence of fluorescent signals.(2)The ICG-Rituximab solution(containing 0.12 ?g of ICG)was subcutaneously injected into the hindlimb instep of each mouse.The mice were divided into three subgroups,and were sacrificed via cervical dislocation at 6 h,12 h,or 24 h post-injection.Subsequently,the popliteal space was dissected.Five minutes prior to dissection,methylene blue was injected into the mice using the same procedure as described above.The stained SLNs,secondary lymph nodes,and tertiary lymph nodes were collected,and the MDM-I fluorescence vascular imager was then used to detect the presence or absence of fluorescence.Results: The popliteal lymph node(LN)was defined as the SLN for hindlimb lymphatic drainage,with the iliac LN as the secondary and the para-aortic or renal LN as the thirdlevel LN(a few mice lacked a para-aortic lymph node,in such cases,the renal lymph node was adopted as the tertiary node).The initial imaging time and the optimal imaging time of the SLN were shortened with increased doses of ICG-Rituximab,and the imaging rates of the second-and third-level LNs increased accordingly.The optimal dose was 0.12?g(dose of ICG)and its optimal imaging time was 34 min.After 24 h,the SLN imaging rate maintained 100%,and the second-and the third-level LNs imaging rates increased from 0% and 0%(6h)to 10% and 10%(12h)and to 20% and 10%(24h),respectively.Conclusions: The low-cost murine SLN model established in this study is technically simple to set up and could clearly reveal the tracer's drainage in the lymphatic vessels and lymph nodes with developed lymphatic system.The optimal dose of ICG-Rituximab was 0.12?g(dose of ICG),and the optimal interval for SLN detection was from 34 min to 6h after injection without imaging of the second-or third-level LN.This new receptor-targeted tracer has a high value for clinical research and application.