Additionally, our fluorescent targeted agents could be used intraoperatively for image guided surgery to remove involved nodes and leave uninvolved nodes (48). pad (MFP) tumors; and by peritumoral MFP injection of the targeted imaging probe in mice with spontaneous ALN metastases. Fluorescence imaging showed that probe was only retained in positive tumors and metastases. As few as 1000 cells that endogenously express Mobp mammaglobin-A were detected in ALN indicating high sensitivity of this method. Hence, this approach has potential for translation into clinical use for the non-invasive staging of breast cancer. Keywords:breast cancer, imaging, lymph node metastasis, mammaglobin-A, targeted agent == Introduction == In breast cancer, the presence of disease in axillary lymph nodes is an important prognostic factor and drives treatment decision Cyclovirobuxin D (Bebuxine) making (1,2). Currently, most patients with breast cancer undergo axillary sentinel node biopsy for initial staging (3). Sentinel lymph nodes (SLNs) are the nodes that first receive lymph from the area of the breast harboring the tumor and are the nodes most likely to contain metastatic cells. If the SLNs are determined to be free of disease, it is accepted that all other axillary lymph nodes (ALNs) will be negative and axillary dissection can be avoided. SLN are currently identified with Tc-99m-labeled colloids (4) and/or isosulfan blue dye which is injected peri-tumorally and transiently accumulates in SLN (5,6). Then, the sentinel nodes are removed and examined by standard pathological methods for the presence of metastasis. SLN biopsy (SLNB) can be associated with complications including seroma formation, lymphedema and sensory nerve injury (7). Furthermore, SLNB is resource intensive requiring a team with specialized training, and specialized imaging and surgical equipment (8-10). Since there is no therapeutic value to removing uninvolved SLNs (11) development of effective, non-invasive strategies for excluding the presence of metastatic breast cancer in ALNs would represent a major advance and axillary surgery could be avoided in most early stage breast cancer patients. Recently, several groups have investigated non-invasive imaging modalities for SLN evaluation, including single photon emission computed tomography (SPECT) (12,13), multiphoton microscopy (14), magnetic resonance imaging (MRI)(15), optical lymphography with indocyanine green dye or multicolor quantum dots (16-19), photoacoustic tomography (PAT) using nanoparticle-based contrast agents such as carbon nanotubes (20), gold carbon nanotubes (21), gold nanocages (22), gold nanorods (23), methylene blue dye (24) and gold nanobeacons (25). However, these approaches were not targeted and thus were not able to assess SLN status. Untargeted probes distribute randomly across the SLNs with only transient and non-specific visualization of the lymphatic system. Hence, these methods only provide anatomic maps, and do not detect tumor cells present in lymph nodes. Development of imaging agents that selectively target cancer cells with no cross-reactivity to non-tumor cells is a long-term goal for cancer imaging and could allow for the non-invasive staging of breast cancer by detection of tumor cells in ALNs. However, to date, there has been no definitive study for the development of a targeted imaging method for detection of tumor cells specifically in ALNs. Breast cells are typically only present in the ALNs when breast cancer has spread from Cyclovirobuxin D (Bebuxine) the primary tumor. Therefore, cell-surface marker(s) that discriminate breast epithelial cells from lymphoid cells can be used to detect metastasis (26-28). A comprehensive gene expression analysis of breast epithelial cells versus lymphoid cells determined that mRNA encoding mammaglobin-A protein can be used to distinguish epithelial cells from lymphoid cells (29). The mammaglobin-A gene (SCGB2A2) encodes a 10-kDa glycoprotein and is a member of the epithelial secretoglobin family 2A (30,31). Several studies have demonstrated that mammaglobin-A is exclusively expressed in breast tissue and can be used as a marker for the detection of micrometastasis in SLN [for review see (32)]. Mammaglobin-A has also been investigated as a molecular marker for targeted therapy of breast cancer (33,34). In the present study, we applied a high-resolutionin vivofluorescence imaging technique to non-invasively detect lymphatic metastasis of human breast cancer cells in a mouse model. For this Cyclovirobuxin D (Bebuxine) purpose, a monoclonal antibody specific for binding to mammaglobin-A was conjugated to a near-infrared (NIR) fluorescent dye, (termed MamAb-680), and delivered to the lymphatic system by peritumoral injection into the mammary fat pad of nude mice, allowing imaging of mammaglobin-A expressing cells that have spread to the ALN. Thus, we have combined the specificity of a mammaglobin-A specific antibody, which.