Considering the current Breast Cancer Landscape and the Breast Cancer Research Program’s (BCRP’s) vision, relapse is one of the major problems to end breast cancer. The overall goal of the proposal is to develop a unique imaging agent with the ability to identify breast tumors and accurately distinguish tumors and surrounding normal tissues during surgical operation. With our proposed approach, it will potentially prevent breast cancer recurrence by precise surgical resection and improve the outcome of surgical treatment while saving overall cost by reducing rates of re-operation.

With the recent advanced detection procedures, many breast tumors can be found at early stages. For patients with early stages of the disease, surgical removal of local cancer followed by chemotherapy or radiotherapy is the most common breast cancer treatment. The precise surgical resection directly influences patient prognosis. However, it is often very difficult or even impossible for surgeons to distinguish breast tumor from normal tissue during tumor resection. Currently, surgeons during surgical operation only have direct visual perception and palpation along with imaging results taken before surgery as their guide. In fact, cancer cells are sometimes found at the edge of the removed tissue after surgery (positive margin), then additional surgery is needed as breast cancer cells left behind increase risk of cancer recurrence and metastasis.

Prior to any surgical procedure, tumor is usually assessed by the traditional imaging procedures such as mammography, ultrasound, MRI (magnetic resonance imaging), etc. Although these imaging technics are clearly useful to detect breast tumor and determine the extent of resection(s), they are not suitable to use as intraoperative imaging because handling of these imaging-instruments are typically too complicated or unsuitable to use during surgical operation or are incapable of detecting tumor margins with high contrast. Thus, development of new intraoperative imaging to clearly detect tumor margins is needed to completely remove tumor at the initial surgical operation.

Recently, optical imaging is gaining traction in the image-guided surgery field especially when coupled with near-infrared (NIR) fluorescence agent. The advantages of using NIR fluorescent image-guided surgery are real-time detection with high-resolution image and flexible instrument without ionizing radiation. Furthermore, NIR imaging visualizes disease through skin before resection and does not change the appearance of the surgical field of view. Several NIR fluorescence agents have been preclinically developed, among them indocyanin green (ICG), which is a non-toxic NIR fluorescence agent and currently registered by the Food and Drug Administration for clinical applications. Although ICG is useful as cardiac output or hepatic function marker, it is a non-specific contrast agent limiting in discerning tumor from normal tissue. In order to use ICG as an intraoperative tumor marker, development of a tumor-targeting carrier is essential.

Many attempts have been made in the development of carrier molecules; in particular, cell-penetrating peptides have shown to be a promising strategy for improving the delivery and intracellular uptake of diagnostic and therapeutic agents. We have identified that a 28 amino acids peptide fragment, p28, preferentially enters and is retained significantly longer in breast cancer cells. In addition to cancer-targeting property, p28 also has anticancer activity. Safety and toxicity of p28 as a single therapeutic agent, but not imaging carrier/agent, has been tested in two Phase I clinical trials in the US. Preclinical and clinical studies showed that p28 preferentially penetrates cancer cells without toxicity and immunogenicity in animals and humans.

Based on the above facts, we hypothesize that developing p28 as a nontoxic tumor-targeting carrier for ICG will provide an ideal intraoperative imaging agent to clearly distinguish tumor from surrounding normal tissue. Preliminary data with this new imaging molecule (p28 chemically conjugated to ICG, ICG-p28) support the hypothesis. Here, we will take a strategic approach to test our hypothesis in clinically relevant setting in multiple breast cancer animal models. We will initially focus on relatively simpler breast cancer animal models including patient-derived xenografts to validate the preliminary data with an NIR imaging system, which has been used in clinic. Then, we will test out hypothesis on transgenic mouse model that histologically resembling human breast cancer. We will perform animal surgery in clinically relevant setting with ICG-p28 to test whether precise resection of primary tumor using ICG-p28 prevent cancer recurrence. These preclinical tests as well as toxicity/biodistribution of the agent proposed in the application are essential for moving to early phase clinical trial for an image-guided surgery. It potentially provides breakthrough based on unique approach to prevent recurrence and re-operation while reducing overall treatment cost that will be a major milestone and relevant to the BCRP focus area.