Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival

Abstract

The completeness of tumor removal during surgery is dependent on the surgeon's ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative wholebody tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.intraoperative fluorescence imaging | molecular navigation | long-term survival | molecular imaging | surgical margin

Administration de molécules actives dans les cellules

“…Ce complexe ne pénètre pas efficacement dans les cellules non tumorales, mais le clivage du linker par des protéases spécifiques de la tumeur libère le CPP-Cy5 qui pénètre dans les cellules tumorales, marquant ainsi très efficacement la tumeur in vivo. L'équipe a ainsi pu montrer que quand la chirurgie est effectuée en présence de ce complexe de pénétration cellulaire, il y a dix fois moins de cellules tumorales résiduelles que lors d'une chirurgie classique [43]. Tsien a obtenu le prix Nobel de chimie en 2008 pour l'ensemble de ses travaux sur la conception de molécules fluorescentes et photolabiles [57].…”

Fluorescence and Glioma Surgery

“…In a preclinical study on the efficacy of resection surgery guided by ACPPDs linked to Cy5, a fluorescent dye, and gadolinium, the authors found that this procedure on mice suffering from melanoma yielded a better prognosis (100% higher eight month progression-free survival and 50% higher overall survival) than mice subjected to white light surgery [15]. The drastic improvement in prognosis is due to a 10-fold increase in ACPPDs in subjects with complete resection compared to the group that underwent white light surgery and the procedure's 93% specificity rate [15]. ACPPs can also aid in evaluating the presence of cancer in lymph nodes during surgery, which is vital to the progression of the procedure.…”

“…In order for ACPPs to enter their active state and translocate into tumor cells, the bond between the polyanionic peptide and the polycationic peptide, which is coupled to the cargo, must undergo proteolytic cleavage by matrix metalloproteinases (MMP). MMP2 and MMP9, which are proteases overexpressed in several types of cancer, are integral for tumorigenesis due to their ability to degrade extracellular matrices, induce angiogenesis, and promote growth factors [15]. The degree of MMP2 and MMP9 expression is contingent on the magnitude of tumor malignancy (the higher the grade of tumor, the higher expression of these proteins), and the enzymes have highly specific binding sites for substrates, thus making them robust targets for identifying cancer [15].…”

“…ACPPs consist of a region containing a polycationic cell-penetrating peptide linked to a polyanionic peptide, which neutralizes the positive charge, by a cleavable bond [15]. In order for ACPPs to enter their active state and translocate into tumor cells, the bond between the polyanionic peptide and the polycationic peptide, which is coupled to the cargo, must undergo proteolytic cleavage by matrix metalloproteinases (MMP).…”

“…MMP2 and MMP9, which are proteases overexpressed in several types of cancer, are integral for tumorigenesis due to their ability to degrade extracellular matrices, induce angiogenesis, and promote growth factors [15]. The degree of MMP2 and MMP9 expression is contingent on the magnitude of tumor malignancy (the higher the grade of tumor, the higher expression of these proteins), and the enzymes have highly specific binding sites for substrates, thus making them robust targets for identifying cancer [15]. ACCPs can be conjugated to dendrimers (ACPPDs), which increases the degree of tumor fluorescence and contrast between the background and tumor, thus creating a superior delineation of tumor margins compared to ACPPs.…”