Real-time In Vivo Molecular Detection of Primary Tumors and Metastases with Ratiometric Activatable Cell-Penetrating Peptides

Abstract

Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathologic examination. We report a family of novel ratiometric activatable cell-penetrating peptides, which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 reemission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 and 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40-fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information about cancer invasion.

Ratiometric Activatable Cell‐Penetrating Peptides Provide Rapid In Vivo Readout of Thrombin Activation

“…This study reports data from mice that have been injected with either RACPP PPRSFL (with or without hirudin), RACPP peg6 (15), or RACPP PLGC(Me)AG (20). RACPP PLGC(Me)AG is closely related to RACPP-2 described by Savariar et al [29] for imaging MMP-2/-9 activities in tumors and metastases, but RACPP PLGC(Me)AG adds a solubilizing PEG12 chain attached by way of a d-Cys following the polyglutamate sequence. For thrombin inhibition studies, mice were injected subcutaneously with hirudin (2000 U/mouse; n = 3) 20 min prior to probe injection.…”

“…[29] Peptides were purified using preparative HPLC and characterized using analytical HPLC, combined with mass spectrometry. Details of the synthesis and characterizations are shown in the Supporting Information.…”

Label-free Detection of Lymph Node Metastases with US-guided Functional Photoacoustic Imaging

“…Although PET has excellent sensitivity, it requires the use of ionizing radiation and has relatively poor resolution. Recently, optical imaging has been proposed as a tool with which to not only map the SLN but also detect molecular signatures of metastases (10,11). Optical imaging, however, has limited sensitivity at depth, and its resolution quickly degrades at depths of more than 1 mm (12).…”

Fluorescence and Glioma Surgery

“…ACPPs can also aid in evaluating the presence of cancer in lymph nodes during surgery, which is vital to the progression of the procedure. Usually, in order to analyze the presence of cancer in lymph nodes, all lymph nodes in the anatomical vicinity of the primary tumor are surgically removed and pathologically examined in a process called sentinel lymph node (SLN) identification [16]. This strenuous, lengthy procedure is inefficient in providing feedback during surgery.…”

“…Therefore, the fluorescence emitted from dequenching, which allows tumors to fluoresce significantly early during the procedure (one to two hours after injection), in conjunction with the accumulation of Cy-5 labeled polycationic domains within tumor cells provides a more prolonged fluorescence with a higher tumor-background contrast compared to nonratiometric ACPPs, which take six and 24 hours for optimal use [16]. In a study conducted comparing the ability of non-ratiometric ACPPDs to RACPPs to detect metastases, RACPP analysis provided higher contrast between tumor tissue and healthy tissue compared to ACPPD contrast, despite the fact that significantly different intensities between tumor and healthy tissue was observed for both groups (p=.02 compared to p=.0007) [16]. It is apparent that one can manipulate ACPPs in numerous ways by linking different types of cargo to ACPP domains to maximize the efficacy of resection surgeries.…”