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.

A hydrogen peroxide (H2O2)-activated cell-penetrating peptide was developed through incorporation of a boronic acid-containing cleavable linker between polycationic cell-penetrating peptide and polyanionic fragments. Fluorescence labeling of the two ends of the molecule enabled monitoring its reaction with H2O2 through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H2O2 sensor selectively reacts with endogenous H2O2 in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H2O2 has potential applications in imaging and therapy of diseases related to oxidative stress.

Background The aim of this study was to evaluate the efficacy of using matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores to accurately label pancreatic cancer for fluorescence-guided surgery (FGS) in an orthotopic mouse model. Methods Orthotopic mouse models were established using MiaPaCa-2-GFP human pancreatic cancer cells. Two weeks after implantation, tumor-bearing mice were randomized to conventional white light reflectance (WLR) surgery or FGS. FGS was performed at far-red and infrared wavelengths with a customized fluorescence-dissecting microscope 2 h after injection of MMP-2 and MMP-9-cleavable RACPPs. Green fluorescence imaging of the GFP-labeled cancer cells was used to assess the effectiveness of surgical resection and monitor recurrence. At 8 weeks, mice were sacrificed to evaluate tumor burden and metastases. Results Mice in the WLR group had larger primary tumors than mice in the FGS group at termination [1.72 g ± standard error (SE) 0.58 vs. 0.25 g ± SE 0.14; respectively, p = 0.026). Mean disease-free survival was significantly lengthened from 5.33 weeks in the WLR group to 7.38 weeks in the FGS group (p = 0.02). Recurrence rates were lower in the FGS group than in the WLR group (38 vs. 73 %; p = 0.049). This translated into lower local and distant recurrence rates for FGS compared to WLR (31 vs. 67 for local recurrence, respectively, and 25 vs. 60 % for distant recurrence, respectively). Metastatic tumor burden was significantly greater in the WLR group than in the FGS group (96.92 mm2 ± SE 52.03 vs. 2.20 mm2 ± SE 1.43; respectively, χ2 = 5.455; p = 0.02). Conclusions RACPPs can accurately and effectively label pancreatic cancer for effective FGS, resulting in better postresection outcomes than for WLR surgery.

We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

Extracellular proteases including matrix metalloproteinases (MMPs) are speculated to play a significant role in chronic lung diseases, such as asthma. Although increased protease expression has been correlated with lung pathogenesis, the relationship between localized enzyme activity and disease progression remains poorly understood. We report the application of MMP-2/9 activatable cell-penetrating peptides (ACPPs) and their ratiometric analogs (RACPPs) for in vivo measurement of protease activity and distribution in the lungs of mice that were challenged with the allergen ovalbumin. MMP-2/9 activity was increased greater than twofold in whole, dissected lungs from acutely challenged mice compared with control mice (P=1.8×10(-4)). This upregulation of MMP-2/9 activity was localized around inflamed airways with 1.6-fold higher protease-dependent ACPP uptake surrounding diseased airways compared with adjacent, pathologically normal lung parenchyma (P=0.03). MMP-2/9 activity detected by ACPP cleavage colocalized with gelatinase activity measured with in situ dye-quenched gelatin. For comparison, neutrophil elastase activity and thrombin activity, detected with elastase- and thrombin-cleavable RACPPs, respectively, were not significantly elevated in acutely allergen-challenged mouse lungs. The results demonstrate that ACPPs, like the MMP-2/9-activated and related ACPPs, allow for real-time detection of protease activity in a murine asthma model, which should improve our understanding of protease activation in asthma disease progression and help elucidate new therapy targets or act as a mechanism for therapeutic drug delivery.

Surgical resection with complete removal of tumor remains one of the most effective modalities in the treatment of cancer. The aim of this study was to evaluate the efficacy of using activatable cell penetrating peptides (ACPPs) conjugated to Cy5 fluorophores to accurately label primary pancreatic tumor for fluorescence-guided surgery (FGS) in orthotopic mouse models. Orthotopic mouse models of human pancreatic cancer using surgical implantation of tumor fragments of the MiaPaca-2-GFP pancreatic cancer cell line were established. Two weeks after tumor implantation, the tumors were resected with bright light surgery (BLS) or FGS. FGS was performed with a florescence-dissecting microscope (MVX10, Olympus) 2 hours after periorbital injection of ACPPs. Completeness of resection was assessed from postoperative images obtained with the Maestro CRI Small Imaging System. Postoperatively, we performed whole body imaging of the mice to assess for recurrence. At eight weeks, mice were sacrificed to evaluate pancreatic tumor burden, as well as local and distant recurrence. Cy-5-conjugated ACPPs, cleavable by MMP-2 and MMP-9 permitted clear visualization of tumor margins for fluorescence-guided resection. With FGS, no residual tumor was detected in the postoperative surgical site in all 16 mice. One mouse out of 16 undergoing BLS had evidence of residual tumor postoperatively. At 8 weeks, mice in the BLS group demonstrated larger primary tumors compared to mice in the FGS group (1.72 g ± SE 0.58 vs 0.25 g ± SE 0.14, p=0.026). Mean disease-free survival (DFS) was significantly lengthened from 5.33 weeks in the BLS group to 7.38 weeks in the FGS group (p=0.02). Furthermore, recurrence rates were lower in the FGS group (38% vs 73%, p=0.049). This translated into a lower local and distant recurrence rate for FGS (31% vs 67% and 25% vs 60%, respectively). ACPPs can provide an alternative method to accurately and effectively label cancer cells for improved resection of tumors that do not express specific tumor antigens and result in better outcomes post-resection. Citation Format: Cristina A. Metildi, Sharmeela Kaushal, Csilla N. Felsen, Quyen T. Nguyen, Robert M. Hoffman, Roger Y. Tsien, Michael Bouvet. Fluorescence-guided surgery of pancreatic cancer using activatable cell penetrating peptides (ACPPs) in orthotopic mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4311. doi:10.1158/1538-7445.AM2014-4311

<p>PDF file - 40K</p>

<p>PDF file - 835K, Supplementary Fig 1. Structures of RACPP1,2,3 and uncleavable control. Supplementary Fig 2. Structures of synthesized RACPPs with varying acceptor fluorophores. Supplementary Fig 3. Characterization of RACPP2. Supplementary Fig 4. Ratiometric RACPP images show superior contrast compared to Cy5 fluorescence alone. Supplementary Fig 5. RACPPs show specificity for MMP2,9 cleavage in transgenic animal models. Supplementary Fig 6. RACPPs show tumor specific cleavage and uptake. Supplementary Fig 7. Non-ratiometric ACPP has poor contrast in liver metastases. Supplementary Fig 8. Ratiometric analyses using RACPPs show sensitive detection of metastatic lymph nodes. Supplementary Fig 9. LCMS characterization of RACPPs. Supplementary Fig 10. Synthetic schemes for RACPPs.</p>