Ratiometric Activatable Cell-Penetrating Peptides Label Pancreatic Cancer, Enabling Fluorescence-Guided Surgery, Which Reduces Metastases and Recurrence in Orthotopic Mouse Models

Abstract

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.

Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell

“…Similar designs have subsequently been published utilizing matrix metalloproteases, caspases or other enzymes [75]. Activatable PTDs/CPPs coupled to fluorescent probes have potential to assist as molecular navigation tools during surgery [76,77]. Furthermore, when combined to classical targeting peptides, such as RGD or GalNac, they can become a very useful addition to enhance specific uptake at precise target sites.…”

Early detection of squamous cell carcinoma in carcinogen induced oral cancer rodent model by ratiometric activatable cell penetrating peptides

“…Diagnosis of HNSCC is often made at an advanced stage, partly due to the lack of reliable non-invasive methods to accurately identify malignant lesions [26–30]. Our lab has established the use of MMP sensitive RACPPs for image-guided surgery of tumors in various animal xenograft and transgenic models of breast cancer [20, 23], pancreatic cancer [21], melanoma and HNSCC [22]. In this study, we have successfully generated oral cavity SCC in an immunocompetent rodent model as previously described [10] and used established MMP responsive ratiometric fluorescence imaging, for comparative detection of cancerous lesions using white light inspection.…”

“…We have previously reported the use of MMP activatable cell penetrating peptides (ACPPs) probes for image guided surgery of tumors in various animal models of breast cancer [20], pancreatic cancer[21], melanoma and head and neck xenografts[22, 23]. We have also demonstrated significant correlation of ratiometric fluorescence (Cy5:Cy7) from the MMP cleavable RACPP with tumor burden in a pancreatic tumor mouse model [21]. Concentration of other MMPs including, MMP 1 and 3 in human saliva and oral cancer are strong diagnostic markers oforal SCCA [8].…”

Current status and future perspectives of fluorescence-guided surgery for cancer

“…Nguyen et al reported significantly improved outcomes when ACPP guidance was compared to bright light surgery in mouse models of human fibrosarcoma and melanoma [48]. Metildi et al demonstrated that MMP-2 and MMP-9-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores could effectively label pancreatic cancer in an orthotopic mouse model, and that FGS using this method reduced metastasis and recurrence [49]. …”