Abstract A61: Detection of ovarian cancer micrometastasis using nanoparticle-delivered probe targeted towards tumor-associated neovasculature.
Authors: Alvero Ayesha , Lima Eydis , Kim Dongin , Orton Sean , Pitruzzello Mary , Yang-Hartwich Yang , Silasi Dan-Arin 
Published: May 2016

Background: Ovarian cancer causes more death than all other gynecologic malignancies combined. In patients, the best predictor of overall survival is the amount of residual disease following surgical debulking. Indeed, studies have shown that patients have the best prognosis when maximal surgical effort is accomplished. However, despite numerous technological advances, surgery still primarily relies on white-light reflectance and the surgeon's vision. As such, micrometastasis are usually missed and most patients clinically classified as a complete responder carry residual tumor burden, eventually recur, and succumb to the disease. We hypothesize that we can utilize the specific phenotype of tumor-associated neovasculature to target optical enhancers to locate and delineate micrometastasis. Specific targeting is achieved by encapsulating deep infrared dye (DIR) in FDA-approved PLGA-based nanoparticle (NP) coated with the peptide sequence arginine-glycine-aspartate (RGD). RGD binds with high affinity to αVβ3 integrins, which are minimally expressed in quiescent blood vessels and normal cells but overexpressed in tumor-associated neovasculature and cancer cells of various origins, including ovarian cancer. Thus our objective is to develop specific tumor-targeting optical enhancers that can aid surgeons in the performance of microscopic tumor debulking with the goal of minimizing residual disease.

Materials and methods: Intra- peritoneal (i.p.) tumors are established in nude mice using ovarian cancer cells that stably express mCherry fluorescent protein, which allows the colocalization of DIR signal and mCherry+ micrometastatic lesions. Using this model we compared the ability of soluble DIR, DIR encapsulated in naked NP (DIR-NP), and DIR encapsulated in RGD-coated NP (DIR-RGD-NP) to detect ovarian cancer micrometastasis. Formulations were delivered i.p. and fluorescence signal were measured ex vivo following necropsy. More importantly, the ability of the probe to locate and delineate micrometastatic lesions was determined using fluorescent dissection microscope.

Results: Colocalization analysis of DIR and mCherry fluorescent images obtained at necropsy demonstrated the sensitivity of DIR-RGD-NP. In these animals, we observed 75% colocalization of DIR and mCherry signals compared to 26% in animals administered DIR-NP and 0% in those given soluble DIR. Ex vivo analysis of mean DIR intensity in tumors less than 2 mm showed mean fluorescent intensity (MFI) of 1209 in animals administered DIR-RGD-NP versus 155 MFI in animals administered DIR-NP. Morphological examination under the dissection microscope show that in animals administered DIR-RGD-NP, we can locate 81% of the mCherry+ micrometastasis based on DIR staining. In these animals, tumors less than 1 mm were detectable due to a halo of DIR staining around each micrometastatic lesion. In these foci, DIR signal was observed to stain the vasculature surrounding the small tumor implants especially those in the mesentery and diaphragm. In contrast, we were able to detect only 18% of the mCherry+ micrometastasis in animals administered DIR-NP.

Conclusion: We demonstrate that we can visualize micrometastasis by using RGD as a tool to target the unique phenotype of ovarian cancer-associated neovasculature. RGD-coated nanoparticles are able to carry probes to the tumor microenvironment leading to optimal staining of micrometastasis. Our results highlight the use of this nanotechnology platform in microscopic surgical debulking to assure maximal surgical effort, minimize residual disease, and improve patient survival. Furthermore, delineation of tumor borders will also reduce inadvertent injury to vital structures and decrease surgery-associated morbidities.

Citation Format: Ayesha Alvero, Eydis Lima, Dongin Kim, Sean Orton, Mary Pitruzzello, Yang Yang-Hartwich, Dan-Arin Silasi. Detection of ovarian cancer micrometastasis using nanoparticle-delivered probe targeted towards tumor-associated neovasculature. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A61.