Novel approach for the detection of intraperitoneal micrometastasis using an ovarian cancer mouse model

Abstract

Patients with epithelial ovarian cancer have the best overall survival when maximal surgical effort is accomplished. However, despite numerous technological advances, surgery still relies primarily on white-light reflectance and the surgeon's vision. As such, micrometastases are usually missed and most patients clinically classified as a complete responder eventually recur and succumb to the disease. Our objective is to develop optical enhancers which can aid in the visualization of ovarian cancer micrometastasis. To this end we developed a nanoparticle (NP) platform, which is specifically targeted to the tumor microenvironment. Targeting is achieved by coating FDA-approved PLGA-PEG NP with the peptide sequence RGD, which binds with high affinity to αVβ3 integrins present in both the tumor-associated neovasculature and on the surface of ovarian cancer cells. Administration of the NP platform carrying fluorescent dyes to mice bearing intraperitoneal ovarian cancer allowed visualization of tumor-associated vasculature and its contrast against normal blood vessels. More importantly, we demonstrate the visualization of intraperitoneal ovarian cancer micrometastasis as small as 100 μm with optimal resolution. Finally, we demonstrate that the fluorescent dye cargo was able to penetrate intra-tumorally. Such modality could be used to allow microscopic surgical debulking to assure maximal surgical effort.

Figure 1. Fluorescent probes encapsulated RGD Nanoparticles.

(A) Synthesis of RGD-NP composed of PLGA-PEG and its self-assembled NP. (B) NPs particle size was determined by DLC (upper panel) and TEM (down panel). (C) Surface charges of NP with and without RGD were compared. (D) Encapsulation amount of each dye within NP was also determined. (E) The release kinetic of each dye from NP was measured.

Figure 2. Staining and delineation of tumors and tumor-associated vasculature by DIR-RGD-NP.

Upon establishment of tumors (ROI~40,000), mice were given four doses of DIR-RGD-NP (‘lower dose” indicated in Table 1) given every other day (n = 12). (A–D) Gross tumors appear distinctly stained compared to the intestines. Staining is specific to tumor-associated vasculature (white arrow), which can be easily contrasted to the normal vasculature (yellow arrow); (E,F) Tumors under the diaphragm are likewise stained; (F) Micrometastasis (blue arrow) is visible due to DIR-stained vessels (white arrow) and is contrasted against normal vessels (yellow arrow).

Figure 3. Enhanced retention and better colocalization in vivo with DIR-RGD-NP.

Upon establishment of tumors (ROI~40,000), mice were given four doses of soluble DIR, DIR-NP, or DIR-RGD-NP given every other day. mCherry (red) and DIR (green) fluorescent images were obtained in live animals at designated time points 24 h after the 4^th dose. Images shown are merged images demonstrating the best colocalization of mCherry and DIR signals (yellow) in animals that received DIR-RGD-NP. A representative animal for each delivery system is shown; (n = 4 for soluble DIR and DIR-NP; n = 12 for DIR-RGD-NP).

Figure 4. Staining of microtumors with DIR-RGD-NP.

Fluorescence spectral images from dissected intestines and the attached mesentery. Images shown are from mCherry channel (red; left column) and DIR channel (green; middle column). The merged images (right column) demonstrate the best colocalization of mCherry and DIR signals (white arrow) in animals that received DIR-RGD-NP. A representative animal for each delivery system is shown; (n = 4 for soluble DIR and DIR-NP; n = 12 for DIR-RGD-NP).

Figure 5. Delineation of tumor-associated vasculature with DIR-RGD-NP.

(A) Delineation of tumor vascularity. White light image of tumors showing the outline of tumor-associated vasculature only when dye was administered in RGD-coated nanoparticles; (B) Ex vivo imaging and colocalization of mCherry and DIR signals in dissected tumors (from top: 2 mm, 5 mm, and 10 mm in size); (C) Quantification of DIR MFI, data shows mean ± SEM, *p < 0.002 compared to soluble DIR and p < 0.0237 compared to DIR-NP; (D) Quantification of DIR intensity and penetration in cross-sectioned tumors; a representative animal for each delivery system is shown in A, B, and D; (n = 4 for soluble DIR and DIR-NP; n = 12 for DIR-RGD-NP).

Figure 6. Identification of micrometastasis by white light.

Stereoscopic images comparing specificity and sensitivity of the described nanoparticle platform (full data in Table 2). Squared area and white arrows point to micrometastasis. M, mesentery; I, intestines; a representative animal for each delivery system is shown; (n = 4 for soluble DIR and DIR-NP; n = 12 for DIR-RGD-NP).

Figure 7. Specificity of detection is maintained with one time dosing of DIR-RGD-NP.

(A) Upon establishment of tumors (ROI~40,000), mice were given a single dose of DIR-RGD-NP (“higher dose” indicated in Table 1; n = 4). Stereoscopic images show detection of micrometastasis by white light. White arrows point to micrometastasis. M, mesentery; I, instestines. (B) i, Ex vivo imaging and colocalization of mCherry and DIR signals in dissected tumors; ii, quantification of DIR intensity and penetration in cross-sectioned tumors.

Figure 8. Biodistribution of C6-RGD-NP in the tumor microenvironment.

Upon establishment of tumors (ROI~40,000), mice were given a single dose Cu6-RGD-NP and sacrificed after 2 h (n = 4). Fluorescence imaging demonstrates the spatial relationship between the DIR and mCherry signals; scale bar = 180 μm.

Figure 9. Visualization of micrometastasis using the ICG probe and the PINPOINT system.

(A) Images from the PINPOINT system demonstrating the visualization of tumor-associated vasculature and micrometastasis (arrows); (B) quantification of ICG intensity showing most intense staining in tumors less than 5 mm compared to the rest of the abdomen, data shows mean ± SEM,*p < 0.0001 compared to abdomen, heart, and liver (n = 4).