Harnessing competing endocytic pathways for overcoming the tumor-blood barrier: magnetic resonance imaging and near-infrared imaging of bifunctional contrast media

Abstract

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)-based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells.

Figure 1. Specific binding and endocytosis of Daidzein-BSA-FAM by MLS ovarian carcinoma cells

Two-photon fluorescence microscopy of MLS human ovarian carcinoma cells incubated (1 h in 37°C) in presence of Daidzein-BSA-FAM (200 μg/ml) or BSA-FAM (200 μg/ml), in the presence or absence of a blocking dose of nystatin (50 μg/ml). Blue, DAPI nuclear staining; green, Daidzein-BSA-FAM or BSA-FAM.

Figure 2. Specific binding and endocytosis of BSA-ROX and Daidzein-BSA-FAM by MLS ovarian carcinoma cells

Two-photon fluorescence microscopy of MLS human ovarian carcinoma incubated for 30 min at 37°C in the presence of (200 μg/ml) Daidzein-BSA-FAM and BSA-ROX (200 μg/ml) with or without inhibition of caveolae mediated uptake using nystatin (50 μg/ml). Green, Daidzein-BSA-FAM; red, BSA-ROX; blue, DAPI nuclear staining.

Figure 3. Binding and endocytosis of BSA-ROX and Daidzein-BSA-FAM by MLS ovarian carcinoma cells

Flow cytometry of MLS human ovarian carcinoma incubated for 30 min at 37°C in the presence of (200 μg/ml) Daidzein-BSA-FAM or/and BSA-ROX (200 μg/ml) with or without inhibition of caveolae mediated uptake using nystatin (50 μg/ml) (green-Daidzein-BSA-FAM, red-BSA-ROX). A) Histogram of a) Daidzein-BSA-FAM uptake (grey-control, blue-Daidzein-BSA-FAM uptake, green- Daidzein-BSA-FAM uptake in presence of nystatin), b) BSA-ROX uptake (grey-control, blue- BSA-ROX uptake, red- BSA-FAM uptake in presence of nystatin). B) Statistics median of uptake histogram analysis. C) Competition of Daidzein-BSA-FAM (green) by BSA-ROX (red) in the presence and absence of nystatin (FL1-FAM fluorescence, FL2- ROX fluorescence). D) Statistical analysis of competition assay. Change on percent of positively stained fluorescent cells in absence and presence of nystatin (green-Daidzein-BSA-FAM, red-BSA-ROX).

Figure 4. In Vivo NIR Imaging of targeted delivery of daidzein-BSA-CyTE-777 to ovarian carcinoma tumors

A) CD-1 nude mice were inoculated subcutaneously with 2.5*10⁶ cells. When the tumor was visible the mice were administered intravenously with 1) BSA-CyTE-777; 2) daidzein-BSA-CyTE-777. The NIR signal 48 hours after injection is shown. B) Elimination of the NIR signal, tumor to background ratio over time. p=0.047

Figure 5. Ex-vivo characterization of contrast material distribution inside the tumor

MLS bearing mice were injected with BSA-FAM or Daidzein-BSA-FAM or combination of Daidzein-BSA-FAM and BSA-ROX. Tumors were isolated 24h after injection, fixed in Carnoy and imbedded in paraffin blocks. A) H&E staining of the tumor. Histological sections were stained with DAPI and visualized by fluorescence microscopy. B) Section of a tumor from a mouse injected with BSA-FAM or with Daidzein-BSA-FAM (green, FAM; blue, DAPI). C) Tumor sections derived from a mouse injected with daidzein-BSA-FAM and BSA-ROX (green, FAM; red, ROX; blue, DAPI).

Figure 6. Daidzein-BSA-GdDTPA as a contrast material to MRI

A) The specific R₁ relaxivity of daidzein-BSA-GdDTPA was measured to be 194 mM⁻¹s⁻¹ per BSA. B), C) Specific localization and retention of the contrast material inside the tumor: MLS bearing mice were administered intravenously with 12mg BSA-GdDTPA (left) or daidzein-BSA-GdDTPA (right). T1 weighted MRI images and R₁ maps were obtained 24, 48 and 72h after injection, and used for derivation of mean R₁ values for the tumor. ROI analysis of MLS tumors of mice injected with BSA-GdDTPA (left) or daidzein-BSA-GdDTPA (right) and control (preinjection). * Significant differences in comparison of treatments (Student ttest p<0.05). D) Specific localization and retention of the contrast material inside the tumor: MLS tumor bearing mice were administered intravenously with 4.5mg daidzein-BSA-GdDTPA. R₁ maps were obtained 24h and 7d after injection.