Labeling fibroblasts with biotin-BSA-GdDTPA-FAM for tracking of tumor-associated stroma by fluorescence and MR imaging

Abstract

Fibroblasts at the tumor-host interface can differentiate into myofibroblasts and pericytes, and contribute to the guidance and stabilization of endothelial sprouts. After intravenous administration of biotin-BSA-GdDTPA-FAM in mice with subcutaneous MLS human ovarian carcinoma tumors, the distribution of the macromolecular MRI/optical contrast material was confined to blood vessels in normal tissues, while it co-registered with alphaSMA-positive stroma tracks within the tumor. These alphaSMA-positive tumor-associated myofibroblasts and pericytes showed uptake of the contrast material into intracellular granules. We evaluated the use of this contrast material for in vitro labeling of tumor fibroblasts as an approach for tracking their involvement in angiogenesis. Fluorescence microscopy demonstrated internalization of the contrast material, and MRI revealed a significant increase in the R(1) relaxation rate of labeled fibroblasts. R(1) not only remained elevated for 2 weeks in culture, it also increased with cell proliferation, indicating prolonged retention of the contrast material and subsequent intracellular processing and redistribution of the material, and thereby enhancing MR contrast. Moreover, cells that were labeled ex vivo with MR contrast material and co-inoculated with tumor cells in mice were detected in vivo by MRI. Uptake of the contrast material was suppressed by nystatin, suggesting internalization by caveolae-mediated endocytosis. This study shows that labeling of fibroblasts with biotin-BSA-GdDTPA-FAM is feasible and would allow noninvasive in vivo tracking of fibroblasts during tumor angiogenesis and vessel maturation.

Figure 1. Distribution of the contrast material in MLS tumors

Fluorescence microscopy of representative tumors initiated by co-inoculation of MLS cells and PF42T fibroblasts. Biotin-BSA-GdDTPA was administered intravenously into the tail vein of a tumor bearing mice (16 days after implantation; 30 minutes prior to tumor retrieval). The distribution of biotin-BSA-GdDTPA was detected by staining with Avidin-FITC. Tumor borders are illustrated by arrowheads (a).

Figure 2. Internalization of contrast material into myofibroblasts in vivo

Immunohistochemical fluorescence microscopy of representative images acquired from four individual tumors initiated by co-inoculation of MLS cells and PF42T fibroblasts. Biotinylated contrast material (green; avidin-FITC; a, d, g, j) was intravenously administrated into the tail vein of tumor bearing mice (16 days after implantation; 30 minutes prior to tumor retrieval). αSMA staining of pericytes, vascular smooth muscle cells and myofibroblasts (red; b, e, h, k), merged images (c, f, i, l), and nuclear staining (blue; a-f). Arrowheads indicate intracellular uptake.

Figure 3. Viability of fibroblasts after ex-vivo labeling

a) Confocal microscopy of PFN2 fibroblasts double labeled with biotin-BSA-GdDTPA-FAM (48 hours; 10 mg/ml) and propidium iodide (nuclear staining). Arrowheads indicate proliferating cells. b) Neutral red incorporation was used to measure cell viability in control unlabeled cells and in labeled cells. Optical density was measured at 570 nm (mean ± SEM; n=9; p=0.12, t-test un-paired, two tail).

Figure 4. Detectable increase in R₁ relaxation rate of fibroblasts labeled with biotin-BSA-GdDTPA

T₁ weighted images of control and labeled cells were acquired on a 400 MHz spectrometer. (a) Representative R₁ maps of unlabeled cells and (b) cells labeled with biotin-BSA-GdDTPA contrast material (48 hours; 10 mg/ml). (c) Non linear single exponential fittings of signal intensity as a function of TR, used for derivation of R₁ for unlabeled cells (close squares) and for labeled cells (open squares). (d) MRI detection of the change in R₁ (ΔR₁=R₁(labeled)−R₁(unlabeled), obtained from ROI analysis) of fibroblasts labeled with biotin-BSA-GdDTPA compared with that of unlabeled cells (PFN2 n=4; PF40/42T n=3; N1 n=2; * significant change in R₁ of labeled versus unlabeled cells p<0.03, t-test paired, one tail).

Figure 5. Nystatin inhibits internalization of biotin-BSA-GdDTPA into fibroblasts

a) Various primary fibroblasts were labeled with biotin-BSA-GdDTPA (48 hours; 10 mg/ml) in the presence (close bars) or absence (open bars) of the caveolae inhibitor nystatin (50 μM). (PFN2 n=3; PF40/42T n=2; N1 n=2; * p=0.006 for nystatin effect t-test paired, one tail). b) Neutral red incorporation was used to measure cell viability in cos-7 cells after incubation with CM and nystatin. Optical density was measured at 570 nm (mean ± SEM; n=10 p>0.5, t-test un-paired, two tail).

Figure 6. Prolonged intracellular retention and enhanced visibility of the contrast material during cell proliferation

a) PFN2 and PF42T fibroblasts were labeled with biotin-BSA-GdDTPA (48 hours; 10 mg/ml), and incubated in fresh medium for up to two weeks (PFN2 2w; PF42T 2w), or harvested prior to additional incubation (PFN2 2w re-plating). ΔR₁ was measured after two weeks of further culturing (* p<0.01 for labeling retention; t-test paired, one tail). b) Cos-7 cells were labeled with the contrast material (24 hours; 10 mg/ml). ΔR₁ was measured daily, each time for new 1×10⁶ cells harvested from log phase monolayer culture (* p<0.03 for labeling retention- ΔR₁>0 relative to unlabeled cells; t-test paired, one tail). c) Effective relaxivity (R_ef) of the contrast material was derived from ΔR₁ (b) according to the equation: R_ef α ΔR₁ * 2ⁿ (where n is the number of cell divisions)