Novel nano-sized MR contrast agent mediates strong tumor contrast enhancement in an oncogene-driven breast cancer model

Abstract

The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r1) of approximately 30 (mM Mn)-1 s-1 (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2-4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.

Figure 1. Plasma concentrations of Mn (solid line) and Si (dotted line) in plasma of SD rats injected with Spago Pix (n = 3).

Figure 2. Illustration of mammary gland localization in the mouse and T ₁-weighted MR images of pre-injection and 1 h and 4.5 h post injection, respectively, of Spago Pix.

The 1 h image has tumors (T), liver (L) and gall bladder (G) indicated with white arrows. Note that the pre-injection image is without applied fat saturation.

Figure 3. T ₁-weighted MR images of the torso of a mouse where enlargements of the indicated tumor site are shown in Panel A before administration of Spago Pix and at five time points thereafter.

Panels B and C show enlargements of ten tumor sites (labeled 1–10) in different animals before administration of Spago Pix and 30 min (B) and 2–4 h (C) thereafter.

Figure 4. Boxplot of CNR values for pooled tumors at T  = 0 min (24 tumors in 7 animals), 30 min (29 tumors in 7 animals) and >2 h (31 tumors in 6 animals).

P_{0–30 min} <0.0001, P_{0 min–2 h} <0.0001, P_{30 min–2 h}  = 0.0074.

Figure 5. Boxplots of manganese and silicon content in muscle and tumor tissue ∼40 min to 4.5 h post injection of Spago Pix in MRI study 1 (n = 3 for ∼40 min, n = 1 for 4 h). p<0.001 for manganese and p>0.05 for silicon.

Background concentrations (n = 1) are indicated by stars (tumor) and circles (muscle). Note the interrupted y-axis in the boxplot for silicon content.

Figure 6. Immunohistochemistry images of anti-PEG (brown) and hematoxylin counterstaining (blue) of tumor (A1–A3 and B1–B3) and muscle tissue (A4 and B4) 30 minutes (A) and 2 h (B) post injection of Spago Pix.

Tumor tissue at both time points is shown at three different enlargements where the enlarged areas are indicated by the square boxes. Vessels (V), fibroblasts (Fb) and fat cells (Fc) are indicated with rounded lines. Note that the tumor tissue in panel B1–B3 originates from a mouse injected with a dose of 10 µmol Mn/kg, whereas the other tissues originate from animals administered 20 µmol Mn/kg.

Figure 7. Immunohistochemistry image of anti-PEG (brown) and hematoxylin counterstaining (blue) of tumor tissue 30 min post injection of Spago Pix.

The dense PEG-staining corresponds to vessels. Note the staining pattern radiating from the vessel to the right in the figure (see text for details).

Figure 8. PEG surface area in tumor and muscle at 30 minutes and 2–4 hours post-injection of Spago Pix. p<0.01 for tumor at 2–4 h compared to muscle at 30 min and 2–4 h and compared to tumor at 30 min.

The difference between tumor at 30 min and the muscle groups was not statistically significant.