Imaging Tumor Necrosis with Ferumoxytol

Abstract

Objective: Ultra-small superparamagnetic iron oxide nanoparticles (USPIO) are promising contrast agents for magnetic resonance imaging (MRI). USPIO mediated proton relaxation rate enhancement is strongly dependent on compartmentalization of the agent and can vary depending on their intracellular or extracellular location in the tumor microenvironment. We compared the T1- and T2-enhancement pattern of intracellular and extracellular USPIO in mouse models of cancer and pilot data from patients. A better understanding of these MR signal effects will enable non-invasive characterizations of the composition of the tumor microenvironment.

Materials and methods: Six 4T1 and six MMTV-PyMT mammary tumors were grown in mice and imaged with ferumoxytol-enhanced MRI. R1 relaxation rates were calculated for different tumor types and different tumor areas and compared with histology. The transendothelial leakage rate of ferumoxytol was obtained by our measured relaxivity of ferumoxytol and compared between different tumor types, using a t-test. Additionally, 3 patients with malignant sarcomas were imaged with ferumoxytol-enhanced MRI. T1- and T2-enhancement patterns were compared with histopathology in a descriptive manner as a proof of concept for clinical translation of our observations.

Results: 4T1 tumors showed central areas of high signal on T1 and low signal on T2 weighted MR images, which corresponded to extracellular nanoparticles in a necrotic core on histopathology. MMTV-PyMT tumors showed little change on T1 but decreased signal on T2 weighted images, which correlated to compartmentalized nanoparticles in tumor associated macrophages. Only 4T1 tumors demonstrated significantly increased R1 relaxation rates of the tumor core compared to the tumor periphery (p<0.001). Transendothelial USPIO leakage was significantly higher for 4T1 tumors (3.4±0.9x10-3 mL/min/100cm3) compared to MMTV-PyMT tumors (1.0±0.9x10-3 mL/min/100 cm3). Likewise, ferumoxytol imaging in patients showed similar findings with high T1 signal in areas of tumor necrosis and low signal in areas of intracellularly compartmentalized iron.

Conclusion: Differential T1- and T2-enhancement patterns of USPIO in tumors enable conclusions about their intracellular and extracellular location. This information can be used to characterize the composition of the tumor microenvironment.

Fig 1. Ferumoxytol nanoparticles delineate early necrosis on MR images.

Axial T1- and T2*-weighted MR images of 4T1 and PymT tumors in mice before, 1 hour and 24 hours post injection (p.i.) of ferumoxytol at a dose of 0.5 mmol Fe/kg. Note homogenous tumors on pre-contrast images with relatively uniform blood pool enhancement at 1 h p.i.. 4T1 tumors demonstrate marked central T1-enhancement at 24 h p.i..

Fig 2. Gadopentetate does not delineate early necrosis.

Axial T1 weighted MR images of the same 4T1 tumor as in Fig 1 at 60 seconds after intravenous injection of Gd-DTPA at a dose of 0.2 mmol/kg. Note homogenous tumor morphology with only slightly increased enhancement of the tumor periphery compared to the tumor center.

Fig 3. 4T1 tumors with central necrosis exhibit significant T1-enhancement.

Difference between pre- and post-contrast R1 relaxation rates (ΔR1 relaxation rates) of 4T1 tumors, PymT tumors as well as skeletal muscle. Data are displayed as mean data of six tumors in each group and standard deviations. Insert shows ΔR1 data of 4T1 tumor core versus periphery at 24 hours. Tumor ΔR1 data at 60 minutes and 24 hours post contrast are significantly different from baseline values (p<0.05).

Fig 4. 4T1 tumor T1-enhancement corresponds to clusters of free iron oxides in central necrosis.

(A) H&E stain of 4T1 tumor shows a necrotic core (square C) and highly cellular tumor periphery (square D, 100x magnification). (B) H&E stain of PymT tumor shows uniformly heterogeneous tumor tissue without central necrosis (100x magnifications). (C) Prussian blue stain (400x magnification) of the necrotic core of the 4T1 tumor shows diffuse distribution blue (iron) staining consistent with iron oxide nanoparticles in a non-compartmentalized, extracellular localization. (D) Prussian blue stain (400x magnifications) of the peripheral area of the 4T1 tumor shows blue iron staining in cells.

Fig 5. GEH121333 Tumor T1-enhancement corresponds to free iron oxides in central necrosis.

(A) Axial T1- and T2*-weighted MR images of a representative PymT tumor before and 48 hours post injection (p.i.) of ferumoxytol at a dose of 0.5 mmol Fe/kg. Note homogenous tumors on pre-contrast images. The tumor center demonstrates hyperintense (bright) T1-enhancement and hypointense (dark) T2*-enhancement while the tumor periphery demonstrates hypointense enhancement on both sequences. (B) Corresponding histopathology (20x magnification) demonstrates areas of apparently free PEG-Cy5 (red) positive iron oxides in central tumor areas. F4/80 positive (green) macrophages outline these central areas. (C) Higher magnification (40x magnification) of the central tumor area confirms free PEG-Cy5 positive iron oxides (red) in central, necrotic tumor areas with low cell quantity (DAPI, blue). (D) Closer evaluation of peripheral tumor areas with high cell content demonstrate PEG-Cy5 (red) positive iron oxides co-localized to abundant F4/80 positive (green) macrophages.

Fig 6. T1-enhancement of ferumoxytol in early necrosis of a Ewing’s sarcoma.

A) T1- and T2 weighted MRI images of a 21 year old patient with metastatic Ewing’s sarcoma in the left sacrum and ilium (arrow). Normal bone marrow demonstrates marked T2-enhancement and minimal or no T1-enhancement at 24 h p.i., consistent with intracellular iron in bone marrow macrophages. Fluid-isointense liquefied necrosis demonstrates minimal peripheral rim enhancement. Blood in iliac vessels and T2-hyperintense tumor necrosis in the sacrum demonstrate marked T2-enhancement and marked T1-enhancement at 24 h p.i., consistent with extracellular iron. B) T1 weighed axial MRI image after intravenous injection of Gd-BOPTA demonstrates peripheral rim enhancement around the liquefied necrosis in the ilium, but interestingly no enhancement of the necrotic tumor area in the sacrum. C) PET image from the same area demonstrates highly cellular bone marrow after induction chemotherapy and relatively mild ¹⁸F-FDG uptake into the tumor (arrow).

Fig 7. T1-enhancement of ferumoxytol in dilated vascular sinusoids of a teleangiectatic osteosarcoma.

T1 weighted pre and post contrast images with corresponding histology. A) Non-contrast, nonfat-saturated, T1 weighted axial image of the femur shows a telangiectatic osteosarcoma (arrow). Tumor appears featureless. B) Fat-saturated, T1 weighted axial image of the same tumor after administration of 5 mg Fe/kg ferumoxytol. Tumor now shows hyperintense fluid-fluid levels in cystic-vascular areas. C) Corresponding histology shows findings compatible with telangiectatic osteosarcoma with numerous vascular sinusoids.