Optical imaging of the peri-tumoral inflammatory response in breast cancer

<p>Abstract</p> <p>Purpose</p> <p>Peri-tumoral inflammation is a common tumor response that plays a central role in tumor invasion and metastasis, and inflammatory cell recruitment is essential to this process. The purpose of this study was to determine whether injected fluorescently-labeled monocytes accumulate within murine breast tumors and are visible with optical imaging.</p> <p>Materials and methods</p> <p>Murine monocytes were labeled with the fluorescent dye DiD and subsequently injected intravenously into 6 transgenic MMTV-PymT tumor-bearing mice and 6 FVB/n control mice without tumors. Optical imaging (OI) was performed before and after cell injection. Ratios of post-injection to pre-injection fluorescent signal intensity of the tumors (MMTV-PymT mice) and mammary tissue (FVB/n controls) were calculated and statistically compared.</p> <p>Results</p> <p>MMTV-PymT breast tumors had an average post/pre signal intensity ratio of 1.8+/- 0.2 (range 1.1-2.7). Control mammary tissue had an average post/pre signal intensity ratio of 1.1 +/- 0.1 (range, 0.4 to 1.4). The p-value for the difference between the ratios was less than 0.05. Confocal fluorescence microscopy confirmed the presence of DiD-labeled cells within the breast tumors.</p> <p>Conclusion</p> <p>Murine monocytes accumulate at the site of breast cancer development in this transgenic model, providing evidence that peri-tumoral inflammatory cell recruitment can be evaluated non-invasively using optical imaging.</p

Imaging Tumor Necrosis with Ferumoxytol.

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.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.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.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

Development of Novel Tumor‐Targeted Theranostic Nanoparticles Activated by Membrane‐Type Matrix Metalloproteinases for Combined Cancer Magnetic Resonance Imaging and Therapy

A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional "theranostic" nanoparticles (TNPs) is described for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death is observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens

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

<p>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).</p

Gadopentetate does not delineate early necrosis.

<p>Axial T1 weighted MR images of the same 4T1 tumor as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142665#pone.0142665.g001" target="_blank">Fig 1</a> 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.</p

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

<p>(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.</p

Average ΔR1 and ΔR2 in tumor and necrosis after 1 hour, 24 hour and 48 hour post injection.

<p>Average ΔR1 and ΔR2 in tumor and necrosis after 1 hour, 24 hour and 48 hour post injection.</p

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

<p>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.</p

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

<p>(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.</p