Immunomodulatory Magnetic Microspheres for Augmenting Tumor-Specific Infiltration of Natural Killer (NK) Cells

The purpose of this research is to develop magnetic resonance imaging (MRI) visible immunomodulatory microspheres (IMM-MS) for efficient image guided cancer immunotherapy. IMM-MS composed of recombinant interferon gamma (IFN-γ), iron oxide nanocubes (IONC), and biodegradable poly­(lactide-<i>co</i>-glycolide) (PLGA) were successfully prepared via a double-emulsion method. The prepared IMM-MS exhibited a sustained IFN-γ release and highly sensitive MR <i>T</i>₂ contrast effects. Finally, in an orthotopic liver tumor VX2 rabbit model, successful hepatic intra-arterial (IA) transcatheter delivery of IMM-MS to liver tumors was confirmed with MR images. The deposition of IMM-MS significantly increased NK-cell infiltration into the liver tumor site

Ultrasound guided implantation.

<p>(A) Using ultrasound guidance, a 17 gauge coaxial introducer (arrow) is advanced into the liver. The sharp inner stylet is removed and a small tumor fragment (3-4mm³) is placed in the hub of the introducer and pushed with the blunt stylet. (B) Post implantation US image reveals a small focus of hyperechogenicity (arrow) representing the tumor fragment and air.</p

Tumor preparation.

<p>(A) Hind limb tumors were placed in a sterile petri dish and sliced into small tumor fragments (3-4mm³). (B) A 17 gauge coaxial introducer has a hollow core (white arrow), with two inner stylets, sharp (black arrow head) and blunt (white arrow head).</p

MRI and Gross Pathology.

<p>(A) Axial T2 weighted MRI image demonstrates two T2 hyperintense lesions in the left hepatic lobe (arrow heads). (B) Explanted gross specimen reveals tumors corresponding to the lesions seen on MRI (arrow heads).</p

Mean tumor growth rates.

<p>Mean tumor growth rates.</p

MRI-Monitored Intra-Tumoral Injection of Iron-Oxide Labeled <i>Clostridium novyi-NT</i> Anaerobes in Pancreatic Carcinoma Mouse Model

<div><p>Objectives</p><p>To validate the feasibility of labeling <i>Clostridium novyi-NT</i> (<i>C.novyi-NT</i>) anaerobes with iron-oxide nanoparticles for magnetic resonance imaging (MRI) and demonstrate the potential to use MRI to visualize intra-tumoral delivery of these iron-oxide labeled <i>C.novyi-NT</i> during percutaneous injection procedures.</p><p>Materials and Methods</p><p>All studies were approved by IACUC. <i>C.novyi-NT</i> were labeled with hybrid iron-oxide Texas red nanoparticles. Growth of labeled and control samples were evaluated with optical density. Labeling was confirmed with confocal fluorescence and transmission electron microscopy (TEM). MRI were performed using a 7 Tesla scanner with T2*-weighted (T2*W) sequence. Contrast-to-noise ratio (CNR) measurements were performed for phantoms and signal-to-noise ratio (SNR) measurements performed in C57BL/6 mice (n = 12) with Panc02 xenografts before and after percutaneous injection of iron-oxide labeled <i>C.novyi-NT.</i> MRI was repeated 3 and 7 days post-injection. Hematoxylin-eosin (HE), Prussian blue and Gram staining of tumor specimens were performed for confirmation of intra-tumoral delivery.</p><p>Results</p><p>Iron-oxide labeling had no influence upon <i>C.novyi-NT</i> growth. The signal intensity (SI) within T2*W images was significantly decreased for iron-oxide labeled <i>C.novyi-NT</i> phantoms compared to unlabeled controls. Under confocal fluorescence microscopy, the iron-oxide labeled <i>C.novyi-NT</i> exhibited a uniform red fluorescence consistent with observed regions of DAPI staining and overall labeling efficiency was 100% (all DAPI stained <i>C.novyi-NT</i> exhibited red fluorescence). Within TEM images, a large number iron granules were observed within the iron-oxide labeled <i>C.novyi-NT</i>; these were not observed within unlabeled controls. Intra-procedural MRI measurements permitted <i>in</i><i>vivo</i> visualization of the intra-tumoral distribution of iron-oxide labeled <i>C.novyi-NT</i> following percutaneous injection (depicted as punctate regions of SI reductions within T2*-weighted images); tumor SNR decreased significantly following intra-tumoral injection of <i>C.novyi-NT</i> (p<0.05); these SNR reductions were maintained at 3 and 7 day follow-up intervals. Prussian blue and Gram staining confirmed presence of the iron-oxide labeled anaerobes.</p><p>Conclusions</p><p><i>C.novyi-NT</i> can be labeled with iron-oxide nanoparticles for MRI visualization of intra-tumoral deposition following percutaneous injection during bacteriolytic therapy.</p></div

Histology.

<p>H&E staining was performed confirming tumors within the liver.</p

Tumor SNR measurements within T2*-weighted images before and after percutaneous injection of iron-oxide labeled <i>C.novyi-NT</i> into cystic Panc02 tumors with extensive zones of liquefactive necrosis (a, n = 6) and into solid Panc02 tumors (b, n = 6) with follow-up measurements performed immediately after injection and at 3-day and 7-day intervals post-injection.

<p>*p<0.05 for all pre- <i>vs</i> post-injection SNR measurements.</p

OD₆₀₀ of <i>C.novyi-NT</i> at different time points after iron-oxide labeling.

<p>There was no significance between iron-oxide labeled and unlabeled <i>C.novyi-NT</i> OD₆₀₀ measurements (p>0.05 for comparisons at each time point, n = 3).</p

Parameters for T1W, T2W and T2*W Sequences.

<p>Note: T1W = T1-weighted, T2W = T2-weighted, T2*W = T2*-weighted, GRE = Gradient echo, TSE = Turbo spine echo, TR = Repetition time, TE = Echo time, FOV = Field of view, FA = Flip angle.</p><p>Parameters for T1W, T2W and T2*W Sequences.</p