A. Sheu

Poly(lactide-co-glycolide) microspheres for MRI-monitored transcatheter delivery of sorafenib to liver tumors.

The multi-kinase inhibitor (MKI) sorafenib can be an effective palliative therapy for patients with hepatocellular carcinoma (HCC). However, patient tolerance is often poor due to common systemic side effects following oral administration. Local transcatheter delivery of sorafenib to liver tumors has the potential to reduce systemic toxicities while increasing the dose delivered to targeted tumors. We developed sorafenib-eluting PLG microspheres for delivery by intra-hepatic transcatheter infusion in an orthotropic rodent HCC model. The particles also encapsulated iron-oxide nanoparticles permitting magnetic resonance imaging (MRI) of intra-hepatic biodistributions. The PLG microspheres (diameter≈1μm) were loaded with 18.6% (w/w) sorafenib and 0.54% (w/w) ferrofluid and 65.2% of the sorafenib was released within 72h of media exposure. In vitro studies demonstrated significant reductions in HCC cell proliferation with increasing doses of the sorafenib-eluting microspheres, where the estimated IC50 was a 29μg/mL dose of microspheres. During in vivo studies, MRI permitted intra-procedural visualization of intra-hepatic microsphere delivery. At 72h after microsphere infusion, microvessel density was significantly reduced in tumors treated with the sorafenib-eluting microspheres compared to both sham control tumors (by 35%) and controls (by 30%). These PLG microspheres offer the potential to increase the efficacy of molecularly targeted MKI therapies while reducing systemic exposures via selective catheter-directed delivery to HCC.

MRI-monitored transcatheter intra-arterial delivery of SPIO-labeled natural killer cells to hepatocellular carcinoma: Preclinical studies in a rodent model

ObjectivesThe objective of this study was to test the hypotheses that intra-arterial infusion allows for targeted natural killer (NK) lymphocyte delivery to hepatocellular carcinoma (HCC) and that iron oxide labeling allows for quantitative visualization of intra-arterial NK delivery with magnetic resonance imaging (MRI). Materials and MethodsExperiments received approval from the institutional animal care and use committee. NK-92MI cells were labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Cell viability, labeling efficacy, and cell phantom imaging studies were performed. Eighteen rats were each implanted with HCC tumors. Catheter was placed in proper hepatic artery for either NK lymphocyte (12 rats) or saline (6 rats) infusion. For the 6 rats, MRI T2* measurements for tumor and normal liver were compared before and after the NK infusion and correlated with histologic measurements. Prussian blue staining was used for labeled NK identification. The remaining rats survived for 8 days after the infusion to compare tumor size changes in the rats that received NK cell (6 rats) or saline (6 rats) infusions. Spearman correlation coefficients and t tests were calculated for statistical analyses. ResultsIncreasing SPIO incubation concentration decreased cell viability. Labeling efficacy mean (SD) was 88.0% (3.1%) across samples. The spatial extent of T2*-weighted contrast and R2* relaxivity values increased for cell phantom samples incubated with increasing SPIO concentrations. T2* measurements decreased in the tumor and normal liver tissues after the NK infusion (P < 0.001); &Dgr;T2* was greater in the tumors than in the normal liver tissue (P < 0.001). Histologic measurements demonstrated increased NK delivery to the tumor compared with the normal liver (P < 0.001). &Dgr;T2* was well correlated with histologic NK measurements (&rgr; = 0.70). Changes in tumor diameter 8 days after the infusion were significantly different between those rats that received NK cell infusions (−2.49 [0.86] mm) and those that received sham saline infusion (5.23 [0.66] mm). ConclusionsIntra-arterial infusion permitted selective delivery of NK cells to HCC. Transcatheter delivery of SPIO-labeled NK cells can be quantitatively visualized with MRI. Transcatheter NK cell delivery limited tumor size progression compared with controls.

Antigen-loaded Dendritic Cell Migration: MR Imaging in a Pancreatic Carcinoma Model

PURPOSE To test the following hypotheses in a murine model of pancreatic cancer: (a) Vaccination with antigen-loaded iron-labeled dendritic cells reduces T2-weighted signal intensity at magnetic resonance (MR) imaging within peripheral draining lymph nodes ( LN lymph node s) and (b) such signal intensity reductions are associated with tumor size changes after dendritic cell vaccination. MATERIALS AND METHODS The institutional animal care and use committee approved this study. Panc02 cells were implanted into the flanks of 27 C57BL/6 mice bilaterally. After tumors reached 10 mm, cell viability was evaluated, and iron-labeled dendritic cell vaccines were injected into the left hind footpad. The mice were randomly separated into the following three groups (n = 9 in each): Group 1 was injected with 1 million iron-labeled dendritic cells; group 2, with 2 million cells; and control mice, with 200 mL of phosphate-buffered saline. T1- and T2-weighted MR imaging of labeled dendritic cell migration to draining LN lymph node s was performed before cell injection and 6 and 24 hours after injection. The signal-to-noise ratio ( SNR signal-to-noise ratio ) of the draining LN lymph node s was measured. One-way analysis of variance ( ANOVA analysis of variance ) was used to compare Prussian blue-positive dendritic cell measurements in LN lymph node s. Repeated-measures ANOVA analysis of variance was used to compare in vivo T2-weighted SNR signal-to-noise ratio LN lymph node measurements between groups over the observation time points. RESULTS Trypan blue assays showed no significant difference in mean viability indexes (unlabeled vs labeled dendritic cells, 4.32% ± 0.69 [standard deviation] vs 4.83% ± 0.76; P = .385). Thirty-five days after injection, the mean left and right flank tumor sizes, respectively, were 112.7 mm(2) ± 16.4 and 109 mm(2) ± 24.3 for the 1-million dendritic cell group, 92.2 mm(2) ± 9.9 and 90.4 mm(2) ± 12.8 for the 2-million dendritic cell group, and 193.7 mm(2) ± 20.9 and 189.4 mm(2) ± 17.8 for the control group (P = .0001 for control group vs 1-million cell group; P = .00007 for control group vs 2-million cell group). There was a correlation between postinjection T2-weighted SNR signal-to-noise ratio decreases in the left popliteal LN lymph node 24 hours after injection and size changes at follow-up for tumors in both flanks (R = 0.81 and R = 0.76 for left and right tumors, respectively). CONCLUSION MR imaging approaches can be used for quantitative measurement of accumulated iron-labeled dendritic cell-based vaccines in draining LN lymph node s. The amount of dendritic cell-based vaccine in draining LN lymph node s correlates well with observed protective effects.

Seven-tesla magnetic resonance imaging accurately quantifies intratumoral uptake of therapeutic nanoparticles in the McA rat model of hepatocellular carcinoma: Preclinical study in a rodent model

ObjectivesAfter inducing McA tumors in Sprague-Dawley rats (McA-SD), the following hypotheses were tested: first, that hypervascular McA tumors grown in Sprague-Dawley rats provide a suitable platform to investigate drug delivery; and second, that high-field MRI can be used to measure intratumoral uptake of DOX-SPIOs. Materials and MethodsMcA cells were implanted into the livers of 18 Sprague-Dawley rats. In successfully inoculated animals, 220-&mgr;L DOX-SPIOs were delivered to tumors via the intravenous or intra-arterial route. Pretreatment and posttreatment T2*-weighted images were obtained using 7-T MRI, and change in R2* value (&Dgr;R2*) was obtained from mean signal intensities of tumors in these images. Tumor iron concentration ([Fe]), an indicator of DOX-SPIO uptake, was measured using mass spectroscopy. The primary outcome variable was the Pearson correlation between &Dgr;R2* and [Fe]. ResultsTumors grew successfully in 13 of the 18 animals (72%). Mean (SD) maximum tumor diameter was 0.83 (0.25) cm. The results of phantom studies revealed a strong positive correlation between &Dgr;R2* and [Fe], with r = 0.98 (P < 0.01). The results of in vivo drug uptake studies demonstrated a positive correlation between &Dgr;R2* and [Fe], with r = 0.72 (P = 0.0004). ConclusionsThe McA tumors grown in the Sprague-Dawley rats demonstrated uptake of nanoparticle-based therapeutic agents. Magnetic resonance imaging quantification of intratumoral uptake strongly correlated with iron concentrations in pathological specimens, suggesting that MRI may be used to quantify uptake of iron-oxide nanotherapeutics.

Abstract 4289: MRI-guided intra-arterial delivery of SPIO-labeled natural killer cells to hepatocellular carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Adoptive immunotherapy with natural killer (NK) lymphocytes is a promising approach for treatment of HCC; however, intravenous (IV) infusion may lead to insufficient NK dose delivery to tumors. Also, quantification of intra-tumoral NK delivery may be crucial to optimize therapy or to predict response. We hypothesize that: a) transcatheter intra-arterial (IA) infusion allows for targeted delivery of NK cells to HCC and b) iron oxide labeling methods allow for visualization of IA NK delivery with MRI. 4.0×106 NK-92 cells (ATCC; Manassas, VA) were labeled overnight with 10 pg/cell of Texas Red iron oxide nanoparticles (GENOVIS AB, Sweden) using 4.5 mg/ml protamine sulfate as a transfection agent. Cell viability was measured using a cell counter, and labeling efficacy was measured by fluorescence microscopy with DAPI and by Prussian blue iron staining. With IACUC approval, 6 Sprague-Dawley rats were implanted with 4.0×106 McA-RH7777 HCC cells divided between the left lateral and median lobes to simulate a metastatic tumor. After 8 days of tumor growth, a 24G catheter (Terumo Medical Co., Somerset, NJ) was placed in the proper hepatic artery via laparotomy, and digital subtraction angiography (DSA) confirmed placement. 7.0T MRI scanner (Bruker, Billerica, MA) was used for T2*W scans pre- and post- NK infusion. T2* measurements in tumor and normal liver were compared pre- and post-infusion using paired t-test. Livers were harvested for Prussian blue histology to confirm NK delivery; percentage of cells in a high-powered (20×) field identified as NK (%HPF) were compared between tumor and normal tissues. NK cell viability was >90% before and after labeling. Labeling efficacy was >95%. DSA confirmed successful catheter placement in each animal. Transcatheter NK infusions led to significant reductions in tumor T2* (mean±SD: pre 11.6±0.7 msec, post 9.8±1.1 msec, p=0.04) but no significant reductions in normal liver T2* (mean±SD: pre 7.5±0.1 msec, post 7.6±0.3 msec, p=0.36) during intra-procedural MRI scans. Histologic %HPF measurements were significantly higher in tumor (1.51%) than surrounding normal liver tissues (0.06%) (p<0.01). Transcatheter infusion permitted selective delivery of NK cells to HCC. The intra-hepatic distribution of iron oxide labeled NK cells was quantitatively visualized with MRI. We will perform studies in the future to assess relationships between therapeutic outcomes and the delivered NK dose, comparing IV and IA administration routes. Clinicians could one day use these methods to adjust patient-specific therapeutic regimens during adoptive immunotherapies for the treatment of HCC. In vivo quantification of the delivery of therapeutic agents is a powerful concept that offers the potential to address many unmet needs in both pre-clinical and translational research settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4289. doi:1538-7445.AM2012-4289