Development of an Electroporation and Nanoparticle-based Therapeutic Platform for Bone Metastases.

Purpose To assess for nanopore formation in bone marrow cells after irreversible electroporation (IRE) and to evaluate the antitumoral effect of IRE, used alone or in combination with doxorubicin (DOX)-loaded superparamagnetic iron oxide (SPIO) nanoparticles (SPIO-DOX), in a VX2 rabbit tibial tumor model. Materials and Methods All experiments were approved by the institutional animal care and use committee. Five porcine vertebral bodies in one pig underwent intervention (IRE electrode placement without ablation [n = 1], nanoparticle injection only [n = 1], and nanoparticle injection followed by IRE [n = 3]). The animal was euthanized and the vertebrae were harvested and evaluated with scanning electron microscopy. Twelve rabbit VX2 tibial tumors were treated, three with IRE, three with SPIO-DOX, and six with SPIO-DOX plus IRE; five rabbit VX2 tibial tumors were untreated (control group). Dynamic T2*-weighted 4.7-T magnetic resonance (MR) images were obtained 9 days after inoculation and 2 hours and 5 days after treatment. Antitumor effect was expressed as the tumor growth ratio at T2*-weighted MR imaging and percentage necrosis at histologic examination. Mixed-effects linear models were used to analyze the data. Results Scanning electron microscopy demonstrated nanopores in bone marrow cells only after IRE (P , .01). Average volume of total tumor before treatment (503.1 mm3 ± 204.6) was not significantly different from those after treatment (P = .7). SPIO-DOX was identified as a reduction in signal intensity within the tumor on T2*-weighted images for up to 5 days after treatment and was related to the presence of iron. Average tumor growth ratios were 103.0% ± 75.8 with control treatment, 154.3% ± 79.7 with SPIO-DOX, 77% ± 30.8 with IRE, and -38.5% ± 24.8 with a combination of SPIO-DOX and IRE (P = .02). The percentage residual viable tumor in bone was significantly less for combination therapy compared with control (P = .02), SPIO-DOX (P , .001), and IRE (P = .03) treatment. The percentage residual viable tumor in soft tissue was significantly less with IRE (P = .005) and SPIO-DOX plus IRE (P = .005) than with SPIO-DOX. Conclusion IRE can induce nanopore formation in bone marrow cells. Tibial VX2 tumors treated with a combination of SPIO-DOX and IRE demonstrate enhanced antitumor effect as compared with individual treatments alone. © RSNA, 2017 Online supplemental material is available for this article.

Safety and Efficacy of an Absorbable Filter in the Inferior Vena Cava to Prevent Pulmonary Embolism in Swine.

Purpose To evaluate the immediate and long-term safety as well as thrombus-capturing efficacy for 5 weeks after implantation of an absorbable inferior vena cava (IVC) filter in a swine model. Materials and Methods This study was approved by the institutional animal care and use committee. Eleven absorbable IVC filters made from polydioxanone suture were deployed via a catheter in the IVC of 11 swine. Filters remained in situ for 2 weeks (n = 2), 5 weeks (n = 2), 12 weeks (n = 2), 24 weeks (n = 2), and 32 weeks (n = 3). Autologous thrombus was administered from below the filter in seven swine from 0 to 35 days after filter placement. Fluoroscopy and computed tomography follow-up was performed after filter deployment from weeks 1-6 (weekly), weeks 7-20 (biweekly), and weeks 21-32 (monthly). The infrarenal IVC, lungs, heart, liver, kidneys, and spleen were harvested at necropsy. Continuous variables were evaluated with a Student t test. Results There was no evidence of IVC thrombosis, device migration, caval penetration, or pulmonary embolism. Gross pathologic analysis showed gradual device resorption until 32 weeks after deployment. Histologic assessment demonstrated neointimal hyperplasia around the IVC filter within 2 weeks after IVC filter deployment with residual microscopic fragments of polydioxanone suture within the caval wall at 32 weeks. Each iatrogenic-administered thrombus was successfully captured by the filter until resorbed (range, 1-4 weeks). Conclusion An absorbable IVC filter can be safely deployed in swine and resorbs gradually over the 32-week testing period. The device is effective for the prevention of pulmonary embolism for at least 5 weeks after placement in swine. © RSNA, 2017.

Irreversible Electroporation in the Epidural Space of the Porcine Spine: Effects on Adjacent Structures.

Purpose To determine the effects of irreversible electroporation (IRE) on the neural tissues after ablation in the epidural space of the porcine spine. Materials and Methods The institutional animal care and use committee approved this study. With the IRE electrode positioned in the right lateral recess of the spinal epidural space, 20 IRE ablations were performed with computed tomographic (CT) guidance by using different applied voltages in four animals that were euthanized immediately after magnetic resonance (MR) imaging of the spine, performed 6 hours after IRE (terminal group). Histopathologic characteristics of the neural tissues were assessed and used to select a voltage for a survival study. Sixteen CT-guided IRE ablations in the epidural space were performed by using 667 V in four animals that were survived for 7 days (survival group). Clinical characteristics, MR imaging findings (obtained 6 hours after IRE and before euthanasia), histopathologic characteristics, and simulated electric field strengths were assessed. A one-way analysis of variance was used to compare the simulated electric field strength to histologic findings. Results The mean distance between the IRE electrode and the spinal cord and nerve root was 1.71 mm ± 0.90 and 8.47 mm + 3.44, respectively. There was no clinical evidence of paraplegia after IRE ablation. MR imaging and histopathologic examination showed no neural tissue lesions within the spinal cord; however, five of 16 nerve roots (31.2%) demonstrated moderate wallerian degeneration in the survival group. The severity of histopathologic injury in the survival group was not significantly related to either the simulated electric field strength or the distance between the IRE electrode and the neural structure (P > .05). Conclusion Although the spinal cord appears resistant to the toxic effects of IRE, injury to the nerve roots may be a limiting factor for the use of IRE ablation in the epidural space. © RSNA, 2016 Online supplemental material is available for this article.

Irreversible electroporation of the lumbar vertebrae in a porcine model: is there clinical-pathologic evidence of neural toxicity?

PURPOSE: To evaluate the effects of irreversible electroporation (IRE) in the porcine spine. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Twenty computed tomographically guided IRE ablations in either a transpedicular location or directly over the posterior cortex were performed in the lumbar vertebrae of 10 pigs by a single operator. T1- and T2-weighted magnetic resonance (MR) imaging was performed with and without contrast material 2 or 7 days after ablation. Mathematical modeling was performed to estimate the extent of ablation. Clinical, radiologic, pathologic, and simulation findings were analyzed. The Miller low-bias back transformation was used to construct 95% confidence intervals for the mean absolute percentage difference between the maximum length and width of the ablation zone on MR images and pathologic measurements by using square-root-transformed data. RESULTS: Bipolar IRE electrode placement and ablation were successful in all cases. The mean distances from the IRE electrode to the posterior wall of the vertebral body or the exiting nerve root were 2.93 mm ± 0.77 (standard deviation) and 7.87 mm ± 1.99, respectively. None of the animals had neurologic deficits. Well-delineated areas of necrosis of bone, bone marrow, and skeletal muscle adjacent to the vertebral body were present. Histopathologic changes showed outcomes that matched with simulation-estimated ablation zones. The percentage absolute differences in the ablation measurements between MR imaging and histopathologic examination showed the following average errors: 24.2% for length and 28.8% for width measurements on T2-weighted images, and 26.1% for length and 33.3% for width measurements on T1-weighted contrast material-enhanced images. CONCLUSION: IRE ablation in the porcine spine is feasible and safe and produces localized necrosis with minimal neural toxicity. Signal intensity changes on images acquired with standard MR imaging sequences demonstrate the ablation zone to be larger than that at histopathologic examination.

Rabbit hepatic arterial anatomy variations: implications on experimental design.

BACKGROUND: The VX2 rabbit model of liver cancer is commonly used to evaluate the efficacy of locoregional anticancer therapy and knowledge of the hepatic arterial anatomy in the rabbit is important for catheter-directed experiments. PURPOSE: To describe the normal anatomy and anatomic variations of the celiac axis and hepatic artery in the rabbit. MATERIAL AND METHODS: Angiograms of 222 rabbits were retrospectively reviewed. The branching pattern of the celiac axis was classified and the diameters of the major branches were measured. Paired t-tests were used to compare the difference between the average sizes of arteries. RESULTS: Variant celiac axis or hepatic artery anatomy was noted in 25.9% of angiograms, with the gastric branches arising from the proper hepatic artery in 23.3% of cases. The celiac axis could be successfully classified into one of five distinct branching patterns in 193 (86.9%) cases. The mean diameters of the right and left hepatic arteries were 0.67 mm (95% CI [0.64, 0.7]) and 1.25 mm (95% CI [1.19, 1.31]), respectively. The mean diameters of the medial and lateral branches of the left hepatic artery were 0.63 mm (95% CI [0.6, 0.67]) and 0.91 mm (95% CI [0.86, 0.96]), respectively. The right hepatic artery was significantly smaller than the left hepatic artery and the lateral branch of the left hepatic artery (all P values <0.0001). CONCLUSION: Arterial variants in the rabbit are not uncommon. The proper hepatic artery often gives origin to gastric artery branches. To facilitate superselective intra-arterial intervention, the left lateral lobe of the liver should be targeted for tumor implantation because of the significant size difference between the right and left hepatic arteries.

Core Needle Biopsy Guidance Based on Tissue Morphology Assessment with AI-OCT Imaging.

This paper presents a combined optical imaging/artificial intelligence (OI/AI) technique for the real-time analysis of tissue morphology at the tip of the biopsy needle, prior to collecting a biopsy specimen. This is an important clinical problem as up to 40% of collected biopsy cores provide low diagnostic value due to high adipose or necrotic content. Micron-scale-resolution optical coherence tomography (OCT) images can be collected with a minimally invasive needle probe and automatically analyzed using a computer neural network (CNN)-based AI software. The results can be conveyed to the clinician in real time and used to select the biopsy location more adequately. This technology was evaluated on a rabbit model of cancer. OCT images were collected with a hand-held custom-made OCT probe. Annotated OCT images were used as ground truth for AI algorithm training. The overall performance of the AI model was very close to that of the humans performing the same classification tasks. Specifically, tissue segmentation was excellent (~99% accuracy) and provided segmentation that closely mimicked the ground truth provided by the human annotations, while over 84% correlation accuracy was obtained for tumor and non-tumor classification.

Immune modulation by molecularly targeted photothermal ablation in a mouse model of advanced hepatocellular carcinoma and cirrhosis.

Immunotherapy is a promising new treatment approach for hepatocellular carcinoma (HCC), but there are numerous barriers to immunotherapy in HCC, including an immunosuppressive microenvironment and the "immunotolerance" of the liver. Hyperthermia treatment modalities are standard of care for early stage HCC, and hyperthermia is known to have immunomodulatory effects. We have developed a molecularly targeted photothermal ablation (MTPA) technology that provides thermally tunable, tumor-specific heat generation. The purpose of this study was to evaluate the morphologic and immunologic effects of MTPA in an immunotherapy-resistant syngeneic mouse model of HCC in a background of toxin-induced cirrhosis. We found that the anatomic, cellular, and molecular features of this model recapitulate the characteristics of advanced human HCC. MTPA as a monotherapy and in combination with immune checkpoint therapy significantly increased intratumoral CD3+ and activated CD8+ T cells while decreasing regulatory T cells relative to control or immune checkpoint therapy alone based on immunohistochemistry, flow cytometry, and single cell RNA sequencing data. Furthermore, we identified evidence of MTPA's influence on systemic tumor immunity, with suppression of remote tumor growth following treatment of orthotopic tumors. The results of this study suggest that tumor-specific hyperthermia may help overcome resistance mechanisms to immunotherapy in advanced HCC.

Influence of injection technique, drug formulation and tumor microenvironment on intratumoral immunotherapy delivery and efficacy.

BACKGROUND: Intratumoral delivery of immunotherapeutics represents a compelling solution to directly address local barriers to tumor immunity. However, we have previously shown that off-target delivery is a substantial problem during intratumoral injections; this can lead to diminished drug efficacy and systemic toxicities. We have identified three variables that influence intratumoral drug delivery: injection technique, drug formulation and tumor microenvironment. The purpose of this study was to characterize the impact of modifications in each variable on intratumoral drug delivery and immunotherapy efficacy. METHODS: Intratumoral injections were performed in a hybrid image-guided intervention suite with ultrasound, fluoroscopy and CT scanning capabilities in both rat and mouse syngeneic tumor models. Intratumoral drug distribution was quantified by CT volumetric imaging. The influence of varying needle design and hydrogel-based drug delivery on the immune response to a stimulator of interferon genes (STING) agonist was evaluated using flow cytometry and single cell RNA sequencing. We also evaluated the influence of tumor stiffness on drug injection distribution. RESULTS: Variations in needle design, specifically with the use of a multiside hole needle, led to approximately threefold improvements in intratumoral drug deposition relative to conventional end-hole needles. Likewise, delivery of a STING agonist through a multiside hole needle led to significantly increased expression of type I interferon-associated genes and 'inflammatory' dendritic cell gene signatures relative to end-hole STING agonist delivery. A multidomain peptide-based hydrogel embedded with a STING agonist led to substantial improvements in intratumoral deposition; however, the hydrogel was noted to generate a strong immune response against itself within the target tumor. Evaluation of tumor stroma on intratumoral drug delivery revealed that there was a greater than twofold improvement in intratumoral distribution in soft tumors (B16 melanoma) compared with firm tumors (MC38 colorectal). CONCLUSIONS: Injection technique, drug formulation and tumor stiffness play key roles in the accurate delivery of intratumoral immunotherapeutics.

Molecularly targeted photothermal ablation improves tumor specificity and immune modulation in a rat model of hepatocellular carcinoma.

Thermal ablation is a standard therapy for patients with hepatocellular carcinoma (HCC). Contemporary ablation devices are imperfect, as they lack tumor specificity. An ideal ablation modality would generate thermal energy only within tumoral tissue. Furthermore, as hyperthermia is known to influence tumor immunity, such a tumor-specific ablation modality may have the ability to favorably modulate the tumor immune landscape. Here we show a clinically relevant thermal ablation modality that generates tumor-specific hyperthermia, termed molecularly targeted photothermal ablation (MTPA), that is based upon the excellent localization of indocyanine green to HCC. In a syngeneic rat model, we demonstrate the tumor-specific hyperthermia generated by MTPA. We also show through spatial and transcriptomic profiling techniques that MTPA favorably modulates the intratumoral myeloid population towards tumor immunogenicity and diminishes the systemic release of oncogenic cytokines relative to conventional ablation modalities.

Investigation of tissue cellularity at the tip of the core biopsy needle with optical coherence tomography.

We report the development and the pre-clinical testing of a new technology based on optical coherence tomography (OCT) for investigating tissue composition at the tip of the core biopsy needle. While ultrasound, computed tomography, and magnetic resonance imaging are routinely used to guide needle placement within a tumor, they still do not provide the resolution needed to investigate tissue cellularity (ratio between viable tumor and benign stroma) at the needle tip prior to taking a biopsy core. High resolution OCT imaging, however, can be used to investigate tissue morphology at the micron scale, and thus to determine if the biopsy core would likely have the expected composition. Therefore, we implemented this capability within a custom-made biopsy gun and evaluated its capability for a correct estimation of tumor tissue cellularity. A pilot study on a rabbit model of soft tissue cancer has shown the capability of this technique to provide correct evaluation of tumor tissue cellularity in over 85% of the cases. These initial results indicate the potential benefit of the OCT-based approach for improving the success of the core biopsy procedures.

Imaging Intratumoral Nanoparticle Uptake After Combining Nanoembolization with Various Ablative Therapies in Hepatic VX2 Rabbit Tumors.

Combining image-guided therapy techniques for the treatment of liver cancers is a strategy that is being used to improve local tumor control rates. Here, we evaluate the intratumoral uptake of nanoparticles used in combination with radiofrequency ablation (RFA), irreversible electroporation (IRE), or laser induced thermal therapy (LITT). Eight rabbits with VX2 tumor in the liver underwent one of four treatments: (i) nanoembolization (NE) with radiolabeled, hollow gold nanoparticles loaded with doxorubicin (64Cu-PEG-HAuNS-DOX); (ii) NE + RFA; (iii) NE + IRE; (iv) NE +LITT. Positron emission tomography/computed tomography (PET/CT) imaging was obtained 1-hr or 18-hrs after intervention. Tissue samples were collected for autoradiography and transmission electron microscopy (TEM) analysis. PET/CT imaging at 1-hr showed focal deposition of oil and nanoparticles in the tumor only after NE+ RFA but at 18-hrs, all animals had focal accumulation of oil and nanoparticles in the tumor region. Autoradiograph analysis demonstrated nanoparticle deposition in the tumor and in the ablated tissues adjacent to the tumor when NE was combined with ablation. TEM results showed the intracellular uptake of nanoparticles in tumor only after NE + IRE. Nanoparticles demonstrated a structural change, suggesting direct interaction, potentially leading to drug release, only after NE + LITT. The findings demonstrate that a combined NE and ablation treatment technique for liver tumors is feasible, resulting in deposition of nanoparticles in and around the tumor. Depending on the ablative energy applied, different effects are seen on nanoparticle localization and structure. These effects should be considered when designing nanoparticles for use in combination with ablation technologies.

Paclitaxel induces inactivation of p70 S6 kinase and phosphorylation of Thr421 and Ser424 via multiple signaling pathways in mitosis.

The 70 kDa ribosomal S6 kinase (p70S6K) is important for cell growth and survival. Activation of p70S6K requires sequential phosphorylation of multiple serine and threonine sites often triggered by growth factors and hormones. Here, we report that paclitaxel, a microtubule-damaging agent, induces phosphorylation of p70S6K at threonine 421 and serine 424 (T421/S424) in a concentration- and time-dependent manner in multiple breast and ovarian cancer cell lines demonstrated by a T421/S424 phospho-p70S6K antibody. Phosphoamino-acid analysis and Western blot analysis by serine-/threonine-specific antibodies further confirms that both serine and threonine residues are phosphorylated in p70S6K following treatment with paclitaxel. Paclitaxel-induced p70S6K(T421/S424) phosphorylation requires both de novo RNA and protein synthesis via multiple signaling pathways including ERK1/2 MAP kinase, JNK, PKC, Ca(++), PI3K, and mammalian target of rapamycin (mTOR). Despite phosphorylation of p70S6K(T421/S424), paclitaxel inactivates this kinase in a concentration- and time-dependent manner as illustrated by in vitro kinase assay. Inhibitors of mTOR, PI3K, and Ca(++) impair p70S6K activity, whereas inhibitors of JNK and PKC stimulate p70S6K activity. Inhibition of PKC and JNK prevents paclitaxel-induced p70S6K inactivation. Moreover, the paclitaxel-induced phosphorylation and low activity of p70S6K mainly occurs during mitosis. In summary, paclitaxel is able to induce p70S6K(T421/S424) phosphorylation and decrease its activity in mitotic cells via multiple signaling pathways. Our data suggest that paclitaxel-induced p70S6K(T421/S424) phosphorylation and kinase inactivation are differentially regulated. Our data also indicate that paclitaxel may exert its antitumor effect, at least in part, via inhibition of p70S6K.

Interleukin-1 alpha increases the cytotoxic activity of etoposide against human osteosarcoma cells.

The recurrence of pulmonary metastases resistant to salvage chemotherapy continues to be a major problem in osteosarcoma patients. Our goal is to identify novel combinations of biologic response modifiers plus chemotherapeutic agents that can be translated into clinical trials. Response rates of relapsed osteosarcoma patients to etoposide have been extremely low. The present investigation demonstrated that IL-1 alpha dramatically increased the sensitivity of MG-63, SAOS-2, and TE-85 osteosarcoma cells to etoposide when the two agents were used simultaneously. The cytostatic activity of 1 microM etoposide was increased from 35 to 70%, 30 to 65%, and 4 to 90%, respectively, by 5.0 U/ml IL-1 alpha. Analysis using the colony-forming assay to quantify cytotoxicity showed that the percentage of cell survival following exposure to etoposide decreased from 0.81 to 0.56, 0.55 to 0.2, and 0.4 to 0.05 when the combination treatment was used. Increased sensitivity was not seen when etoposide treatment preceded IL-1 alpha treatment. IL-1 alpha also increased the sensitivity of these cells to doxorubicin but not to cisplatin or topotecan. The mechanism of this enhanced activity is independent of p-glycoprotein, drug-uptake, or effects on topoisomerase II.

In vitro model of normal, immortalized ovarian surface epithelial and ovarian cancer cells for chemoprevention of ovarian cancer.

BACKGROUND: Epithelial ovarian cancer has the highest mortality rate among the gynecologic cancers. The synthetic retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR), has been used in the chemoprevention of ovarian cancer. However, the effectiveness of its application for different populations has been questioned because of the genetic differences among normal, high risk, and women with cancer. OBJECTIVE: To explore the similarities and the differences in 4-HPR effects on different ovarian epithelial cells which mimic different populations of women, normal ovarian surface epithelium to represent the normal population of women, immortalized ovarian surface epithelium to represent premalignant changes, and cells derived from ovarian cancer cells to represent malignant changes were used as in vitro models. METHODS: Normal ovarian surface epithelial cells, immortalized ovarian surface epithelial cells, and ovarian cancer cells were incubated for different intervals with increasing concentrations of 4-HPR. Growth inhibition, the fraction of apoptotic cells, the expression of apoptosis-related genes, including p53, p16, p21, and caspase-3, and mitochondrial permeability transition were measured before and after 4-HPR treatment. RESULTS: Treatment with 4-HPR produced growth inhibition and apoptosis in a dose-dependent manner for all 3 cell types. 4-HPR produced the strongest activation of the p53 pathway in normal ovarian epithelial (NOE) cells, while it caused the largest increase in MPT in the cancer cells, suggesting a different mechanism for growth inhibition and/or apoptosis in these cell lines. 4-HPR, at a concentration of 10 muM, had a maximal effect on caspase-3 activity at 72 h in normal cells and at 48 h in immortalized and cancer cells, although the effects were modest. CONCLUSIONS: Normal ovarian surface epithelial cells, immortalized ovarian surface epithelial cells, and ovarian cancer cells showed a differential response to 4-HPR. Although the same endpoints of growth inhibition and apoptosis induction were present in response to 4-HPR, these endpoints may be regulated through different pathways. IMPLICATIONS: Clinical trials with higher concentrations of 4-HPR should prove beneficial.