Experimental and Numerical Investigation of Parameters Affecting High-Frequency Irreversible Electroporation for Prostate Cancer Ablation.

While the primary goal of focal therapy for prostate cancer (PCa) is conserving patient quality of life by reducing oncological burden, available modalities use thermal energy or whole-gland radiation which can damage critical neurovascular structures within the prostate and increase risk of genitourinary dysfunction. High-frequency irreversible electroporation (H-FIRE) is a promising alternative ablation modality that utilizes bursts of pulsed electric fields (PEFs) to destroy aberrant cells via targeted membrane damage. Due to its nonthermal mechanism, H-FIRE offers several advantages over state-of-the-art treatments, but waveforms have not been optimized for treatment of PCa. In this study, we characterize lethal electric field thresholds (EFTs) for H-FIRE waveforms with three different pulse widths as well as three interpulse delays in vitro and compare them to conventional irreversible electroporation (IRE). Experiments were performed in non-neoplastic and malignant prostate cells to determine the effect of waveforms on both targeted (malignant) and adjacent (non-neoplastic) tissue. A numerical modeling approach was developed to estimate the clinical effects of each waveform including extent of nonthermal ablation, undesired thermal damage, and nerve excitation. Our findings indicate that H-FIRE waveforms with pulse durations of 5 and 10 µs provide large ablations comparable to IRE with tolerable levels of thermal damage and minimized muscle contractions. Lower duration (2 µs) H-FIRE waveforms exhibit the least amount of muscle contractions but require increased voltages which may be accompanied by unwanted thermal damage.

Properties of tissue within prostate tumors and treatment planning implications for ablation therapies.

Irreversible electroporation (IRE) is a promising alternative therapy for the local treatment of prostate tumors. The procedure involves the direct insertion of needle electrodes into the target zone, and subsequent delivery of short but high-voltage pulses. Successful outcomes rely on adequate exposure of the tumor to a threshold electrical field. To aid in predicting this exposure, computational models have been developed, yet often do not incorporate the appropriate tissue-specific properties. This work aims to quantify electrical conductivity behavior during IRE for three types of tissue present in the target area of a prostate cancer ablation: the tumor tissue itself, the surrounding healthy tissue, and potential areas of necrosis within the tumor. Animal tissues were used as a stand-in for primary samples. The patient-derived prostate tumor tissue showed very similar responses to healthy porcine prostate tissue. An examination of necrotic tissue inside the tumors revealed a large difference, however, and a computational model showed that a necrotic core with differing electrical properties can cause unexpected inhomogeneities within the treatment region.

Multi-Tissue Analysis on the Impact of Electroporation on Electrical and Thermal Properties.

OBJECTIVE: Tissue electroporation is achieved by applying a series of electric pulses to destabilize cell membranes within the target tissue. The treatment volume is dictated by the electric field distribution, which depends on the pulse parameters and tissue type and can be readily predicted using numerical methods. These models require the relevant tissue properties to be known beforehand. This study aims to quantify electrical and thermal properties for three different tissue types relevant to current clinical electroporation. METHODS: Pancreatic, brain, and liver tissue were harvested from pigs, then treated with IRE pulses in a parallel-plate configuration. Resulting current and temperature readings were used to calculate the conductivity and its temperature dependence for each tissue type. Finally, a computational model was constructed to examine the impact of differences between tissue types. RESULTS: Baseline conductivity values (mean 0.11, 0.14, and 0.12 S/m) and temperature coefficients of conductivity (mean 2.0, 2.3, and 1.2 % per degree Celsius) were calculated for pancreas, brain, and liver, respectively. The accompanying computational models suggest field distribution and thermal damage volumes are dependent on tissue type. CONCLUSION: The three tissue types show similar electrical and thermal responses to IRE, though brain tissue exhibits the greatest differences. The results also show that tissue type plays a role in the expected ablation and thermal damage volumes. SIGNIFICANCE: The conductivity and its changes due to heating are expected to have a marked impact on the ablation volume. Incorporating these tissue properties aids in the prediction and optimization of electroporation-based therapies.

Patient Derived Xenografts Expand Human Primary Pancreatic Tumor Tissue Availability for ex vivo Irreversible Electroporation Testing.

New methods of tumor ablation have shown exciting efficacy in pre-clinical models but often demonstrate limited success in the clinic. Due to a lack of quality or quantity in primary malignant tissue specimens, therapeutic development and optimization studies are typically conducted on healthy tissue or cell-line derived rodent tumors that don't allow for high resolution modeling of mechanical, chemical, and biological properties. These surrogates do not accurately recapitulate many critical components of the tumor microenvironment that can impact in situ treatment success. Here, we propose utilizing patient-derived xenograft (PDX) models to propagate clinically relevant tumor specimens for the optimization and development of novel tumor ablation modalities. Specimens from three individual pancreatic ductal adenocarcinoma (PDAC) patients were utilized to generate PDX models. This process generated 15-18 tumors that were allowed to expand to 1.5 cm in diameter over the course of 50-70 days. The PDX tumors were morphologically and pathologically identical to primary tumor tissue. Likewise, the PDX tumors were also found to be physiologically superior to other in vitro and ex vivo models based on immortalized cell lines. We utilized the PDX tumors to refine and optimize irreversible electroporation (IRE) treatment parameters. IRE, a novel, non-thermal tumor ablation modality, is being evaluated in a diverse range of cancer clinical trials including pancreatic cancer. The PDX tumors were compared against either Pan02 mouse derived tumors or resected tissue from human PDAC patients. The PDX tumors demonstrated similar changes in electrical conductivity and Joule heating following IRE treatment. Computational modeling revealed a high similarity in the predicted ablation size of the PDX tumors that closely correlate with the data generated with the primary human pancreatic tumor tissue. Gene expression analysis revealed that IRE treatment resulted in an increase in biological pathway signaling associated with interferon gamma signaling, necrosis and mitochondria dysfunction, suggesting potential co-therapy targets. Together, these findings highlight the utility of the PDX system in tumor ablation modeling for IRE and increasing clinical application efficacy. It is also feasible that the use of PDX models will significantly benefit other ablation modality testing beyond IRE.

Ultrarapid Inductive Rewarming of Vitrified Biomaterials with Thin Metal Forms.

Arteries with 1-mm thick walls can be successfully vitrified by loading cryoprotective agents (CPAs) such as VS55 (8.4 M) or less concentrated DP6 (6 M) and cooling at or beyond their critical cooling rates of 2.5 and 40 °C/min, respectively. Successful warming from this vitrified state, however, can be challenging. For example, convective warming by simple warm-bath immersion achieves 70 °C/min, which is faster than VS55's critical warming rate of 55 °C/min, but remains far below that of DP6 (185 °C/min). Here we present a new method that can dramatically increase the warming rates within either a solution or tissue by inductively warming commercially available metal components placed within solutions or in proximity to tissues with non-invasive radiofrequency fields (360 kHz, 20 kA/m). Directly measured warming rates within solutions exceeded 1000 °C/min with specific absorption rates (W/g) of 100, 450 and 1000 for copper foam, aluminum foil, and nitinol mesh, respectively. As proof of principle, a carotid artery diffusively loaded with VS55 and DP6 CPA was successfully warmed with high viability using aluminum foil, while standard convection failed for the DP6 loaded tissue. Modeling suggests this approach can improve warming in tissues up to 4-mm thick where diffusive loading of CPA may be incomplete. Finally, this technology is not dependent on the size of the system and should therefore scale up where convection cannot.

Biomaterial scaffolds for non-invasive focal hyperthermia as a potential tool to ablate metastatic cancer cells.

Currently, there are very few therapeutic options for treatment of metastatic disease, as it often remains undetected until the burden of disease is too high. Microporous poly(ε-caprolactone) biomaterials have been shown to attract metastasizing breast cancer cells in vivo early in tumor progression. In order to enhance the therapeutic potential of these scaffolds, they were modified such that infiltrating cells could be eliminated with non-invasive focal hyperthermia. Metal disks were incorporated into poly(ε-caprolactone) scaffolds to generate heat through electromagnetic induction by an oscillating magnetic field within a radiofrequency coil. Heat generation was modulated by varying the size of the metal disk, the strength of the magnetic field (at a fixed frequency), or the type of metal. When implanted subcutaneously in mice, the modified scaffolds were biocompatible and became properly integrated with the host tissue. Optimal parameters for in vivo heating were identified through a combination of computational modeling and ex vivo characterization to both predict and verify heat transfer dynamics and cell death kinetics during inductive heating. In vivo inductive heating of implanted, tissue-laden composite scaffolds led to tissue necrosis as seen by histological analysis. The ability to thermally ablate captured cells non-invasively using biomaterial scaffolds has the potential to extend the application of focal thermal therapies to disseminated cancers.

Improved tissue cryopreservation using inductive heating of magnetic nanoparticles.

Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica-coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes.

Quantification and biodistribution of iron oxide nanoparticles in the primary clearance organs of mice using T1 contrast for heating.

PURPOSE: To use contrast based on longitudinal relaxation times (T1 ) or rates (R1 ) to quantify the biodistribution of iron oxide nanoparticles (IONPs), which are of interest for hyperthermia therapy, cell targeting, and drug delivery, within primary clearance organs. METHODS: Mesoporous silica-coated IONPs (msIONPs) were intravenously injected into 15 naïve mice. Imaging and mapping of the longitudinal relaxation rate constant at 24 h or 1 week postinjection were performed with an echoless pulse sequence (SWIFT). Alternating magnetic field heating measurements were also performed on ex vivo tissues. RESULTS: Signal enhancement from positive T1 contrast caused by IONPs was observed and quantified in vivo in liver, spleen, and kidney at concentrations up to 3.2 mg Fe/(g tissue wt.) (61 mM Fe). In most cases, each organ had a linear correlation between the R1 and the tissue iron concentration despite variations in intra-organ distribution, degradation, and IONP surface charge. Linear correlation between R1 and volumetric SAR in hyperthermia therapy was observed. CONCLUSION: The linear dependence between R1 and tissue iron concentration in major organs allows quantitative monitoring of IONP biodistribution in a dosage range relevant to magnetic hyperthermia applications, which falls into the concentration gap between CT and conventional MRI techniques. Magn Reson Med 78:702-712, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Development of a computational simulation tool to design a protocol for treating prostate tumours using transurethral laser photothermal therapy.

OBJECTIVES: The objective of this study was to design laser treatment protocols to induce sufficient thermal damage to a tumour embedded in a prostate model, while protecting the surrounding healthy tissue. METHODS: A computational Monte Carlo simulation algorithm of light transport in a spherical prostatic tumour containing gold nanorods was developed to determine laser energy deposition. The laser energy absorption was then used to simulate temperature elevations in the tumour embedded in an elliptical human prostate model. The Arrhenius integral was coupled with the heat transfer model to identify heating protocols to induce 100% damage to the tumour, while resulting in less than 5% damage to the surrounding sensitive prostatic tissue. RESULTS: Heating time to achieve 100% damage to the tumour was identified to be approximately 630 s when using a laser irradiance of 7 W/cm2 incident on the prostatic urethral surface. Parametric studies were conducted to show how the local blood perfusion rate and urethral surface cooling affect the heating time to achieve the same thermal dosage. The heating time was shorter when cooling at the urethra was not applied and/or with heat-induced vasculature damage. The identified treatment protocols were acceptable since the calculated percentages of the damaged healthy tissue volume to the healthy prostatic volume were approximately 2%, less than the threshold of 5%. The approach and results from this study can be used to design individualised treatment protocols for patients suffering from prostatic cancer.

MicroCT image-generated tumour geometry and SAR distribution for tumour temperature elevation simulations in magnetic nanoparticle hyperthermia.

OBJECTIVES: The objective of this study was to develop and test computer algorithms to export micro computed tomography (microCT) images and to generate tumour geometry and specific absorption rate (SAR) distribution for heat transfer simulation in magnetic nanoparticle hyperthermia. METHODS: Computer algorithms were written to analyse and export microCT images of 3D tumours containing magnetic nanoparticles. MATLAB(®) and ProE(®) programs were used to generate a prototype of the tumour geometry. The enhancements in the microCT pixel index number due to presence of nanoparticles in the tumours were first converted into corresponding SAR values. The SAR data were then averaged over three-dimensional clusters of pixels using the SAS(®) program. This greatly decreased the size of the SAR file, while in the meantime it ensured that the amount of total energy deposited in the tumour was conserved. Both the tumour geometry and the SAR file were then imported into the COMSOL(®) software package to simulate temperature elevations in the tumour and their surrounding tissue region during magnetic nanoparticle hyperthermia. RESULTS: A linear relationship was obtained to relate individual pixel index numbers in the microCT images to the SAR values under a specific magnetic field. The generated prototype of the tumour geometry based on only 30 slices of microCT images resembled the original tumour shape and size. The tumour geometry and the simplified SAR data set were successfully accepted by the COMSOL software for heat transfer simulation. Up to 20 °C temperature elevations from its baseline temperature were found inside the tumours, implying possible thermal damage to the tumour during magnetic nanoparticle hyperthermia.

Computational simulation of temperature elevations in tumors using Monte Carlo method and comparison to experimental measurements in laser photothermal therapy.

Accurate simulation of temperature distribution in tumors induced by gold nanorods during laser photothermal therapy relies on precise measurements of thermal, optical, and physiological properties of the tumor with or without nanorods present. In this study, a computational Monte Carlo simulation algorithm is developed to simulate photon propagation in a spherical tumor to calculate laser energy absorption in the tumor and examine the effects of the absorption (µ(a)) and scattering (µ(s)) coefficients of tumors on the generated heating pattern in the tumor. The laser-generated energy deposition distribution is then incorporated into a 3D finite-element model of prostatic tumors embedded in a mouse body to simulate temperature elevations during laser photothermal therapy using gold nanorods. The simulated temperature elevations are compared with measured temperatures in PC3 prostatic tumors in our previous in vivo experimental studies to extract the optical properties of PC3 tumors containing different concentrations of gold nanorods. It has been shown that the total laser energy deposited in the tumor is dominated by µ(a), while both µ(a) and µ(s) shift the distribution of the energy deposition in the tumor. Three sets of µ(a) and µ(s) are extracted, representing the corresponding optical properties of PC3 tumors containing different concentrations of nanorods to laser irradiance at 808 nm wavelength. With the injection of 0.1 cc of a 250 optical density (OD) nanorod solution, the total laser energy absorption rate is increased by 30% from the case of injecting 0.1 cc of a 50 OD nanorod solution, and by 125% from the control case without nanorod injection. Based on the simulated temperature elevations in the tumor, it is likely that after heating for 15 min, permanent thermal damage occurs in the tumor injected with the 250 OD nanorod solution, while thermal damage to the control tumor and the one injected with the 50 OD nanorod solution may be incomplete.

Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be harvested at a low cost without an invasive procedure. However, there has been no report on comparing hUCMSCs with human bone marrow MSCs (hBMSCs) for bone regeneration in vivo. The aim of this study was to investigate hUCMSC and hBMSC seeding on macroporous calcium phosphate cement (CPC), and to compare their bone regeneration in critical-sized cranial defects in rats. Cell attachment, osteogenic differentiation and mineral synthesis on RGD-modified macroporous CPC were investigated in vitro. Scaffolds with cells were implanted in 8-mm defects of athymic rats. Bone regeneration was investigated via micro-CT and histological analysis at 4, 12, and 24 weeks. Three groups were tested: CPC with hUCMSCs, CPC with hBMSCs, and CPC control without cells. Percentage of live cells and cell density on CPC in vitro were similarly good for hUCMSCs and hBMSCs. Both cells had high osteogenic expressions of alkaline phosphatase, osteocalcin, collagen I, and Runx2. Bone mineral density and trabecular thickness in hUCMSC and hBMSC groups in vivo were greater than those of CPC control group. New bone amount for hUCMSC-CPC and hBMSC-CPC constructs was increased by 57% and 88%, respectively, while blood vessel density was increased by 15% and 20%, than CPC control group at 24 weeks. hUCMSC-CPC and hBMSC-CPC groups generally had statistically similar bone mineral density, new bone amount and vessel density. In conclusion, hUCMSCs seeded on CPC were shown to match the bone regeneration efficacy of hBMSCs in vivo for the first time. Both hUCMSC-CPC and hBMSC-CPC constructs generated much more new bone and blood vessels than CPC without cells. Macroporous RGD-grafted CPC with stem cell seeding is promising for craniofacial and orthopedic repairs.

Diffusion of dimethyl sulfoxide in tissue engineered collagen scaffolds visualized by computer tomography.

Cryopreservation is a convenient method for long-term preservation of natural and engineered tissues in regenerative medicine. Homogeneous loading of tissues with CPAs, however, forms one of the major hurdles in tissue cryopreservation. In this study, computer tomography (CT) as a non-invasive imaging method was used to determine the effective diffusion of Me2SO in tissue-engineered collagen scaffolds. The dimensions of the scaffolds were 30 x 30 x 10 mm3 with a homogeneous pore size of 100 microm and a porosity of 98%. CT images were acquired after equilibrating the scaffolds in phosphate buffered saline (PBS) and transferring them directly in 10% (v/v)Me2SO. The Me2SO loading process of the scaffold could thus be measured and visualized in real time. The experimental data were fitted using a diffusion equation. The calculated effective diffusion constant for Me2SO in the PBS loaded scaffold was determined from experimental diffusion studies to be 2.4 x 10(-6) cm2/s at 20 degrees C.