Towards Realistic 3D Models of Tumor Vascular Networks.

For reliable in silico or in vitro investigations in, for example, biosensing and drug delivery applications, accurate models of tumor vascular networks down to the capillary size are essential. Compared to images acquired with conventional medical imaging techniques, digitalized histological tumor slices have a higher resolution, enabling the delineation of capillaries. Volume rendering procedures can then be used to generate a 3D model. However, the preparation of such slices leads to misalignments in relative slice orientation between consecutive slices. Thus, image registration algorithms are necessary to re-align the slices. Here, we present an algorithm for the registration and reconstruction of a vascular network from histologic slices applied to 169 tumor slices. The registration includes two steps. First, consecutive images are incrementally pre-aligned using feature- and area-based transformations. Second, using the previous transformations, parallel registration for all images is enabled. Combining intensity- and color-based thresholds along with heuristic analysis, vascular structures are segmented. A 3D interpolation technique is used for volume rendering. This results in a 3D vascular network with approximately 400-450 vessels with diameters down to 25-30 µm. A delineation of vessel structures with close distance was limited in areas of high structural density. Improvement can be achieved by using images with higher resolution and or machine learning techniques.

Vessel Delineation Using U-Net: A Sparse Labeled Deep Learning Approach for Semantic Segmentation of Histological Images.

Semantic segmentation is an important imaging analysis method enabling the identification of tissue structures. Histological image segmentation is particularly challenging, having large structural information while providing only limited training data. Additionally, labeling these structures to generate training data is time consuming. Here, we demonstrate the feasibility of a semantic segmentation using U-Net with a novel sparse labeling technique. The basic U-Net architecture was extended by attention gates, residual and recurrent links, and dropout regularization. To overcome the high class imbalance, which is intrinsic to histological data, under- and oversampling and data augmentation were used. In an ablation study, various architectures were evaluated, and the best performing model was identified. This model contains attention gates, residual links, and a dropout regularization of 0.125. The segmented images show accurate delineations of the vascular structures (with a precision of 0.9088 and an AUC-ROC score of 0.9717), and the segmentation algorithm is robust to images containing staining variations and damaged tissue. These results demonstrate the feasibility of sparse labeling in combination with the modified U-Net architecture.

Application of magnetic iron oxide nanoparticles: Thrombotic activity, imaging and cytocompatibility of silica-coated and carboxymethyl dextrane-coated particles.

Coated iron oxide nanoparticles (IONs) are promising candidates for various applications in nanomedicine, including imaging, magnetic hyperthermia, and drug delivery. The application of IONs in nanomedicine is influenced by factors such as biocompatibility, surface properties, agglomeration, degradation behavior, and thrombogenicity. Therefore, it is essential to investigate the effects of coating material and thickness on the behavior and performance of IONs in the human body. In this study, IONs with a carboxymethyl dextran (CMD) coating and two thicknesses of silica coating (TEOS0.98, and TEOS3.91) were screened and compared to bare iron oxide nanoparticles (BIONs). All three coated particles showed good cytocompatibility (>70%) when tested with smooth muscle cells over three days. To investigate their potential long term behavior inside the human body, the Fe2+ release and hydrodynamic diameters of silica-coated and CMD (carboxymethyl dextrane)-coated IONs were analyzed in simulated body fluids for 72 h at 37 °C. The ION@CMD showed moderate agglomeration of around 100 nm in all four simulated fluids and dissolved faster than the silica-coated particles in artificial exosomal fluid and artificial lysosomal fluid. The particles with silica coating agglomerated in all tested simulated media above 1000 nm. Increased thickness of the silica coating led to decreased degradation of particles. Additionally, CMD coating resulted in nanoparticles with the least prothrombotic activity, and the thick silica coating apparently decreased the prothrombotic properties of nanoparticles compared to BIONs and ION@TEOS0.98. For magnetic resonance applications, ION@CMD and ION@TEOS3.91 showed comparatively high relaxation rates R2 values. In magnetic particle imaging experiments ION@TEOS3.91 yielded the highest normalized signal to noise ratio values and in magnetic hyperthermia studies, ION@CMD and ION@TEOS0.98 showed similar specific loss power. These findings demonstrate the potential of coated IONs in nanomedicine and emphasize the importance of understanding the effect of coating material and thickness on their behavior and performance in the human body.

Iron Oxide Nanoparticle-Based Hyperthermia as a Treatment Option in Various Gastrointestinal Malignancies.

Iron oxide nanoparticle-based hyperthermia is an emerging field in cancer treatment. The hyperthermia is primarily achieved by two differing methods: magnetic fluid hyperthermia and photothermal therapy. In magnetic fluid hyperthermia, the iron oxide nanoparticles are heated by an alternating magnetic field through Brownian and Néel relaxation. In photothermal therapy, the hyperthermia is mainly generated by absorption of light, thereby converting electromagnetic waves into thermal energy. By use of iron oxide nanoparticles, this effect can be enhanced. Both methods are promising tools in cancer treatment and are, therefore, also explored for gastrointestinal malignancies. Here, we provide an extensive literature research on both therapy options for the most common gastrointestinal malignancies (esophageal, gastric and colorectal cancer, colorectal liver metastases, hepatocellular carcinoma, cholangiocellular carcinoma and pancreatic cancer). As many of these rank in the top ten of cancer-related deaths, novel treatment strategies are urgently needed. This review describes the efforts undertaken in vitro and in vivo.

3D-Printed Replica and Porcine Explants for Pre-Clinical Optimization of Endoscopic Tumor Treatment by Magnetic Targeting.

BACKGROUND: Animal models have limitations in cancer research, especially regarding anatomy-specific questions. An example is the exact endoscopic placement of magnetic field traps for the targeting of therapeutic nanoparticles. Three-dimensional-printed human replicas may be used to overcome these pitfalls. METHODS: We developed a transparent method to fabricate a patient-specific replica, allowing for a broad scope of application. As an example, we then additively manufactured the relevant organs of a patient with locally advanced pancreatic ductal adenocarcinoma. We performed experimental design investigations for a magnetic field trap and explored the best fixation methods on an explanted porcine stomach wall. RESULTS: We describe in detail the eight-step development of a 3D replica from CT data. To guide further users in their decisions, a morphologic box was created. Endoscopies were performed on the replica and the resulting magnetic field was investigated. The best fixation method to hold the magnetic field traps stably in place was the fixation of loops at the stomach wall with endoscopic single-use clips. CONCLUSIONS: Using only open access software, the developed method may be used for a variety of cancer-related research questions. A detailed description of the workflow allows one to produce a 3D replica for research or training purposes at low costs.

FEM based simulation of magnetic drug targeting in a multibranched vessel model.

BACKGROUND AND OBJECTIVE: Magnetic drug targeting (MDT) is a promising technology to improve cancer therapy. MDT describes the accumulation of drug loaded superparamagnetic iron oxide nanoparticles (SPIONs) at a desired location, e. g. a tumor, by application of a magnetic field. Here, we evaluate the effectivity of MDT for an endoscopic placement of two different configurations of magnet arrays, i. e. six magnets with same poles facing each other and a Halbach array. Compared to conventional magnet setups outside the body, this endoscopic placement gives the possibility to achieve higher magnetic field gradients inside the tumor. METHODS: For such a MDT concept, we present FEM based simulations of MDT tracing single SPIONs in a 3D geometry of eight multibranched vessels with sizes in the range of capillaries. In these simulations, the effect of the magnetic field gradient as well as of magnet distance to the vessel geometry, magnetic flux density of the magnets, SPIONs hydrodynamic diameter and magnetic moment on the MDT effectivity is calculated. The blood flow is modelled as an incompressible Newtonian fluid and the SPIONs are suspended in the blood flow. Statistical significance of the targeting effectivity results is tested with the Mann-Whitney-U-Test. RESULTS: The results show that the magnetic targeting effectivity is up to 32 % higher than the one calculated without the presence of a magnetic field. In the investigated vessel network, this effect on the targeting effectivity is dependent on the number of local magnetic field maxima that are approached with a high gradient and is noticeable up to 200 µm distance of the magnet to the vessel geometry. CONCLUSIONS: We conclude that for an effective application of MDT, the magnet configuration needs to be placed close to the tumor and should yield a large number of magnetic field maxima that are approached with a high gradient.

Magnetic Fluid Hyperthermia as Treatment Option for Pancreatic Cancer Cells and Pancreatic Cancer Organoids.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with a meager prognosis due to its chemotherapy resistance. A new treatment method may be magnetic fluid hyperthermia (MFH). Magnetoliposomes (ML), consisting of superparamagnetic iron oxide nanoparticles (SPION) stabilized with a phospholipid-bilayer, are exposed to an alternating magnetic field (AMF) to generate heat. To optimize this therapy, we investigated the effects of MFH on human PDAC cell lines and 3D organoid cultures. MATERIAL AND METHODS: ML cytotoxicity was tested on Mia PaCa-2 and PANC-1 cells and on PDAC 3D organoid cultures, generated from resected tissue of patients. The MFH was achieved by AMF application with an amplitude of 40-47 kA/m and a frequency of 270 kHz. The MFH effect on the cell viability of the cell lines and the organoid cultures was investigated at two different time points. Clonogenic assays evaluated the impairment of colony formation. Altering ML set-ups addressed differences arising from intra- vs extracellular ML locations. RESULTS: Mia PaCa-2 and PANC-1 cells showed no cytotoxic effects at ML concentrations up to 300 µg(Fe)/mL and 225 µg(Fe)/mL, respectively. ML at a concentration of 225 µg(Fe)/mL were also non-toxic for PDAC organoid cultures. MFH treatment using exclusively extracellular ML presented the highest impact on cell viability. Clonogenic assays demonstrated remarkable impairment as long-term outcome in MFH-treated PDAC cell lines. Additionally, we successfully treated PDAC organoids with extracellular ML-derived MFH, resulting in notably reduced cell viabilities 2h and 24 h post treatment. Still, PDAC organoids seem to partly recover from MFH after 24 h as opposed to conventional 2D-cultures. CONCLUSION: Treatment with MFH strongly diminished pancreatic cancer cell viability in vitro, making it a promising treatment strategy. As organoids resemble the more advanced in vivo conditions better than conventional 2D cell lines, our organoid model holds great potential for further investigations.

Nanomagnetic Actuation of Hybrid Stents for Hyperthermia Treatment of Hollow Organ Tumors.

This paper describes a magnetic nanotechnology that locally enables hyperthermia treatment of hollow organ tumors by using polymer hybrid stents with incorporated magnetic nanoparticles (MNP). The hybrid stents are implanted and activated in an alternating magnetic field to generate therapeutically effective heat, thereby destroying the tumor. Here, we demonstrate the feasibility of nanomagnetic actuation of three prototype hybrid stents for hyperthermia treatment of hollow organ tumors. The results show that the heating efficiency of stent filaments increases with frequency from approximately 60 W/gFe (95 kHz) to approximately 250 W/gFe (270 kHz). The same trend is observed for the variation of magnetic field amplitude; however, heating efficiency saturates at approximately 30 kA/m. MNP immobilization strongly influences heating efficiency showing a relative difference in heating output of up to 60% compared to that of freely dispersed MNP. The stents showed uniformly distributed heat on their surface reaching therapeutically effective temperatures of 43 °C and were tested in an explanted pig bile duct for their biological safety. Nanomagnetic actuation of hybrid stents opens new possibilities in cancer treatment of hollow organ tumors.

Size-isolation of superparamagnetic iron oxide nanoparticles improves MRI, MPI and hyperthermia performance.

Superparamagnetic iron oxide nanoparticles (SPION) are extensively used for magnetic resonance imaging (MRI) and magnetic particle imaging (MPI), as well as for magnetic fluid hyperthermia (MFH). We here describe a sequential centrifugation protocol to obtain SPION with well-defined sizes from a polydisperse SPION starting formulation, synthesized using the routinely employed co-precipitation technique. Transmission electron microscopy, dynamic light scattering and nanoparticle tracking analyses show that the SPION fractions obtained upon size-isolation are well-defined and almost monodisperse. MRI, MPI and MFH analyses demonstrate improved imaging and hyperthermia performance for size-isolated SPION as compared to the polydisperse starting mixture, as well as to commercial and clinically used iron oxide nanoparticle formulations, such as Resovist® and Sinerem®. The size-isolation protocol presented here may help to identify SPION with optimal properties for diagnostic, therapeutic and theranostic applications.

Modeling of magnetoliposome uptake in human pancreatic tumor cells in vitro.

The internalization kinetics resulting from magnetic nanoparticle interactions with tumor cells play an important role in nanoparticle-based cancer treatment efficiency. Here, the uptake kinetics of magnetoliposomes (ML) into human pancreatic tumor cells (MiaPaCa-2 and BxPC-3) are quantified using magnetic particle spectrometry. A comparison to the uptake kinetics for healthy L929 cells is given. The experimental results are used for the development of an uptake kinetics model describing the three relevant internalization processes: ML adsorption to the cell membrane, endo- and exocytosis. By fitting of experimental data, the rate constant of each internalization process is determined enabling the prediction of internalized ML at any incubation time. After seven hours incubation time, MiaPaCa-2 internalized three times more ML than BxPC-3 and L929 cells even though their ML adsorption rate constants were nearly the same. As the interaction of the ML with the cell membrane is non-specific, the uptake kinetics mirror the individual cell response to ML internalization. With a new mathematical term to cover the exocytosis contribution to the overall internalization process, the extended uptake kinetics model offers new possibilities to analyze the specific internalization mechanism for other nanoparticle and cell types.

Combining Bulk Temperature and Nanoheating Enables Advanced Magnetic Fluid Hyperthermia Efficacy on Pancreatic Tumor Cells.

Many efforts are made worldwide to establish magnetic fluid hyperthermia (MFH) as a treatment for organ-confined tumors. However, translation to clinical application hardly succeeds as it still lacks of understanding the mechanisms determining MFH cytotoxic effects. Here, we investigate the intracellular MFH efficacy with respect to different parameters and assess the intracellular cytotoxic effects in detail. For this, MiaPaCa-2 human pancreatic tumor cells and L929 murine fibroblasts were loaded with iron-oxide magnetic nanoparticles (MNP) and exposed to MFH for either 30 min or 90 min. The resulting cytotoxic effects were assessed via clonogenic assay. Our results demonstrate that cell damage depends not only on the obvious parameters bulk temperature and duration of treatment, but most importantly on cell type and thermal energy deposited per cell during MFH treatment. Tumor cell death of 95% was achieved by depositing an intracellular total thermal energy with about 50% margin to damage of healthy cells. This is attributed to combined intracellular nanoheating and extracellular bulk heating. Tumor cell damage of up to 86% was observed for MFH treatment without perceptible bulk temperature rise. Effective heating decreased by up to 65% after MNP were internalized inside cells.

Establishment of a biophysical model to optimize endoscopic targeting of magnetic nanoparticles for cancer treatment.

Superparamagnetic iron oxide nanoparticles (SPION) may be used for local tumor treatment by coupling them to a drug and accumulating them locally with magnetic field traps, that is, a combination of permanent magnets and coils. Thereafter, an alternating magnetic field generates heat which may be used to release the thermosensitively bound drug and for hyperthermia. Until today, only superficial tumors can be treated with this method. Our aim was to transfer this method into an endoscopic setting to also reach the majority of tumors located inside the body. To find the ideal endoscopic magnetic field trap, which accumulates the most SPION, we first developed a biophysical model considering anatomical as well as physical conditions. Entities of choice were esophageal and prostate cancer. The magnetic susceptibilities of different porcine and rat tissues were measured with a superconducting quantum interference device. All tissues showed diamagnetic behavior. The evaluation of clinical data (computed tomography scan, endosonography, surgical reports, pathological evaluation) of patients gave insight into the topographical relationship between the tumor and its surroundings. Both were used to establish the biophysical model of the tumors and their surroundings, closely mirroring the clinical situation, in which we could virtually design, place and evaluate different electromagnetic coil configurations to find optimized magnetic field traps for each tumor entity. By simulation, we could show that the efficiency of the magnetic field traps can be enhanced by 38-fold for prostate and 8-fold for esophageal cancer. Therefore, our approach of endoscopic targeting is an improvement of the magnetic drug-targeting setups for SPION tumor therapy as it holds the possibility of reaching tumors inside the body in a minimal-invasive way. Future animal experiments must prove these findings in vivo.

Mapping of proton relaxation near superparamagnetic iron oxide particle-loaded polymer threads for magnetic susceptibility difference quantification.

OBJECTIVES: Conventional radiological methods, including magnetic resonance imaging (MRI), fail to visualize polymeric surgical mesh implants because of small thread dimensions and material characteristics. For MRI delineation of such meshes, superparamagnetic iron oxide particles (SPIOs) are integrated in the mesh polymer. Usually, if SPIOs are used as an intravenous contrast agent, they increase the R1 and R2 of adjacent protons. It can be assumed that embedding SPIOs in polymers alters their molecular dynamics. The aim of this study was to investigate the influence of SPIO integration in polymer on the relaxation of adjacent protons. MATERIALS AND METHODS: Polymer threads were placed in an agarose phantom. At 1.5 T, R1, R2, and R2* maps were calculated from multi inversion-recovery spin echo, multi-spin echo, and multi-gradient echo images, respectively. The threads were aligned parallel or orthogonal to B(0). RESULTS: No impact of SPIO on proton R1 and R2 was observed. R2* was increased by the SPIO-loaded threads. R1 and R2 amplitude maps showed a magnetic susceptibility difference of 0.97 ppm/(mg SPIO/g polymer) around SPIO-loaded threads. CONCLUSIONS: In contrast to SPIO in aqueous solutions, polymer-embedded SPIO do not affect proton R1 and R2. However, embedded SPIO generate strong local static magnetic field gradients. Thus, SPIO integration is suitable to control the magnetic susceptibility of polymer threads. This can be exploited to visualize implanted polymer-based meshes in MRI using R2* susceptible sequences. Because no impact on R1 and R2 of adjacent protons by SPIO embedded in mesh threads was observed, structures adjacent to implanted meshes will be observable in R1 and R2 maps.

Fluorescent magnetoliposomes as a platform technology for functional and molecular MR and optical imaging.

Here, we present a detailed characterisation of rhodamine B-containing magnetoliposomes (FLU-ML), emphasising the dependence of their fluorescence properties on the presence of iron oxide cores, and the molar fraction of the fluorophore. The magnetoliposome types used exist as colloidally stable, negatively charged clusters with an average hydrodynamic diameter of 95 nm. The molar rhodamine B fractions were 0.67 % and 1.97 %. Rhodamine B normalised fluorescence, quantum yields and fluorescence lifetimes were substantially reduced by inner filter effects as the magnetoliposome concentration is increased, by increasing molar rhodamine B fraction, and by quenching originating from the iron oxide cores. MR relaxometry at 3 T revealed extremely high r2 relaxivities (440 to 554 s-1mM-1) and moderately high r1 values (2.06 to 3.59 s-1mM-1). Upon incubating human prostate carcinoma (PC-3) cells with FLU-ML, a dose-dependent particle internalisation was found by MR relaxometry. In addition, the internalised FLU-ML were clearly visible by fluorescence microscopy. At the FLU-ML concentrations used (up to 3 × 10³ M Fe) cell viability was not substantially impaired. These results provide valuable insights on the fluorescence properties of bimodal magnetoliposomes and open promising perspectives for the use of these materials as a platform technology for advanced functional and molecular MR and optical imaging applications.

In vivo MRI visualization of mesh shrinkage using surgical implants loaded with superparamagnetic iron oxides.

BACKGROUND: Prosthetic mesh implants are widely used in hernia surgery. To show long-term mesh-related complications such as shrinkage or adhesions, a precise visualization of meshes and their vicinity in vivo is important. By supplementing mesh fibers with ferro particles, magnetic resonance imaging (MRI) can help to delineate the mesh itself. This study aimed to demonstrate and quantify time-dependent mesh shrinkage in vivo by MRI. METHODS: Polyvinylidenfluoride (PVDF) meshes with incorporated superparamagnetic iron oxides (SPIOs) were implanted as an abdominal wall replacement in 30 rats. On days 1, 7, 14, or 21, MRI was performed using a gradient echo sequence with repetition time (TR)/echo time (TE) of 50/4.6 and a flip angle of 20°. The length, width, and area of the device were measured on axial, coronal, and sagittal images, and geometric deformations were assessed by surgical explantation. RESULTS: In all cases, the meshes were visualized and their area estimated by measuring the length and width of the mesh. The MRI presented a mean area shrinkage in vivo of 13% on day 7, 23% on day 14, and 23% on day 21. Postmortem measurements differed statistically from MRI, with a mean area shrinkage of 23% on day 7, 28% on day 14, and 30% on day 21. Ex vivo measurements of shrinkage showed in vivo measurements to be overestimated approximately 8%. Delineation of the mesh helped to show folding or adhesions close to the intestine. CONCLUSION: Loading of surgical meshes with SPIOs allows their precise visualization during MRI and guarantees an accurate in vivo assessment of their shrinkage. The authors' observation clearly indicates that shrinkage in vivo is remarkably less than that shown by illustrated explantation measurements. The use of MRI with such meshes could be a reliable technique for checking on proper operation of implanted meshes and showing related complications, obviating the need for exploratory open surgical revision.