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Marcus Stapf, Ulf Teichgräber, Ingrid Hilger Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology, Jena University Hospital, Friedrich-Schiller University Jena, Jena, Germany Abstract: Heat-based approaches have been considered as promising tools due to their ability to directly eradicate tumor cells and/or increase the sensitivity of tumors to radiation- or chemotherapy. In particular, the heating of magnetic nanoparticles (MNPs) via an alternating magnetic field can provide a handy alternative for a localized tumor treatment. To amplify the efficacy of magnetically induced thermal treatments, we elucidated the superior tumor-destructive effect of methotrexate-coupled MNPs (MTX/MNPs) in combination with magnetic heating (nanochemothermia) over the thermal treatment alone. Our studies in a murine bladder xenograft model revealed the enormous potential of nanochemothermia for a localized and relapse-free destruction of tumors which was superior to the thermal treatment alone. Nanochemothermia remarkably fostered the reduction of tumor volume. It impaired proapoptotic signaling (eg, p-p53), cell survival (eg, p-ERK1/2), and cell cycle (cyclins) pathways. Additionally, heat shock proteins (eg, HSP70) were remarkably affected. Moreover, nanochemothermia impaired the induction of angiogenic signaling by decreasing, for example, the levels of VEGF-R1 and MMP9, although an increasing tumor hypoxia was indicated by elevated Hif-1α levels. In contrast, tumor cells were able to recover after the thermal treatments alone. In conclusion, nanochemothermia on the basis of MTX/MNPs was superior to the thermal treatment due to a modification of cellular pathways, particularly those associated with the cellular survival and tumor vasculature. This allowed very efficient and relapse-free destruction of tumors. Keywords: bladder cancer, magnetic heating, magnetic nanoparticles, methotrexate, hyperthermia, mouse xenograft

Marcus Stapf,* Nadine Pömpner,* Ulf Teichgräber, Ingrid HilgerInstitute of Diagnostic and Interventional Radiology, Department of Experimental Radiology, Jena University Hospital, Friedrich-Schiller University, Jena, Germany*These authors contributed equally to this workAbstract: Today, the therapeutic efficacy of cancer is restricted by the heterogeneity of the response of tumor cells to chemotherapeutic drugs. Since those therapies are also associated with severe side effects in nontarget organs, the application of drugs in combination with nanocarriers for targeted therapy has been suggested. Here, we sought to assess whether the coupling of methotrexate (MTX) to magnetic nanoparticles (MNP) could serve as a valuable tool to circumvent the heterogeneity of tumor cell response to MTX by the combined treatment with hyperthermia. To this end, we investigated five breast cancer cell lines of different origin and with different mutational statuses, as well as a bladder cancer cell line in terms of their response to exposure to MTX as a free drug or after its coupling to MNP as well as in presence/absence of hyperthermia. We also assessed whether the effects could be connected to the cell line-specific expression of proteins related to the uptake and efflux of MTX and MNP. Our results revealed a very heterogeneous and cell line-dependent response to an exposure with MTX-coupled MNP (MTX–MNP), which was almost comparable to the efficacy of free MTX in the same cell line. Moreover, a cell line-specific and preferential uptake of MTX–MNP compared with MNP alone was found (probably by receptor-mediated endocytosis), agreeing with the observed cytotoxic effects. Opposed to this, the expression pattern of several cell membrane transport proteins noted for MTX uptake and efflux was only by tendency in agreement with the cellular toxicity of MTX–MNP in different cell lines. Higher cytotoxic effects were achieved by exposing cells to a combination of MTX–MNP and hyperthermal treatment, compared with MTX or thermotherapy alone. However, the heterogeneity in the response of the tumor cell lines to MTX could not be completely abolished – even after its combination with MNP and/or hyperthermia – and the application of higher thermal dosages might be necessary.Keywords: magnetic nanoparticles, SPION, in vitro, methotrexate, hyperthermia, breast cancer, bladder cancer

Jenny Domey, Ulf Teichgräber, Ingrid Hilger Institute of Diagnostic and Interventional Radiology, University Hospital Jena, Jena, Germany Abstract: Due to their high X-ray attenuation, gold nanoparticles (GNPs) emerged as preclinical contrast agents by giving high vasculature contrast. For this reason, GNPs are regularly applied for computed tomography (CT) imaging of tumors but not for the visualization of inflammation. The aim of this study was to evaluate the biocompatibility and applicability of preclinical GNPs (AuroVist™) of two different sizes (1.9 nm and 15 nm) for the detection of inflammation-associated phagocytes in early-stage edema. Both GNP variants were stable under in vitro conditions and achieved high micro-CT (mCT) contrast after embedment into agarose. Fifteen-nanometer GNPs were detected after uptake into macrophages via mCT imaging exhibiting higher X-ray contrast than cells treated with 1.9 nm GNPs and untreated ones. Both 1.9 nm and 15 nm GNPs exhibited good biocompatibility on murine macrophages according to ATP and cellular dehydrogenase levels. Reactive oxygen species levels produced by phagocytic cells decreased significantly (P≤0.05) after co-incubation with GNPs regardless of the size of the nanoparticle (NP) in comparison to untreated control cells. In vivo mCT studies of inflammation imaging revealed that GNPs with a diameter of 15 nm accumulated within subcutaneous edema 2 hours after injection with a maximum signaling 8 hours postinjection and could be detected up to 48 hours within the edema region. In contrast, 1.9 nm GNPs were not shown to accumulate at the site of the inflammation region and were mostly excreted via the renal system 2–4 hours after injection. In conclusion, our study demonstrated that both GNP variants (1.9 nm and 15 nm) were stable and biocompatible under in vitro conditions. However, only 15 nm NPs have the potential as contrast agent for phagocyte labeling and applications in CT imaging of inflammation on a cellular level. Keywords: inflammation, GNP, phagocytes, AuroVist™, biocompatibility