Deep-tissue localization of magnetic field hyperthermia using pulse sequencing.

OBJECTIVE: Deep-tissue localization of thermal doses is a long-standing challenge in magnetic field hyperthermia (MFH), and remains a limitation of the clinical application of MFH to date. Here, we show that pulse sequencing of MFH leads to a more persistent inhibition of tumor growth and less systemic impact than continuous MFH, even when delivering the same thermal dose. METHODS: We used an in vivo orthotopic murine model of pancreatic PANC-1 cancer, which was designed with a view to the forthcoming 'NoCanTher' clinical study, and featured MFH alongside systemic chemotherapy (SyC: gemcitabine and nab-paclitaxel). In parallel, in silico thermal modelling was implemented. RESULTS: Tumor volumes 27 days after the start of MFH/SyC treatment were 53% (of the initial volume) in the pulse MFH group, compared to 136% in the continuous MFH group, and 337% in the non-treated controls. Systemically, pulse MFH led to ca. 50% less core-temperature increase in the mice for a given injected dose of magnetic heating agent, and inflicted lower levels of the stress marker, as seen in the blood-borne neutrophil-to-lymphocyte ratio (1.7, compared to 3.2 for continuous MFH + SyC, and 1.2 for controls). CONCLUSION: Our data provided insights into the influence of pulse sequencing on the observed biological outcomes, and validated the nature of the improved thermal dose localization, alongside significant lowering of the overall energy expenditure entailed in the treatment.

Hyperthermia affects collagen fiber architecture and induces apoptosis in pancreatic and fibroblast tumor hetero-spheroids in vitro.

Desmoplasia, an aberrant production of extracellular matrix (ECM), is considered as one predictive marker of malignancy of pancreatic cancer. In this paper, we study the effect of mild hyperthermia on fibrillary collagen architecture in murine Achilles tendons and in a pancreatic cancer model, in vitro, i.e. 3D hetero-type tumor spheroids, consisting of pancreatic cancer (Panc-1) cells and fibroblasts (WI-38), producing collagen fibers. We clearly demonstrate that i) mild hyperthermia (40 °C, 42 °C) damages the collagen architecture in murine Achilles tendons. ii) Mild extrinsic (hot air) and iron oxide nanoparticle based magnetic hyperthermia reduce the level of collagen fiber architecture in the generated hetero-type tumor spheroids. iii) Mild magnetic hyperthermia reduces cell vitality mainly through apoptotic and necrotic processes in the generated tumor spheroids. In conclusion, hetero-type 3D tumor spheroids are suitable for studying the effect of hyperthermia on collagen fibers, in vitro.

Iron Oxide Nanoparticles Carrying 5-Fluorouracil in Combination with Magnetic Hyperthermia Induce Thrombogenic Collagen Fibers, Cellular Stress, and Immune Responses in Heterotopic Human Colon Cancer in Mice.

In this study we looked for the main protein pathway regulators which were responsible for the therapeutic impact on colon cancers when combining magnetic hyperthermia with the chemotherapeutic agent 5-fluorouracil (5FU). To this end, chitosan-coated magnetic nanoparticles (MNP) functionalized with 5FU were intratumorally injected into subcutaneous human colon cancer xenografts (HT-29) in mice and exposed to an alternating magnetic field. A decreased tumor growth was found particularly for the combined thermo-chemotherapy vs. the corresponding monotherapies. By using computational analysis of the tumor proteome, we found upregulated functional pathway categories termed "cellular stress and injury", "intracellular second messenger and nuclear receptor signaling", "immune responses", and "growth proliferation and development". We predict TGF-beta, and other mediators, as important upstream regulators. In conclusion, our findings show that the combined thermo-chemotherapy induces thrombogenic collagen fibers which are able to impair tumor nutrient supply. Further on, we associate several responses to the recognition of damage associated molecular patterns (DAMPs) by phagocytic cells, which immigrate into the tumor area. The activation of some pathways associated with cell survival implies the necessity to conduct multiple therapy sessions in connection with a corresponding monitoring, which could possibly be performed on the base of the identified protein regulators.

Effect of Matrix-Modulating Enzymes on The Cellular Uptake of Magnetic Nanoparticles and on Magnetic Hyperthermia Treatment of Pancreatic Cancer Models In Vivo.

Magnetic hyperthermia can cause localized thermal eradication of several solid cancers. However, a localized and homogenous deposition of high concentrations of magnetic nanomaterials into the tumor stroma and tumor cells is mostly required. Poorly responsive cancers such as the pancreatic adenocarcinomas are hallmarked by a rigid stroma and poor perfusion to therapeutics and nanomaterials. Hence, approaches that enhance the infiltration of magnetic nanofluids into the tumor stroma convey potentials to improve thermal tumor therapy. We studied the influence of the matrix-modulating enzymes hyaluronidase and collagenase on the uptake of magnetic nanoparticles by pancreatic cancer cells and 3D spheroids thereof, and the overall impact on magnetic heating and cell death. Furthermore, we validated the effect of hyaluronidase on magnetic hyperthermia treatment of heterotopic pancreatic cancer models in mice. Treatment of cultured cells with the enzymes caused higher uptake of magnetic nanoparticles (MNP) as compared to nontreated cells. For example, hyaluronidase caused a 28% increase in iron deposits per cell. Consequently, the thermal doses (cumulative equivalent minutes at 43 °C, CEM43) increased by 15-23% as compared to heat dose achieved for cells treated with magnetic hyperthermia without using enzymes. Likewise, heat-induced cell death increased. In in vivo studies, hyaluronidase-enhanced infiltration and distribution of the nanoparticles in the tumors resulted in moderate heating levels (CEM43 of 128 min as compared to 479 min) and a slower, but persistent decrease in tumor volumes over time after treatment, as compared to comparable treatment without hyaluronidase. The results indicate that hyaluronidase, in particular, improves the infiltration of magnetic nanoparticles into pancreatic cancer models, impacts their thermal treatment and cell depletion, and hence, will contribute immensely in the fight against pancreatic and many other adenocarcinomas.

The Treatment of Heterotopic Human Colon Xenograft Tumors in Mice with 5-Fluorouracil Attached to Magnetic Nanoparticles in Combination with Magnetic Hyperthermia Is More Efficient than Either Therapy Alone.

Magnetic nanoparticles (MNPs) have shown promising features to be utilized in combinatorial magnetic hyperthermia and chemotherapy. Here, we assessed if a thermo-chemotherapeutic approach consisting of the intratumoral application of functionalized chitosan-coated MNPs (CS-MNPs) with 5-fluorouracil (5FU) and magnetic hyperthermia prospectively improves the treatment of colorectal cancer. With utilization of a human colorectal cancer (HT29) heterotopic tumor model in mice, we showed that the thermo-chemotherapeutic treatment is more efficient in inactivating colon cancer than either tumor treatments alone (i.e., magnetic hyperthermia vs. the presence of 5FU attached to MNPs). In particular, the thermo-chemotherapeutic treatment significantly (p < 0.01) impacts tumor volume and tumor cell proliferation (Ki67 expression, p < 0.001) compared to the single therapy modalities. The thermo-chemotherapeutic treatment: (a) affects DNA replication and repair as measured by H2AX and phosphorylated H2AX expression (p < 0.05 to 0.001), (b) it does not distinctly induce apoptosis nor necroptosis in target cells, since expression of p53, PARP cleaved-PARP, caspases and phosphorylated-RIP3 was non-conspicuous, (c) it renders tumor cells surviving therapy more sensitive to further therapy sessions as indicated by an increased expression of p53, reduced expression of NF-κB and HSPs, albeit by tendency with p > 0.05), and (d) that it impacts tumor vascularity (reduced expression of CD31 and αvß3 integrin (p < 0.01 to 0.001) and consequently nutrient supply to tumors. We further hypothesize that tumor cells die, at least in parts, via a ROS dependent mechanism called oxeiptosis. Taken together, a very effective elimination of colon cancers seems to be feasible by utilization of repeated thermo-chemotherapeutic therapy sessions in the long-term.