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• Quantitative iron estimation in MNP: Ferrozine based colorimetric method• Qualitative iron identification in cells: Prussian blue staining, TEM• Cell toxicity assays: Trypan blue counting, MTT assay• Flow analysis of cells: Guava Flow Express Plus flow cytometer• Cell lines: B16F10 melanoma cells, PanO2 pancreatic adenocarcinoma cells, C17.2 neural progenitor cells (NPCs), RAW264.7 monocytes/macrophages (Mo/Ma)• Mouse tumor models: subcutaneous melanoma model; disseminated peritoneal pancreatic cancer model.•10 KW inductive heater to generate AMF• Gaussia luciferase-mediated bioluminescence was measured using an IVIS Lumina II• Histology: Prussian blue staining and PKH26 fluorescence detection.

CELL-MEDIATED MAGNETIC HYPERTHERMIA OF PRECLINICAL TUMORS

Using iron nanoparticles (MNPs) to absorb alternating magnetic field (AMF) energy as a method of generating localized hyperthermia has shown potential as a cancer treatment. Many attempts have been made to increase the localization of MNPs, for example by attaching antibodies recognizing tumor-specific epitopes or peptides binding receptors on tumor cells or neovasculature. Several research groups have shown reliable results using tumor homing cells as delivery cells for different therapeutics. Here we hypothesized that tumor homing cells can carry MNPs specifically to the tumor site, and tumor burden will decrease after AMF exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs).We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After AMF exposure, the targeted delivery of MNPs by the NPCs resulted in a significant decrease in tumor size. Second, monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated magnetic hyperthermia we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors. Based on these observations we concluded that development of localized hyperthermia treatment using tumor tropic cells shows promise as a cancer therapy.

ABSTRACT

Sivasai Balivada†, Rajashekhar Rachakatla†, Matthew T. Basel†, Hongwang Wang‡, Gwi Moon Seo†, Tej B. Shrestha†, Marla Pyle†, Viktor Chikan‡, Stefan H. Bossmann‡, Deryl L. Troyer†

†-Department of Anatomy & Physiology ‡- Department of Chemistry, Kansas State University

HYPOTHESIS

A. Homing of intravenously injected NPCs expressing Gaussia luciferase to metastatic melanoma. B. Tumor volumes in mice injected with B16-F10 melanoma cells (*p < 0.05) when compared with saline control.

RESULTSHistological analysis of tissues from mice treated with MNPs and AMF exposure:

RESULTS

SUMMARY

Increase in survival of mice bearing peritoneal pancreatic tumors by AMF after delivery of MNPs by Mo/Ma:

REFERENCES

•Kaplan-Meier test showed that survival curves were significantly different for the experimental group compared with other groups.(p<0.005)•MNP/AMF treatment mice survived substantially longer, with mice lasting until 33.5 days•Average increase in survival of treatment group is 7 days

ACKNOWLEDGEMENTS

•Attenuation of Mouse Melanoma by A/C Magnetic Field after Delivery of Bi-Magnetic Nanoparticles by Neural Progenitor CellsRaja Shekar Rachakatla*, Sivasai Balivada*, Gwi-Moon Seo, Carl B. Myers, Hongwang Wang, Thilani N. Samarakoon, Raj Dani, Marla Pyle, Franklin O. Kroh, Brandon Walker, Xiaoxuan Leaym, Olga B. Koper, Viktor Chikan, Stefan H. Bossmann, Masaaki Tamura, and Deryl L. Troyer ACS Nano 2010 4 (12), 7093-7104, * co-first authors •Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer modelMatthew T. Basel, Sivasai Balivada, Hongwang Wang, Tej B. Shrestha, Gwi Moon Seo, Marla Pyle, Gayani Abayaweera, Raj Dani, Olga B. Koper, Masaaki Tamura, Viktor Chikan, Stefan H. Bossmann, and Deryl L. Troyer Int J Nanomedicine. 2012; 7: 297–306.I

Tumor homing cells can carry MNPs specifically to the tumor site, and tumor burden will decrease after AMF exposure

RESULTS

BACKGROUND

MATERIALS & METHODS

Inductive heater

MNPs testing on NPCs in vitro:

NPCs migration toward mouse melanoma and attenuation of melanoma growth by AMF after delivery of MNPs by NPCs:

MNPs testing on RAW 264.7 Mo/Ma in vitro:

Homing studies of PKH26-loaded RAW 264.7 Mo/Ma on disseminated peritoneal pancreatic cancer:

A. TEM of Fe/Fe3O4/aminosilaxane/stealth nanoparticles; B. Bright-field image of NPCs loaded with MNPs showing positive Prussian blue staining (magnification 20×); C. TEM image of NPC loaded with MNPs (magnification 30 000×); D. In vitro cell viability of NPCs cultured in medium containing MNPs; E. Loading efficiency of iron MNPs; F. AMF-induced temperature changes in vitro.* p < 0.05, ** p < 0.1 when compared with the control

(A−D) Prussian blue stained tissue sections of melanoma tumor-bearing mice which received NPCs-MNPs followed by AMF treatment: lung (A), liver (B), and tumor (C). Note the absence of blue stained NPCs in the tumor sections. (D) NPCs loaded with MNP (blue color cells) in tumor section of a mouse which received NPCs-MNPs but no AMF treatment. (E,F): green apoptotic cells in a tumor-bearing mouse with NPCs-MNPs+AMF (E) compared to few apoptotic cells in a tumor-bearing mouse with saline only treatment (F).

A. Toxicity of nanoparticles B. Loading of nanoparticles C. Percent of cells loaded D. Bright-field image of cells loaded with MNPs showing positive for Prussian blue staining

Mo/Ma only infiltrate Pan02 tumors. PKH26-labeled mo/ma were injected intraperitoneally into mice bearing intraperitoneal Pan02 tumors. Mice were euthanized 3 days after Mo/Ma injection, and organs were harvested and imaged. Scale bars are 100 μm

This work was supported by NIH 1R21CA135599, NIH-COBRE grant P20 RR0117686, NSF IIP 0930673, HHSN261200800059C from the Small Business Innovation Research Development Center (SBIR) of the National Cancer Institute, the Terry C. Johnson Center for Basic Cancer Research at Kansas State University, Kansas State University Targeted Excellence, Kansas State Legislative Appropriation, and Kansas Agricultural Experiment Station Kansas Bioscience Authority.

•At the concentrations used here, MNPs cause minimal toxicity on NPCs and Mo/Ma; loading of MNPs into cells is concentration dependent.•MNP-loaded NPCs and Mo/Ma can increase the temperature of tumors after AMF exposure.•NPCs can migrate to melanoma tumors after intravenous transplantation.•Mo/Ma can migrate to pancreatic tumors after intraperitoneal transplantation.•AMF exposure resulted in significant regression of melanoma tumor size for a short period of time.•AMF exposure resulted in a significant increase in survival of mice bearing pancreatic tumors after delivery of MNPs by Mo/Ma (average increase: 7 days).

•problem in cancer therapy: metastatic tumors that are smaller than clinically detectable level; inoperable tumors•Therapies that can specifically target to tumors can solve the problem.•Using iron nanoparticles (MNPs) to absorb alternating magnetic field (AMF) energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment.•MNPs were injected directly into the tumor for magnetic hyperthermia treatment in most of the studies reported in the literature.•Several research groups tried to target MNPs to tumors by attaching antibodies recognizing tumor-specific epitopes or peptides binding receptors on tumor cells or neovasculature.•Immune cells and stem cells can migrate towards tumors; using these cells as delivery vehicles for MNPs may help to achieve tumor-targeted magnetic hyperthermia.