Iron-dextran as a thermosensitizer in radiofrequency hyperthermia for cancer treatment
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Radiofrequency hyperthermia is a recently rediscovered oncotherapy rising in popularity. However, lack of a proper thermosensitizer limits current radiofrequency hyperthermia to be only slightly effective, mostly being used as a subsidiary to a standard oncotherapy. Here, we report that iron-dextran delivers iron ion to cancer cells for cancer-selective accumulation of the iron ion, which functions as a thermosensitizer for radiofrequency hyperthermia. Intravenous injection of iron-dextran to tumor-xenografted mice resulted in selective accumulation of iron ion in the targeted cancer cells. The accumulated iron ion in cancer cells dramatically reacted to radiofrequency wave to result in tumor-selective dielectric temperature increment without harming the surrounding normal tissue. The oncotherapeutic effect of was evaluated using tumor-xenografted mice. The overall anticancer efficacy of radiofrequency hyperthermia after injection of iron-dextran as a thermosensitizer in breast cancer-bearing mice was much better than the efficacy of paclitaxel, a standard chemotherapy drug for cancer. Moreover, hyperthermia using iron-dextran as a thermosensitizer completely eradicated cancer in the tumor xenografted mice. This work suggests that iron-dextran is an ideal thermosensitizer for radiofrequency hyperthermia. We believe that the application of iron-dextran as a thermosensitizer would be a major progress in hyperthermia cancer treatments.
KeywordsCancer Hyperthermia Iron-dextran Radiofrequency Thermosensitizer
Treatment of cancer by temperature elevation has been developed from ancient time to modern medicine. The thermal cancer treatment in modern medicine is achieved by ablation and hyperthermia. Ablation uses high heat, typically > 46 °C, to burn cancer tissue, in which laser lights are used. While, hyperthermia uses moderate temperature ranging from 39 to 46 °C. The purpose of hyperthermia is to increase temperature of interbody cancer to induce death of cancer cells either by apoptosis, necrosis or both.
One of the key characteristics of cancer cells is uncontrolled cell division. The uncontrolled cell division of cancer cells leads to the formation of an unorganized piling of cell mass which makes it impossible to properly develop neovascularization as in the case of normal tissues of the body . Thus, cancer tissues are limited of blood circulation, leading to a constant hypoxic condition where glycolysis is facilitated to acidify the environment . In addition to excessive glycolysis, poor blood circulation also makes cancer tissues vulnerable to high temperature. Unlike normal tissues, the excessive heat in the tumor tissues is difficult to be diffused into other parts of body via blood flow . Based on such characteristics of cancer tissues, hyperthermic oncotherapies, in which cancer tissue is heated to high temperatures, were developed (Fig. 2a, b). Although the concept of hyperthermic oncotherapies was developed several decades ago, the therapeutic efficacy and benefit of hyperthermic oncotherapies have not noticed until recently . Hyperthermic oncotherapies have reasonable therapeutic efficacy with almost no side effect so as to be getting popularity currently. Hyperthermic oncotherapies especially in combination with radiotherapy showed excellent oncotherapeutic efficacy.
Elevation of the temperature in cancer can be achieved by various methods such as radiofrequency (RF), vibration of metal nanoparticles in alternative magnetic field, metal nanoparticle in an electromagnetic wave, pyrogenic chemicals, etc. Among these hyperthermal methods, magnetic or metal nanoparticles has got an attention in hyperthermal community and thus most thoroughly investigated so that some of these magnetic or metal nanoparticles even investigated in clinical trials . Although magnetic or metal nanoparticles showed some efficacy, the insoluble nature of the magnetic or metal nanoparticles raised some concern .
Metal ions are known to interact well with electromagnetic waves . Especially, interaction of metal ions which has a very strong dipole moment such as iron ion generates strong dielectric heat when radiated by RF wave . Because of this characteristic, iron ion could be an excellent thermosensitizer in RF thermotherapy if iron ion can be delivered specifically to cancer cells. Considering the fact that X-ray and red light requires sensitizers for an effective oncotherapy, it would be reasonable to speculate that the efficacy of RF thermotherapy could be significantly improved when paired with a sensitizing reagent (Additional file 1: Table S1). To function as a tumor-targeting thermosensitizer, iron ion has to be accumulated more selectively in cancer cells than in normal cells. Iron-dextran, a medication for anemia, is a complex material in which iron ion form a complex with dextran through intermolecular bonding . Because dextran has very strong tumor-targeting characteristics , we speculated whether the injection of iron-dextran would result in accumulation of iron ion in cancer cells. If iron-dextran is able to selectively deliver iron ion to cancer cells, iron-dextran would be an ideal thermosensitizer for RF thermotherapy. In this work, we showed that iron-dextran specifically delivered iron ion to cancer cells and thereby selectively increased the temperature of cancer tissue upon RF hyperthermic treatment. Our results confirmed successful eradication of cancer in a RF thermotherapy of tumor-xenografted animals when iron-dextran was used as a thermosensitizer, making iron-dextran a novel thermosensitizer candidate for RF thermotherapy to cure cancer.
Materials and methods
Cell lines and cell culture
Animal study condition
The protocol was approved by the Ethics Committee of Chonbuk National University Laboratory Animal Center (Permit Number: CBU 2012-0040) in accordance with the ‘Guide for the Care and Use of Laboratory Animals’, published by the National Research Council and endorsed by the ARRIVE Guidelines. The animal care protocol was followed as previously described [11, 12, 13, 14].
Study of generating heat by iron-dextran in vitro
To evaluate the thermal performance of dextran and iron-dextran (Sigma-Aldrich, St. Louis, Missouri, USA), 0.1 mL of 5 mg/mL dextran or iron-dextran solutions were added onto 96-well plates in triplicate. After that, it was exposed for 10 min by the electromagnetic wave-dependent hyperthermia (LAB-EHY 100; Oncotherm, Budaörs, Hungary) at 50 W energy dose. The evaluation of heat capacity of solutions used by the thermal imaging camera (FLIR-E60; FLIR, Wilsonville, Oregon, USA) and the temperature of solutions measured by DirA Program (FLIR Tools; FLIR, Wilsonville, Oregon, USA). The iron-dextran in vitro condition protocol was followed as previously described . The detailed protocol for iron-dextran in vitro condition is described in the Additional file 1. All data are presented as the mean ± SD and were compared using paired Student’s t-tests. P value < 0.05 was considered as the statistical significance level.
Study of in vivo heat generation after iron-dextran injection
Two hundred μL of NCI-H460-luc2 (5 × 106) cells in saline solution was injected into the subcutaneous region of female nude mice with a BALB/c genetic background to generate cancer-bearing mice. Cancer growth was monitored daily. When the tumors reached a size of ~ 100 mm3, the 24 cancer-bearing mice were grouped into 4 groups with 6 mice per group. Two cancer-bearing mice groups were i.v. injected with iron-dextran solution (10 mg/kg/day), and the other two groups were i.v. injected with dextran solution (10 mg/kg/day). Two mouse groups of iron-dextran and dextran were used for local hyperthermia experiment and the other two groups were used for whole-body hyperthermia. The iron-dextran in vivo condition protocol was followed as previously described . The detailed protocol for iron-dextran in vitro condition and temperature measurement method on cancer and normal subcutaneous areas are described Additional file 1.
Organ distribution of iron ion concentration after iron-dextran injection
The organs (liver, heart, lung, kidney, muscle, brain, stomach, and tumor) distribution of iron ion were measured in tumor-bearing mice. A small piece (~ 1 g) of each organ including tumor was isolated from the cancer-bearing mice after injections of dextran or iron-dextran. The iron ion concentrations of each organ were measured by an Inductively Coupled Plasma Mass Spectrometry (Varian 800-MS; Varian Medical Systems, Palo Alto, CA, USA). The preprocessing protocol for measurement on iron ion concentration was followed as previously described .
The anti-tumor efficacy of iron-dextran in vivo
The anti-tumor efficacy of iron-dextran in vivo protocol was followed as previously described . An IVIS imaging system (PerkinElmer, Waltham, MA, USA) was used to generate a bioluminescent image. The detailed preprocessing protocol for measuring in vivo imaging is described Additional file 1.
Histology of tumor tissue
The tumor tissue specimens from the cancer-bearing mice of the experimental groups were surgically isolated. After tumor tissues were fixed at 10% neutral-buffered formalin (Sigma Aldrich, St. Louis, MO, USA), followed by serial sectsion (5 μm) of tumor tissue specimens were cut and stained with hematoxyline and eosin (H&E) as previously described [13, 14]. The stained tissue images were scanned by using a slide scanner, Aperio Scanscope FL (Leica Biosystems, Wetzlar, Germany), and the scanned image was processed by ImageScope Software (Aperio Technologies, Vista, CA, USA).
Iron-dextran generated dielectric heat in RF wave
In addition to generation of dielectric heat, dextran is known to have cancer-targeting characteristics . In order to test whether iron-dextran can selectively deliver iron to cancer cells, cancer and normal cells were exposed to iron-dextran, and heat generation was compared upon treating with RF thermotherapy (Fig. 1a). The exposure of cancer cells to a medium containing iron-dextran boosted temperature elevation in a dose-dependent manner in contrast to normal primary cells (Fig. 1b, Additional file 1: Fig. S1). Overall, these experiments indicated that cancer cells selectively accumulated iron ion after iron-dextran treatment to react with RF wave and induce dielectric heat more strongly. This result suggests that iron-dextran could be a possible candidate for a safe and effective thermosensitizer for RF thermotherapy.
Application of iron-dextran as a thermosensitizer in RF hyperthermia
RF thermotherapy is a hyperthermal therapy that is mostly used as a subsidiary treatment to standard cancer therapies in modern medicine. Even though RF thermotherapy is used as a subsidiary treatment, RF thermotherapy is getting popularity in present clinics because of its safety and a modest efficacy. However, ironically, the main obstacle of RF thermotherapy in cancer treatments is the limitation on temperature elevation. The RF thermotherapy cannot selectively elevate the temperature in cancer tissues, limiting elevation of the temperature of cancer tissues to the safe range of 40–42 °C in which surrounding normal tissues are not damaged. Obviously, such a range of 40–42 °C temperature is not enough to kill cancer cells, instead only causing a few cancer cells to undergo apoptosis. The range of elevated temperature might explain a limit on oncotherapeutic efficacy of current RF thermotherapy.
RF thermotherapy kills cancer cells through apoptosis. It should be noted that this is a different kind of cell death than necrosis which was observed when i.v. injection of iron-dextran was used as a thermosensitizer in RF thermotherapy (Fig. 4c). Necrosis is the premature cell death, which is caused by infection, toxins, trauma, or high heat. Unlike apoptosis, which is triggered by gentle external factor, necrosis is triggered by harsh external factors such as high heat. RF thermotherapy alone elevates the temperature of cancer cells up to 40–42 °C at which only a few cells die through apoptosis. Considering the range of elevated temperature of current RF thermotherapy, it is unrealistic to expect that the current RF thermotherapy is a reasonable choice for an efficient cancer treatment. This work shows that the application of iron-dextran as a thermosensitizer in RF thermotherapy could selectively elevate temperature in cancer tissues up to 47 °C with minimal damage to normal tissues, which cannot be achieved by current RF thermotherapy. It should be noted that, however, IR thermography measurement used in this study has a limit in representing the defined temperatures in heterogeneous curved tumor tissue although it is one of most widely used method for thermotherapy. Consequently, it should be considered as the mean numbers representing 25 spots randomly chosen by program in tumor and normal tissues for comparative analysis.
Dextran is a complex branched polysaccharide made of various lengths of glucose residues, which is manufactured from bacterial fermentation . Because of its higher molecular weight, biocompatibility, biodegradability and hydrophilic nature, dextran has been used in various pharmaceutical purposes such as plasma volume expanders, anticoagulants, drug carriers, a targeting moiety of nanoparticles for cancer, etc. [10, 19]. Especially, the large molecular weight as well as other physical characteristics of dextran made it ideal for tumor-targeting through enhanced and the enhanced permeability and retention effect. Also, glucose itself tends to be drawn by cancerous tissue. Because of these two reasons, dextran is used as one of the favored targeting moieties of current nanoparticles for cancer [20, 21]. In an agreement with those previously known feature of dextran, our experimental data clearly confirmed the cancer-targeting nature of dextran. Because of the excellent cancer-targeting ability, injection of iron-dextran make iron ion targeted into cancer, which resulted in selective accumulation of iron ion in the cancer tissue (Additional file 1: Table S2, Fig. S2).
In this work, we show that i.v. injection of iron-dextran into cancer-bearing mice selectively delivers iron ion to cancer cells, resulting cancer-specific accumulation of iron ion. The accumulated iron ions dramatically boost the cancer-specific dielectric heating in RF thermotherapy, selectively elevating the temperature of cancer cells strong enough to cause necrosis without damaging normal tissues so as to eradicate cancer. Iron-dextran is one of the safest drugs currently used for treating anemia . These characteristics make iron-dextran an ideal choice for RF thermotherapy as a thermosensitizer. Considering current oncotherapeutic efficacy of RF hyperthermia even without a thermosensitizer as well as the oncotherapeutic efficacy showed in this work, the development of iron-dextran as a thermosensitizer in RF hyperthermia should be a major progress in cancer treatment.
HJC and STH analyzed the data and wrote the paper. STH provided technical assistance to HJC and helped with the data collection. HJK edited the manuscript. STH supervised the work. STH and HJC revised and edited the manuscript and supervised the work. All authors read and approved the final manuscript.
This research was supported by the BDRD Research Fund from JINIS Biopharmaceutical Co., Republic of Korea.
The authors declare that they have no competing interests.
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- 4.Roussakow S (2013) The history of hyperthermia rise and decline. Conf Papers Med 2013:140Google Scholar
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