Mol Imaging Biol (2015) DOI: 10.1007/s11307-015-0848-2 * World Molecular Imaging Society, 2015
BRIEF ARTICLE
Improved Hyperthermia Treatment of Tumors Under Consideration of Magnetic Nanoparticle Distribution Using Micro-CT Imaging H. Dähring, J. Grandke, U. Teichgräber, I. Hilger Institute for Diagnostic and Interventional Radiology, Jena University Hospital–Friedrich Schiller University Jena, Bachstraße 18, 07740, Jena, Germany
Abstract Purpose: Heterogeneous magnetic nanoparticle (MNP) distributions within tumors can cause regions of temperature under dosage and reduce the therapeutic efficiency. Here, microcomputed tomography (CT) imaging was used as a tool to determine the MNP distribution in vivo. The therapeutic success was evaluated based on tumor volume and temperature distribution. Procedures: Tumor-bearing mice were intratumorally injected with iron oxide particles. MNP distribution was assessed by micro-CT with a low radiation dose protocol. Results: MNPs were clearly visible, and the exact distribution to nontumor structures was detected by micro-CT. Knowledge of the intratumoral MNP distribution allowed the generation of higher temperatures within the tumor and led to higher temperature values after exposure to an alternating magnetic field (AMF). Consequently, the tumor size after 28 days was reduced to 14 and 73 % of the initial tumor volume for the MNP/AMF/CT and MNP/AMF groups, respectively. Conclusions: The MNP distribution pattern mainly governed the generated temperature spots in the tumor. Knowing the MNP distribution enabled individualized hyperthermia treatment and improved the overall therapeutic efficiency. Key words: Micro-CT imaging, Iron oxide nanoparticles, Magnetic fluid hyperthermia, In vivo, Therapeutic efficiency, Tumor volume, Temperature distribution, Intratumoral MNP distributon
Introduction
C
omputed tomography (CT) is known to be a powerful tool to differentiate between bones and surrounding tissue within body structures. By CT imaging, a three-dimensional (3D) image of the specimen is obtained with a high spatial resolution of the organs. In particular cases, it might be useful to detect iron oxide deposits in the body. For example, high concentrations of iron oxide magnetic nanoparticles (MNPs) are needed for efficient magnetic hyperthermia, a promising
Correspondence to: I. Hilger; e-mail: [email protected]
minimally invasive method for tumor therapy; therefore, the sensitive representation of anatomical structures in combination with the soft tissue contrast allows the noninvasive detection of high MNP doses [1]. Such high MNP concentrations are prone to induce severe susceptibility artifacts in magnetic resonance imaging (MRI). MRI has potentially a better soft tissue contrast [2]; however, these artifacts impair adequate 3D image interpretation. Therefore, the short acquisition time of micro-CT imaging combined with a low radiation dose protocol is a capable tool for the 3D localization of MNP in tumor structures in vivo. Magnetic fluid hyperthermia (MFH) generates thermal energy after deposition of MNPs within tumor tissue and consecutive exposure of the tissue to an alternating magnetic field (AMF) [3, 4]. This procedure allows a localized heating of a small regions of interest (ROI), while the surrounding
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
nontumor tissue is preserved leading to an effective inactivation and/or killing of the tumor cells [4, 5]. In cancer treatment, hyperthermic temperatures between 41 and 46 °C which induce apoptosis, are desired compared to thermo-ablative temperatures above 46 °C which induce unwanted necrosis [4, 5]. The hyperthermic effects are not only dependent on the applied temperature but also on the duration of the treatment [6]. T90 values are the temperatures achieved within 90 % of the tumor area whereas CEM43T90, the cumulative equivalent minutes at a temperature of 43 °C within 90 % of the tumor, is a measure for the temperature dose [7, 8]. The CEM43T90 values for the most effective tumor treatment vary greatly: several studies report low temperature doses (10 CEM43T90) [9, 10], while other studies report promising effects at higher ones (10–100 CEM43T90) [11, 12]. A crucial parameter for the success of intratumorally applied MNP for MFH is the temperature distribution [13]. Therefore, the occurrence of heterogeneous distributions of the MNPs in the tumor area makes it difficult to maintain therapeutically effective temperatures over the whole treatment region. In consequence, regions of temperature under dosage within the tumor might cause insufficient cell death. Therefore, rather homogeneous temperature distributions are desired [14, 15]. Additionally, the placement of the thermal sensors as well as the monitoring of the temperature distribution throughout the MFH is essential. In this context, using CT imaging as an instrument to detect the exact three-dimensional location of MNPs to avoid a destruction of vital nontumor tissues was shown exemplarily for glioblastoma [16, 17]. Nanoparticle distributions and heat generation in gels were correlated before using mathematical algorithms. Also, nanoparticle distributions from tumor that were excised from mice and imaged ex vivo with high-resolution CT [18–20]. However, this approach is not suitable within living animals and is far from clinical translation because of the high radiation dose which has been applied. The goal of this study was to modulate the hyperthermia treatment depending on the position of the MNP, which were determined by micro-CT imaging prior to hyperthermia in vivo. Here, we used micro-CT imaging with a low radiation protocol as an effective tool to increase the efficiency of hyperthermia by monitoring the temperature noninvasively and individually adapting the therapeutic temperatures within the tumor throughout the treatment. We compared the therapeutic outcome of mice after 28 days treated with and without the knowledge of the intratumoral MNP distribution to achieve ideal CEM43T90 values for our breast cancer model.
Material and Methods Xenograft Models and Tumor Implantation All experiments were approved by the regional animal care committee and were in accordance with international guidelines on the ethical use of animals (02-068/11) and carried out under anesthesia with 2.0 % isoflurane as previously described (Aktavis,
Germany) [21]. Briefly, 7- to 8-week-old female immunodeficient nude mice (Harlan Laboratories, The Netherlands) were subcutaneously implanted with human breast adenocarcinoma (2×106 MDA-MB-231 cells (ATCC, Germany)) in 120-μl BD Matrigel™ BMM (BD Biosciences, USA) on their rear back sides. The experiments were started after 48 days of tumor growth, and the tumor volumes were between 70 and 300 mm3.
Treatment Groups Four independent mice treatment groups were formed. The first group (n=3) received MNP, which was exposed to an alternating magnetic field (AMF), and the mice were treated with the knowledge of the nanoparticle distribution by micro-CT imaging BMNP/AMF/CT^. The second group (n=3) received MNP and was exposed to an AMF without the information of the MNP distribution BMNP/AMF^. The third group (n=7) received MNP as well but without exposure to an AMF to investigate the impact of MNP on the tumor volume without heating, and the intratumoral MNP distribution was measured by micro-CT imaging BMNP/CT^. The fourth group (n=6) received no MNP, instead aqua bidest (ddH2O) was injected to monitor the untreated tumor growth and the absence of intratumoral MNPs was confirmed by micro-CT imaging BddH2O/CT^.
Application of MNP and Assessment of MNP Distribution by Micro-CT Imaging One day prior to AMF exposure, the xenograft bearing mice of groups MNP/AMF/CT, MNP/AMF/CT, and MNP/CT were injected intratumorally with 0.24 mg Fe/100 mm3 (MF66: iron oxide, DMSA coated, core diameter 11.7 nm, Liquids Research Limited, UK). The ddH2O/CT group was injected with the same volume of ddH2O relative to the positive group. The mice of the treatment groups MNP/AMF/CT, MNP/CT, and ddH2O/CT were measured via a micro-CT Scanner (TomoScope Synergy Twin, CTImaging, Germany). Measurements were carried out directly after MNP or ddH2O application as well as 7 and 28 days after MNP application to assess the localization and distribution of the MNP within the tumor as well as changes over time. Here, a low radiation dose protocol was used, and spatial resolutions of 114 µm were reached. The ImpactCB acquisition software was used with an acquisition time of 29 s, and the tube voltage was set to 65 kV for both tubes. The image reconstruction was performed with the Impact Recon software. The 3D micro-CT data were analyzed using the Imalytics Research Software (Philips Technologie GmbH, Germany). For the CT images, the following segmentation parameters were chosen after subtraction of an instrument-specific value: the intensity for bones (white) and nanoparticles (red) was set above 376 Hounsfield Units (HU) whereas skin (transparent green) was set between −424 and 176 HU. The animals were exposed to a radiation dose of approximately 500 mGy during the three CT scans with the scanning protocol described above.
Magnetic Fluid Hyperthermia Treatment In brief, 24 h after MNP application, all MNP/AMF/CT and MNP/ AMF mice were exposed twice to an AMF (H=19 mT, f=435 kHz,
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
60 min) within a 7-day interval as described before [21, 22]. Throughout the therapy, temperatures were monitored using an infrared thermography camera (InfRec R300, Nippon Avionics Co., Japan) with fiber optic temperature sensors (TS5 and FOTEMPMK-19, Optocon AG, Germany). The desired temperatures were achieved by individually adapting the power of the AMF generator by the following means: (a) without knowledge of the intratumoral MNP distribution (MNP/AMF), 43 °C in the center of the tumor were used in order to minimize organ damage. (b) With knowledge of the intratumoral MNP distribution (MNP/AMF/CT), it was possible to modulate the power of the AMF generator to reach 43 °C at the tumor border. This strategy led to low thermoablative temperatures in the center of the tumor. If the MNPs were in close vicinity (approximately 2 mm) to vital structures, the hyperthermic temperatures at the tumor border were reduced to 41 °C.
Tumor Volume and Temperature Distribution Within the 28-day period, the tumor volume and the body weight of animals were measured every 3 days. The tumor volume was measured with a caliper and calculated according to the following formula: [23]. The tumor volumes immeadiately before treatment were set to 100 %, and the tumor volume for each animal for each timepoint was normalized to the value before treatment. Then, the mean relative tumor volume was calculated for each treatment group. Data analysis was performed using a t-test for independent samples with SPSS software (IBM SPSS Statistics 20, USA), and the significance level was set to p≤0.01. In order to determine the applied temperature dosages, the tumor surface temperatures of the thermal images taken during MFH were analyzed 10, 30, and 50 min after the beginning of the AMF treatment and the mean values determined. Here, a region of interest (ROI) was placed around each tumor, and the temperature data per pixel was extracted; hence, T90 temperatures and CEM43T90 values were calculated according to Sapareto and Dewey [7]. Additionally, temperature data of each animal were grouped into three categories: temperatures without hyperthermic effect (G43 °C), hyperthermic
temperatures (43–45 °C), and low thermo-ablative temperatures (945 °C) and their relative percentage calculated for each MFH treatment and plotted using Origin® 9.1 (Origin Lab, USA) software.
Results and Discussion Micro-CT Imaging The 3D MNP distribution within tumor and nontumor structures were detected by micro-CT imaging before the first (−1 day) and second (6 days) hyperthermia treatment as well as at the end (27 days) of the treamtent in order to localize their specific position (Fig. 1). In all cases, a heterogeneous iron oxide MNP distribution was clearly detectable as distinct red regions around the outer rim of the tumor tissue independent of the tumor size, since the same scanning protocol with a spatial resolution of 114 μm was used throughout all measurements (MNP/AMF/CT and MNP/CT). Moreover, after the first AMF treatment, a compression of MNP deposits in the tumor was observed for the MNP/AMF/CT and MNP/CT groups. The spatial resolution of the CT system will be the limiting factor with consideration of clinical applications. Therefore, the dimensions of the tumor have to be significantly larger than 0.5 mm based on the resolution of most clinical scanners (e.g., 0.5–0.6 mm) in order to detect the MNPs within the tumor. In regard to the cytotoxicity of the radiation from microCT imaging, we did not observe any significant changes within the blood composition (white blood cells, red blood cells, and amount of hemoglobin) and body weight of the animals that were imaged. Therefore, we assume that the effective dose is below the threshold and do not expect that the radiation dose from micro-CT imaging influences the outcome of this study.
Fig. 1 The tumor volume of the MNP/AMF/CT mice was significantly reduced after 28 days. The distribution and localization of the MNPs within the tumor and nontumor structures were monitored by micro-CT imaging over time (−1, 6, 27 days) in all groups (MNP/AMF/CT [dark green], MNP/CT [blue], and ddH2O/CT [orange], n=3). Each image per treatment group and day consists of three information: first, the tumor morphology is displayed; second, the morphology as CT image; and third, the segmented CT image. Within the segmented CT images, the threshold for the segmentation of bone (white) and MNP (red) was set above 376 HU and of skin (transparent green) within the range of −424 to 176 HU.
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
Fig. 2 Improved therapeutic efficiency after CT-imaging based on the parameters temperature distribution and MNP distribution. a The intratumoral MNP distribution is shown exemplarily for one mouse per treatment group at days −1, 6, and 27: segmented CT image (if applicable, right), the tumor morphology (left). b, c The temperature distribution at 90 % of the tumor surface (in percentage) is plotted for each animal after the first (striped bars) and second hyperthermia treatment (plain colored bars) for the b MNP/AMF/CT and c MNP/AMF groups.
Temperature Distribution in Tumors with and without Micro-CT Imaging A tumor growth reduction in combination with a compression of the MNP regions at the end of the therapy was found for the mice from the MNP/AMF/CT group as well (Fig. 2a). However, this was not the case for mice of the MNP/AMF group where the exact localization of the MNPs was not determined. The temperature distribution profiles for both treatment groups were generated from the infrared thermography data for the first [striped bars] and second AMF [plain colored bars] treatment for
all mice (Fig. 2b, c). The knowledge of the intratumoral MNP distribution pattern by micro-CT imaging led to the generation of adequate temperature profiles (i.e., higher temperatures) within the tumor for the first and the second hyperthermia treatment for the MNP/AMF/CT group. This enabled a better therapeutic outcome for the MNP/AMF/CT group of the three treated mice with 12, 13, and 19 % of the relative tumor volume at day 28 compared to 58, 79, and 81 % for the MNP/AMF group. This is also reflected in the higher T90 values for both treatments with 43.3 and 39.1 °C as well as 40.0 and 36.3 °C for the MNP/AMF/ CT as well as the MNP/AMF groups, respectively, when the
Table 1. Characteristic parameters of the therapeutic effects after in vivo magnetic fluid hyperthermia (MFH) MDA-MB-231 MNP/AMF/CT 3
Applied magnetic material [mg Fe/100 mm ] T90 [°C] CEM43T90 [min] Tumor volume growth rate VTMFH-28d/VTMFH-0d VTMFH-28d/VT28d
1st MFH 2nd MFH
0.24 43.3 39.1 77.6 4.8 %/day 14 % 4.4 %
MNP/AMF 40.0 36.3 2.2 73 % 24 %
A total of 0.24 mg Fe/100 mm3 magnetic material was applied for the breast cancer xenograft MDA-MB-231. The temperature within 90 % of the tumor surface (T90) after the first and second MFH treatment as well as the cumulative equivalent minutes at 43 °C (CEM43T90) are displayed for the MNP/AMF/ CT and MNP/AMF groups. The mean relative tumor growth rates in days as well as the mean tumor volumes (in percentage) compared to the tumor volume at day 0 (VT MFH-28d/VT MFH-0d) or to the untreated control after 28 days (VT MFH-28d/VT 28d) are compared for both groups
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
Fig. 3 Magnetic hyperthermia treatment with micro-CT imaging led to a significant reduction of the tumor volume compared to the control groups. a Micro-CT imaging, with exception of the MNP/AMF group, was used to determined the MNP distribution within the tumors of the mice over time. Per treatment group, one mouse is shown exemplarily with its respective intratumoral MNP distribution in the segmented CT image (if applicable, right) and the tumor morphology (left). b The tumor volume was monitored over time and calculated relative to the tumor volume at day 0 for all treatment groups. The asterisk symbolizes a significant statistical difference of p≤0.01.
same amount (0.24 mg Fe per 100 mm3) of magnetic iron oxide MNPs were applied (Table 1). Additionally, the mean CEM43T90 values of approximately 78 min for the MNP/ AMF/CT group indicate that the critical hyperthermia temperature threshold of 43 °C for 60 min was reached, when the exact MNP distribution was determined, compared to only 2.2 min for the MNP/AMF group. Without the knowledge of the MNP distribution, modulation of temperatures at the tumor borders and surrounding tissue is challenging. All in all, the tumor reduction by means of the mean VT MFH-28d/VT MFH-0d for MNP/
AMF/CT group is with 13.5 % significantly smaller than the mean VT MFH-28d/VT MFH-0d for MNP/AMF group with 72.6 % (p≤0.01).
Therapeutic Effects of In Vivo Magnetic Fluid Hyperthermia with Micro-CT Imaging The mean relative tumor volumes after 28 days were determined to be 14 % for MNP/AMF/CT, 73 % for MNP/
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
AMF, 224 % for MNP/CT, and 305 % for ddH2O/CT compared to the tumor volumes at 0 day (Fig. 3). This equals a relative tumor volume of 4.4 and 23.8 % compared to the ddH2O control after 28 days for MNP/AMF/CT and MNP/AMF, respectively. The application of MNP alone had no effect on tumor growth compared to the ddH2O/CT control group. The tumor volume was significantly reduced for both AMF treatment groups, MNP/AMF/CT and MNP/ AMF, compared to the control groups MNP/CT and ddH2O/ CT. However, the usage of micro-CT imaging even further improved the overall outcome of the hyperthermia treatment, as higher temperatures within the critical tumor tissue were achieved and consequently higher CEM43T90 values. Morphologically, the best results were obtained for the MNP/AMF/CT group with almost complete tumor regression in most cases. Insufficient tumor reduction led to regrowth around the border of tumor for the MNP/AMF group. Opposing views on the ideal CEM43T90 values exist. Here, the best therapeutic success on the basis of the parameter tumor volume reduction and temperature distribution were achieved for higher temperature dosages as proposed by Thrall et al. [12]. We have shown in our study an increased therapeutic effect of MFH using micro-CT imaging. A heterogeneous MNP distribution is difficult to circumvent during intratumoral MNP application, but the exact localization of the iron oxide MNPs can easily be detected by micro-CT imaging, and this knowledge can be used for an efficient hyperthermia therapy. By micro-CT imaging, insufficient amounts of MNP can be detected, and an efficient hyperthermia treatment can be achieved by a repeated MNP application.
Conclusions Micro-CT imaging is capable of depicting intratumorally applied MNP amounts prior to the hyperthermia in vivo and consequently allows a modulation of the generated temperatures within the tumor tissue based on the exact localization. A significant tumor reduction and subsequently a higher therapeutic success of the MFH were achieved with knowledge of the intratumoral MNP distribution. Furthermore, the surrounding nontumor tissue was preserved while effectively treating breast cancer. Micro-CT is thus an important tool for the qualitative determination of the particle distribution and enables a localized noninvasive hyperthermia treatment. Thereby, the hyperthermia treatment in our breast cancer model was distinctly improved based on the parameters tumor volume and the temperature distribution. CT imaging for improved therapeutic success is potentially translatable into clinical practices, if the tumor size is larger than the resolution of the instrument and might also be used for personalized hyperthermia therapy. Acknowledgments. The present work was carried out within the project BMultifunctional Nanoparticles for the Selective Detection and Treatment of
Cancer,^ funded by the European Seventh Framework Program (FP7/20072013) under grant agreement no. 262943. We gratefully acknowledge Liquids Research Limited (UK) for providing the magnetic material. The micro-CT instrument was funded by the Federal State Thuringia and the European Union (EFRE; European Fond for regional development). Conflict of Interests. The authors declare that they have no conflict of interest. Ethical Approval. All applicable institutional and/or national guidelines for the care and use of animals were followed.
References 1. Thomas R, Park IK, Jeong YY (2013) Magnetic iron oxide nanoparticles for multimodal imaging and therapy of cancer. Int J Mol Sci 14:15910–15930 2. Hilger I, Hofmann F, Reichenbach JR et al (2002) In vitro imaging of magnetite particles. RoFo : Fortschr Röntgenstr 174:101–103 3. Alvarez-Berrios MP, Castillo A, Rinaldi C, Torres-Lugo M (2014) Magnetic fluid hyperthermia enhances cytotoxicity of bortezomib in sensitive and resistant cancer cell lines. Int J Nanomedicine 9:145–153 4. Kumar CS, Mohammad F (2011) Magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery. Adv Drug Deliv Rev 63:789–808 5. Silva AC, Oliveira TR, Mamani JB et al (2011) Application of hyperthermia induced by superparamagnetic iron oxide nanoparticles in glioma treatment. Int J Nanomedicine 6:591–603 6. Field SB, Morris CC (1983) The relationship between heating time and temperature: its relevance to clinical hyperthermia. Radiother Oncol J Eur Soc Ther Radiol Oncol 1:179–186 7. Sapareto SA, Dewey WC (1984) Thermal dose determination in cancer therapy. Int J Radiat Oncol Biol Phys 10:787–800 8. Dewey WC (2009) Arrhenius relationships from the molecule and cell to the clinic. Int J Hyperther 25:3–20 9. de Bruijne M, van der Holt B, van Rhoon GC, van der Zee J (2010) Evaluation of CEM43 degrees CT90 thermal dose in superficial hyperthermia: a retrospective analysis. Strahlenther Onkol 186:436–443 10. Maguire PD, Samulski TV, Prosnitz LR et al (2001) A phase II trial testing the thermal dose parameter CEM43 degrees T90 as a predictor of response in soft tissue sarcomas treated with pre-operative thermoradiotherapy. Int J Hyperth 17:283–290 11. Jones EL, Oleson JR, Prosnitz LR et al (2005) Randomized trial of hyperthermia and radiation for superficial tumors. J Clin Oncol 23:3079–3085 12. Thrall DE, LaRue SM, Yu D et al (2005) Thermal dose is related to duration of local control in canine sarcomas treated with thermoradiotherapy. Clin Cancer Res 11:5206–5214 13. Perez CA, Sapareto SA (1984) Thermal dose expression in clinical hyperthermia and correlation with tumor response/control. Cancer Res 44:4818s–4825s 14. Van Vulpen M, De Leeuw AA, Van De Kamer JB et al (2003) Comparison of intra-luminal versus intra-tumoural temperature measurements in patients with locally advanced prostate cancer treated with the coaxial TEM system: report of a feasibility study. Int J Hyperth 19:481–497 15. Van Vulpen M, De Leeuw AA, Raaymakers BW et al (2004) Radiotherapy and hyperthermia in the treatment of patients with locally advanced prostate cancer: preliminary results. BJU Int 93:36–41 16. Maier-Hauff K, Rothe R, Scholz R et al (2007) Intracranial thermotherapy using magnetic nanoparticles combined with external beam radiotherapy: results of a feasibility study on patients with glioblastoma multiforme. J Neuro-Oncol 81:53–60 17. Jordan A (2009) Hyperthermia classic commentary: ‘Inductive heating of ferrimagnetic particles and magnetic fluids: Physical evaluation of their potential for hyperthermia’ by Andreas Jordan et al., International Journal of Hyperthermia, 1993;9:51-68. Int J Hyperth 25:512–516 18. Attaluri A, Ma RH, Qiu Y, Li W, Zhu L (2011) Nanoparticle distribution and temperature elevations in prostatic tumours in mice during magnetic nanoparticle hyperthermia. Int J Hyperth 27:491–502 19. Attaluri A, Ma RH, Zhu LA (2011) Using MicroCT imaging technique to quantify heat generation distribution induced by magnetic nanoparticles for cancer treatments. J Heat Transfer 133(1):011003–5.
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
20. LeBrun A, Manuchehrabadi N, Attaluri A, Wang F, Ma RH, Zhu L (2013) MicroCT image-generated tumour geometry and SAR distribution for tumour temperature elevation simulations in magnetic nanoparticle hyperthermia. Int J Hyperth 29:730– 738 21. Kossatz S, Ludwig R, Dähring H et al (2014) High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature dosages in the tumor area. Pharm Res 31:3274–3288
22. Kossatz S, Grandke J, Couleaud P et al (2015) Efficient treatment of breast cancer xenografts with multi-functionalized iron oxide nanoparticles combining mag-netic hyperthermia and anti-cancer drug delivery. Breast Cancer Research, under review 23. Hilger I, Andra W, Hergt R, Hiergeist R, Schubert H, Kaiser WA (2001) Electromagnetic heating of breast tumors in interventional radiology: in vitro and in vivo studies in human cadavers and mice. Radiology 218:570–575
BRIEF ARTICLE
Improved Hyperthermia Treatment of Tumors Under Consideration of Magnetic Nanoparticle Distribution Using Micro-CT Imaging H. Dähring, J. Grandke, U. Teichgräber, I. Hilger Institute for Diagnostic and Interventional Radiology, Jena University Hospital–Friedrich Schiller University Jena, Bachstraße 18, 07740, Jena, Germany
Abstract Purpose: Heterogeneous magnetic nanoparticle (MNP) distributions within tumors can cause regions of temperature under dosage and reduce the therapeutic efficiency. Here, microcomputed tomography (CT) imaging was used as a tool to determine the MNP distribution in vivo. The therapeutic success was evaluated based on tumor volume and temperature distribution. Procedures: Tumor-bearing mice were intratumorally injected with iron oxide particles. MNP distribution was assessed by micro-CT with a low radiation dose protocol. Results: MNPs were clearly visible, and the exact distribution to nontumor structures was detected by micro-CT. Knowledge of the intratumoral MNP distribution allowed the generation of higher temperatures within the tumor and led to higher temperature values after exposure to an alternating magnetic field (AMF). Consequently, the tumor size after 28 days was reduced to 14 and 73 % of the initial tumor volume for the MNP/AMF/CT and MNP/AMF groups, respectively. Conclusions: The MNP distribution pattern mainly governed the generated temperature spots in the tumor. Knowing the MNP distribution enabled individualized hyperthermia treatment and improved the overall therapeutic efficiency. Key words: Micro-CT imaging, Iron oxide nanoparticles, Magnetic fluid hyperthermia, In vivo, Therapeutic efficiency, Tumor volume, Temperature distribution, Intratumoral MNP distributon
Introduction
C
omputed tomography (CT) is known to be a powerful tool to differentiate between bones and surrounding tissue within body structures. By CT imaging, a three-dimensional (3D) image of the specimen is obtained with a high spatial resolution of the organs. In particular cases, it might be useful to detect iron oxide deposits in the body. For example, high concentrations of iron oxide magnetic nanoparticles (MNPs) are needed for efficient magnetic hyperthermia, a promising
Correspondence to: I. Hilger; e-mail: [email protected]
minimally invasive method for tumor therapy; therefore, the sensitive representation of anatomical structures in combination with the soft tissue contrast allows the noninvasive detection of high MNP doses [1]. Such high MNP concentrations are prone to induce severe susceptibility artifacts in magnetic resonance imaging (MRI). MRI has potentially a better soft tissue contrast [2]; however, these artifacts impair adequate 3D image interpretation. Therefore, the short acquisition time of micro-CT imaging combined with a low radiation dose protocol is a capable tool for the 3D localization of MNP in tumor structures in vivo. Magnetic fluid hyperthermia (MFH) generates thermal energy after deposition of MNPs within tumor tissue and consecutive exposure of the tissue to an alternating magnetic field (AMF) [3, 4]. This procedure allows a localized heating of a small regions of interest (ROI), while the surrounding
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
nontumor tissue is preserved leading to an effective inactivation and/or killing of the tumor cells [4, 5]. In cancer treatment, hyperthermic temperatures between 41 and 46 °C which induce apoptosis, are desired compared to thermo-ablative temperatures above 46 °C which induce unwanted necrosis [4, 5]. The hyperthermic effects are not only dependent on the applied temperature but also on the duration of the treatment [6]. T90 values are the temperatures achieved within 90 % of the tumor area whereas CEM43T90, the cumulative equivalent minutes at a temperature of 43 °C within 90 % of the tumor, is a measure for the temperature dose [7, 8]. The CEM43T90 values for the most effective tumor treatment vary greatly: several studies report low temperature doses (10 CEM43T90) [9, 10], while other studies report promising effects at higher ones (10–100 CEM43T90) [11, 12]. A crucial parameter for the success of intratumorally applied MNP for MFH is the temperature distribution [13]. Therefore, the occurrence of heterogeneous distributions of the MNPs in the tumor area makes it difficult to maintain therapeutically effective temperatures over the whole treatment region. In consequence, regions of temperature under dosage within the tumor might cause insufficient cell death. Therefore, rather homogeneous temperature distributions are desired [14, 15]. Additionally, the placement of the thermal sensors as well as the monitoring of the temperature distribution throughout the MFH is essential. In this context, using CT imaging as an instrument to detect the exact three-dimensional location of MNPs to avoid a destruction of vital nontumor tissues was shown exemplarily for glioblastoma [16, 17]. Nanoparticle distributions and heat generation in gels were correlated before using mathematical algorithms. Also, nanoparticle distributions from tumor that were excised from mice and imaged ex vivo with high-resolution CT [18–20]. However, this approach is not suitable within living animals and is far from clinical translation because of the high radiation dose which has been applied. The goal of this study was to modulate the hyperthermia treatment depending on the position of the MNP, which were determined by micro-CT imaging prior to hyperthermia in vivo. Here, we used micro-CT imaging with a low radiation protocol as an effective tool to increase the efficiency of hyperthermia by monitoring the temperature noninvasively and individually adapting the therapeutic temperatures within the tumor throughout the treatment. We compared the therapeutic outcome of mice after 28 days treated with and without the knowledge of the intratumoral MNP distribution to achieve ideal CEM43T90 values for our breast cancer model.
Material and Methods Xenograft Models and Tumor Implantation All experiments were approved by the regional animal care committee and were in accordance with international guidelines on the ethical use of animals (02-068/11) and carried out under anesthesia with 2.0 % isoflurane as previously described (Aktavis,
Germany) [21]. Briefly, 7- to 8-week-old female immunodeficient nude mice (Harlan Laboratories, The Netherlands) were subcutaneously implanted with human breast adenocarcinoma (2×106 MDA-MB-231 cells (ATCC, Germany)) in 120-μl BD Matrigel™ BMM (BD Biosciences, USA) on their rear back sides. The experiments were started after 48 days of tumor growth, and the tumor volumes were between 70 and 300 mm3.
Treatment Groups Four independent mice treatment groups were formed. The first group (n=3) received MNP, which was exposed to an alternating magnetic field (AMF), and the mice were treated with the knowledge of the nanoparticle distribution by micro-CT imaging BMNP/AMF/CT^. The second group (n=3) received MNP and was exposed to an AMF without the information of the MNP distribution BMNP/AMF^. The third group (n=7) received MNP as well but without exposure to an AMF to investigate the impact of MNP on the tumor volume without heating, and the intratumoral MNP distribution was measured by micro-CT imaging BMNP/CT^. The fourth group (n=6) received no MNP, instead aqua bidest (ddH2O) was injected to monitor the untreated tumor growth and the absence of intratumoral MNPs was confirmed by micro-CT imaging BddH2O/CT^.
Application of MNP and Assessment of MNP Distribution by Micro-CT Imaging One day prior to AMF exposure, the xenograft bearing mice of groups MNP/AMF/CT, MNP/AMF/CT, and MNP/CT were injected intratumorally with 0.24 mg Fe/100 mm3 (MF66: iron oxide, DMSA coated, core diameter 11.7 nm, Liquids Research Limited, UK). The ddH2O/CT group was injected with the same volume of ddH2O relative to the positive group. The mice of the treatment groups MNP/AMF/CT, MNP/CT, and ddH2O/CT were measured via a micro-CT Scanner (TomoScope Synergy Twin, CTImaging, Germany). Measurements were carried out directly after MNP or ddH2O application as well as 7 and 28 days after MNP application to assess the localization and distribution of the MNP within the tumor as well as changes over time. Here, a low radiation dose protocol was used, and spatial resolutions of 114 µm were reached. The ImpactCB acquisition software was used with an acquisition time of 29 s, and the tube voltage was set to 65 kV for both tubes. The image reconstruction was performed with the Impact Recon software. The 3D micro-CT data were analyzed using the Imalytics Research Software (Philips Technologie GmbH, Germany). For the CT images, the following segmentation parameters were chosen after subtraction of an instrument-specific value: the intensity for bones (white) and nanoparticles (red) was set above 376 Hounsfield Units (HU) whereas skin (transparent green) was set between −424 and 176 HU. The animals were exposed to a radiation dose of approximately 500 mGy during the three CT scans with the scanning protocol described above.
Magnetic Fluid Hyperthermia Treatment In brief, 24 h after MNP application, all MNP/AMF/CT and MNP/ AMF mice were exposed twice to an AMF (H=19 mT, f=435 kHz,
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
60 min) within a 7-day interval as described before [21, 22]. Throughout the therapy, temperatures were monitored using an infrared thermography camera (InfRec R300, Nippon Avionics Co., Japan) with fiber optic temperature sensors (TS5 and FOTEMPMK-19, Optocon AG, Germany). The desired temperatures were achieved by individually adapting the power of the AMF generator by the following means: (a) without knowledge of the intratumoral MNP distribution (MNP/AMF), 43 °C in the center of the tumor were used in order to minimize organ damage. (b) With knowledge of the intratumoral MNP distribution (MNP/AMF/CT), it was possible to modulate the power of the AMF generator to reach 43 °C at the tumor border. This strategy led to low thermoablative temperatures in the center of the tumor. If the MNPs were in close vicinity (approximately 2 mm) to vital structures, the hyperthermic temperatures at the tumor border were reduced to 41 °C.
Tumor Volume and Temperature Distribution Within the 28-day period, the tumor volume and the body weight of animals were measured every 3 days. The tumor volume was measured with a caliper and calculated according to the following formula: [23]. The tumor volumes immeadiately before treatment were set to 100 %, and the tumor volume for each animal for each timepoint was normalized to the value before treatment. Then, the mean relative tumor volume was calculated for each treatment group. Data analysis was performed using a t-test for independent samples with SPSS software (IBM SPSS Statistics 20, USA), and the significance level was set to p≤0.01. In order to determine the applied temperature dosages, the tumor surface temperatures of the thermal images taken during MFH were analyzed 10, 30, and 50 min after the beginning of the AMF treatment and the mean values determined. Here, a region of interest (ROI) was placed around each tumor, and the temperature data per pixel was extracted; hence, T90 temperatures and CEM43T90 values were calculated according to Sapareto and Dewey [7]. Additionally, temperature data of each animal were grouped into three categories: temperatures without hyperthermic effect (G43 °C), hyperthermic
temperatures (43–45 °C), and low thermo-ablative temperatures (945 °C) and their relative percentage calculated for each MFH treatment and plotted using Origin® 9.1 (Origin Lab, USA) software.
Results and Discussion Micro-CT Imaging The 3D MNP distribution within tumor and nontumor structures were detected by micro-CT imaging before the first (−1 day) and second (6 days) hyperthermia treatment as well as at the end (27 days) of the treamtent in order to localize their specific position (Fig. 1). In all cases, a heterogeneous iron oxide MNP distribution was clearly detectable as distinct red regions around the outer rim of the tumor tissue independent of the tumor size, since the same scanning protocol with a spatial resolution of 114 μm was used throughout all measurements (MNP/AMF/CT and MNP/CT). Moreover, after the first AMF treatment, a compression of MNP deposits in the tumor was observed for the MNP/AMF/CT and MNP/CT groups. The spatial resolution of the CT system will be the limiting factor with consideration of clinical applications. Therefore, the dimensions of the tumor have to be significantly larger than 0.5 mm based on the resolution of most clinical scanners (e.g., 0.5–0.6 mm) in order to detect the MNPs within the tumor. In regard to the cytotoxicity of the radiation from microCT imaging, we did not observe any significant changes within the blood composition (white blood cells, red blood cells, and amount of hemoglobin) and body weight of the animals that were imaged. Therefore, we assume that the effective dose is below the threshold and do not expect that the radiation dose from micro-CT imaging influences the outcome of this study.
Fig. 1 The tumor volume of the MNP/AMF/CT mice was significantly reduced after 28 days. The distribution and localization of the MNPs within the tumor and nontumor structures were monitored by micro-CT imaging over time (−1, 6, 27 days) in all groups (MNP/AMF/CT [dark green], MNP/CT [blue], and ddH2O/CT [orange], n=3). Each image per treatment group and day consists of three information: first, the tumor morphology is displayed; second, the morphology as CT image; and third, the segmented CT image. Within the segmented CT images, the threshold for the segmentation of bone (white) and MNP (red) was set above 376 HU and of skin (transparent green) within the range of −424 to 176 HU.
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
Fig. 2 Improved therapeutic efficiency after CT-imaging based on the parameters temperature distribution and MNP distribution. a The intratumoral MNP distribution is shown exemplarily for one mouse per treatment group at days −1, 6, and 27: segmented CT image (if applicable, right), the tumor morphology (left). b, c The temperature distribution at 90 % of the tumor surface (in percentage) is plotted for each animal after the first (striped bars) and second hyperthermia treatment (plain colored bars) for the b MNP/AMF/CT and c MNP/AMF groups.
Temperature Distribution in Tumors with and without Micro-CT Imaging A tumor growth reduction in combination with a compression of the MNP regions at the end of the therapy was found for the mice from the MNP/AMF/CT group as well (Fig. 2a). However, this was not the case for mice of the MNP/AMF group where the exact localization of the MNPs was not determined. The temperature distribution profiles for both treatment groups were generated from the infrared thermography data for the first [striped bars] and second AMF [plain colored bars] treatment for
all mice (Fig. 2b, c). The knowledge of the intratumoral MNP distribution pattern by micro-CT imaging led to the generation of adequate temperature profiles (i.e., higher temperatures) within the tumor for the first and the second hyperthermia treatment for the MNP/AMF/CT group. This enabled a better therapeutic outcome for the MNP/AMF/CT group of the three treated mice with 12, 13, and 19 % of the relative tumor volume at day 28 compared to 58, 79, and 81 % for the MNP/AMF group. This is also reflected in the higher T90 values for both treatments with 43.3 and 39.1 °C as well as 40.0 and 36.3 °C for the MNP/AMF/ CT as well as the MNP/AMF groups, respectively, when the
Table 1. Characteristic parameters of the therapeutic effects after in vivo magnetic fluid hyperthermia (MFH) MDA-MB-231 MNP/AMF/CT 3
Applied magnetic material [mg Fe/100 mm ] T90 [°C] CEM43T90 [min] Tumor volume growth rate VTMFH-28d/VTMFH-0d VTMFH-28d/VT28d
1st MFH 2nd MFH
0.24 43.3 39.1 77.6 4.8 %/day 14 % 4.4 %
MNP/AMF 40.0 36.3 2.2 73 % 24 %
A total of 0.24 mg Fe/100 mm3 magnetic material was applied for the breast cancer xenograft MDA-MB-231. The temperature within 90 % of the tumor surface (T90) after the first and second MFH treatment as well as the cumulative equivalent minutes at 43 °C (CEM43T90) are displayed for the MNP/AMF/ CT and MNP/AMF groups. The mean relative tumor growth rates in days as well as the mean tumor volumes (in percentage) compared to the tumor volume at day 0 (VT MFH-28d/VT MFH-0d) or to the untreated control after 28 days (VT MFH-28d/VT 28d) are compared for both groups
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
Fig. 3 Magnetic hyperthermia treatment with micro-CT imaging led to a significant reduction of the tumor volume compared to the control groups. a Micro-CT imaging, with exception of the MNP/AMF group, was used to determined the MNP distribution within the tumors of the mice over time. Per treatment group, one mouse is shown exemplarily with its respective intratumoral MNP distribution in the segmented CT image (if applicable, right) and the tumor morphology (left). b The tumor volume was monitored over time and calculated relative to the tumor volume at day 0 for all treatment groups. The asterisk symbolizes a significant statistical difference of p≤0.01.
same amount (0.24 mg Fe per 100 mm3) of magnetic iron oxide MNPs were applied (Table 1). Additionally, the mean CEM43T90 values of approximately 78 min for the MNP/ AMF/CT group indicate that the critical hyperthermia temperature threshold of 43 °C for 60 min was reached, when the exact MNP distribution was determined, compared to only 2.2 min for the MNP/AMF group. Without the knowledge of the MNP distribution, modulation of temperatures at the tumor borders and surrounding tissue is challenging. All in all, the tumor reduction by means of the mean VT MFH-28d/VT MFH-0d for MNP/
AMF/CT group is with 13.5 % significantly smaller than the mean VT MFH-28d/VT MFH-0d for MNP/AMF group with 72.6 % (p≤0.01).
Therapeutic Effects of In Vivo Magnetic Fluid Hyperthermia with Micro-CT Imaging The mean relative tumor volumes after 28 days were determined to be 14 % for MNP/AMF/CT, 73 % for MNP/
H. Dähring et al.: Micro-CT Imaging for Effective Tumor Therapy After Magnetic Hyperthermia
AMF, 224 % for MNP/CT, and 305 % for ddH2O/CT compared to the tumor volumes at 0 day (Fig. 3). This equals a relative tumor volume of 4.4 and 23.8 % compared to the ddH2O control after 28 days for MNP/AMF/CT and MNP/AMF, respectively. The application of MNP alone had no effect on tumor growth compared to the ddH2O/CT control group. The tumor volume was significantly reduced for both AMF treatment groups, MNP/AMF/CT and MNP/ AMF, compared to the control groups MNP/CT and ddH2O/ CT. However, the usage of micro-CT imaging even further improved the overall outcome of the hyperthermia treatment, as higher temperatures within the critical tumor tissue were achieved and consequently higher CEM43T90 values. Morphologically, the best results were obtained for the MNP/AMF/CT group with almost complete tumor regression in most cases. Insufficient tumor reduction led to regrowth around the border of tumor for the MNP/AMF group. Opposing views on the ideal CEM43T90 values exist. Here, the best therapeutic success on the basis of the parameter tumor volume reduction and temperature distribution were achieved for higher temperature dosages as proposed by Thrall et al. [12]. We have shown in our study an increased therapeutic effect of MFH using micro-CT imaging. A heterogeneous MNP distribution is difficult to circumvent during intratumoral MNP application, but the exact localization of the iron oxide MNPs can easily be detected by micro-CT imaging, and this knowledge can be used for an efficient hyperthermia therapy. By micro-CT imaging, insufficient amounts of MNP can be detected, and an efficient hyperthermia treatment can be achieved by a repeated MNP application.
Conclusions Micro-CT imaging is capable of depicting intratumorally applied MNP amounts prior to the hyperthermia in vivo and consequently allows a modulation of the generated temperatures within the tumor tissue based on the exact localization. A significant tumor reduction and subsequently a higher therapeutic success of the MFH were achieved with knowledge of the intratumoral MNP distribution. Furthermore, the surrounding nontumor tissue was preserved while effectively treating breast cancer. Micro-CT is thus an important tool for the qualitative determination of the particle distribution and enables a localized noninvasive hyperthermia treatment. Thereby, the hyperthermia treatment in our breast cancer model was distinctly improved based on the parameters tumor volume and the temperature distribution. CT imaging for improved therapeutic success is potentially translatable into clinical practices, if the tumor size is larger than the resolution of the instrument and might also be used for personalized hyperthermia therapy. Acknowledgments. The present work was carried out within the project BMultifunctional Nanoparticles for the Selective Detection and Treatment of
Cancer,^ funded by the European Seventh Framework Program (FP7/20072013) under grant agreement no. 262943. We gratefully acknowledge Liquids Research Limited (UK) for providing the magnetic material. The micro-CT instrument was funded by the Federal State Thuringia and the European Union (EFRE; European Fond for regional development). Conflict of Interests. The authors declare that they have no conflict of interest. Ethical Approval. All applicable institutional and/or national guidelines for the care and use of animals were followed.
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