The First Results in Japan

Hindawi Publishing Corporation Conference Papers in Medicine Volume 2013, Article ID 197328, 6 pages http://dx.doi.org/10.1155/2013/197328

Conference Paper Oncothermia Basic Research at In Vivo Level: The First Results in Japan G. Andocs,1 Y. Okamoto,1 K. Kawamoto,1 T. Osaki,1 T. Tsuka,1 T. Imagawa,1 S. Minami,1 L. Balogh,2 N. Meggyeshazi,3 and O. Szasz4 1

Department of Veterinary Clinical Medicine, Faculty of Veterinary Science, Tottori University, 4-101 Koyama Minami, Tottori 680-8553, Japan 2 “Frederic Joliot Curie” National Research Institute for Radiobiology and Radiohygiene, XXII. Ker. Anna u. 5, 1221 Budapest, Hungary 3 ¨ oi u´ t 26, 1085 Budapest, Hungary 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Ull˝ 4 Biotechnics Department, Faculty of Engineering, St. Istv´an University, Pater K. u. 1., 2100 Godollo, Hungary Correspondence should be addressed to G. Andocs; [email protected] Received 17 January 2013; Accepted 28 May 2013 Academic Editors: G. Baronzio, M. Jackson, and A. Szasz This Conference Paper is based on a presentation given by G. Andocs at “Conference of the International Clinical Hyperthermia Society 2012” held from 12 October 2012 to 14 October 2012 in Budapest, Hungary. Copyright © 2013 G. Andocs et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper summarizes the first results of oncothermia basic research conducted in Tottori University, Japan, and had two parts. In the first part C26 murine colorectal cancer model was investigated and oncothermia treatment induced histomorphological and some molecular changes which were examined. In the second study 9L rat glioma model was used to investigate the oncothermia treatment effects on tumor tissue oxygenization. Results of these investigations are very important in oncothermia research because this was the first time when independent research laboratory has repeated oncothermia experiments and proved the significant antitumoral and beneficial effects of oncothermia treatment.

1. Background Oncothermia method (OTM) is a long time (since 1989) applied method in oncology [1] with great clinical success [2]. Oncothermia research group conducts investigations to reveal the basic mechanism of action of this tumor treatment method in basic research level performing a huge number of in vivo studies. The tumor destruction efficacy and the role of temperature independent effects of the OTM were proven earlier and presented elsewhere [3, 4], as well as the recent in vivo results [5, 6]. In this paper we summarize the first results we have achieved in Tottori University, Japan.

2. Materials and Methods 2.1. Study I. In the first study we examine the effect of oncothermia treatment in a mouse tumor model.

2.1.1. Animal Model. Colon 26 (murine colorectal cancer) cell line derived allograft mouse tumor model was used for this study with double tumors (see Figure 1). The use of the mice and the procedures used in this study were approved by the Animal Research Committee of Tottori University. 2.1.2. Experimental Setup and Treatment. A single shot 30 min oncothermia treatment was done reaching maximum 42∘ C intratumoral temperature, using the LabEHY system (Oncotherm Ltd.), under precise tumor temperature control using fluoroptic temperature measurement device (Lumasense m3300) (see Figure 2). 2.1.3. Study Design. Time course study was performed. After a single shot oncothermia treatment, animals were sacrificed at 6H, 24H, 72H, and 120H later and tumors were removed.

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Conference Papers in Medicine

Figure 1: Experimental mouse tumor model. Every animal had two tumors on the femoral regions, the right side was treated and the left side was individual control.

LabEHY device

Temperature (∘ C)

Mouse treating applicator system

45 43 41 39 37 35 33 31 29 27 25 0:00:00

Representative temperature measurement graph of a treatment

0:10:00

Rectal Control tumor

0:20:00 Time (min)

0:30:00

Tumor core Tumor surface

Figure 2: The experimental setup with the LabEHY system and a representative temperature measurement graph of the temperature curve of the tumors.

There were 3 treated animals and 1 untreated control animal in all sampling group in every time point (see Figure 3). 2.1.4. Tumor Sample Processing. All the removed tumors were cut accurately at their centerline. After a standard histological process, the samples were stained with HE and TUNEL reaction and Ki-67 immunohistochemical (IHCH) detection were performed (HE staining and IHCH detection were performed by Sapporo Byori Kensa Center, Japan). Samples were evaluated using complex histomorphological methods. Besides the qualitative analysis, a quantitative microscopical evaluation was also performed in the tumor samples stained with Ki-67. In ten randomly chosen high magnification (400x) microscopic view area of the living part of the tumor tissue samples, the Ki-67 positive cell nuclei were counted, recorded, and evaluated. 2.2. Study II. In the second study we examined the effects of OTM to tumor oxygenization using a rat tumor model.

2.2.1. Animal Model. 9L (rat glioma) cell line derived allograft rat tumor model was used. All animals had 2 tumors on both femoral regions. The use of the rats and the procedures used in this study were approved by the Animal Research Committee of Tottori University. 2.2.2. Oxygen Level Measurement. Tumor tissue oxygenization level was measured using an O2 sensitive electrode system (Eikon Kagaku Ltd. 150D model). 2.2.3. Study Design. In 11 rats, tumor tissue oxigenization level was measured using a pO2 sensitive electrode system right before the treatment. The sensor probe of the system was inserted into the tumor tissue with the help and guidance of a teflon catheter, and then the measured pO2 value was recorded. Then the probe and the catheter were removed and a single shot, 30min oncothermia treatment was performed using a LabEHY system (Oncotherm Ltd.), reaching maximum 42∘ C intratumoral temperature. Right after


3 24 H

6H

72 H

120 H

Figure 3: Oncothermia treated experimental animals in this study.

(a)

(b) Oncothermia treatment: 30 min single shot treatment with 42∘ C tumor core temp. with the LabEHY device, using ﬂexible treatment electrode system.

(c)

(c)

Figure 4: The study design. The 9L glioma cell line derived rat allograft tumor model (a), the oncothermia treatment procedure (b), and the tissue oxygenization measurement system (c).

thetreatment the tumor oxigenization level was measured again (see Figure 4).

3. Results (i) Study I

(a) Histomorphological Changes in a Qualitative and a Quantitative Way (see Figure 5). (b) Histomorphological Changes in Details (see Figure 6). (c) TUNEL Reaction (see Figure 7). (d) Ki-67 Expression Changes (see Figures 8 and 9).

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Conference Papers in Medicine 6H Control Treated

24 H Control Treated



Untreated

Dead tumor area ratio (%)

1

2

CTRL

3

80 70 60 50 40 30 20 10 0

Area of the living part and the dead part of the tumor was measured in a whole tumor cross and living/ dead ratio was calculated and graphed.

Treated

Ratio of dead tumor area

CTRL

24 H 72 H 6H Time after treatment

120 H

Untreated Treated

Figure 5: All the tumor samples involved in this study and the result graph of the quantitative analysis of the living/dead area ratio measurement. Drastic and selective tumor destruction was detected after a single shot oncothermia treatment. The tumor destruction was not immediate; it had a time delay. Samples marked with a red rectangle are evaluated in details.

6H

24 H

72 H

120 H

10x

200x

400x

Figure 6: Detailed morphological analysis of the tumor samples marked with red rectangle in Figure 5. 6 H after the treatment the tumor cells look intact, but 24 H after the treatment, the large part of the tumor is dead, and the cells are shrunk with picnotic cell nuclei. In the 48 H and 72 H samples definite late morphological signs of apoptotic cell death were observed: extremely high number of apoptotic bodies (marked with red arrow). 120 H after the treatment morphological signs of leukocyte (mostly neutrophils, marked with green arrow) invasion can be visible.

(ii) Study II (a) Results of the Tumor pO2 Level Measurement in a Rat Tumor Model (see Figure 10).

4. Conclusions (1) In the mouse study, oncothermia treatment can significantly destroy the tumor tissue in a large volume

of the tumor even with single shot way. Oncothermia treatment induces apoptotic cell death in the destroyed tumor tissue and effectively inhibits cell proliferation in the living part of the tumor. (2) In the rat study, oncothermia treatment can significantly increase the tumor tissue oxygenization which creates the basis of the strong synergism with radiotherapy and some chemotherapy.


5 24 H

Untreated

Treated

72 H

120 H

200x

200x

200x

400x

400x

400x

200x

200x

200x

400x

400x

400x

Figure 7: Result of the qualitative evaluation of TUNEL staining. TUNEL assay enzymatically labels the DNA fragments resulted by apoptotic cell death process. In the dead tumor area a huge number of TUNEL-positive cells were observed after a single shot OTM treatment.

200x

24 H 200x

72 H 200x

120 H 200x

400x

400x

400x

400x

200x

200x

200x

200x

400x

400x

400x

400x

6H

Untreated

Treated

Figure 8: Result of the qualitative evaluation of the Ki-67 staining. The Ki-67 proliferation marker protein is expressed in the nuclear membrane only in the dividing cells. That is why sampling for Ki-67 positive cell analysis and counting was done from the living part of the tumors. The high magnification images from the living part of the tumor samples (marked with red rectangle in the whole cross sections).

Ki-67 positive cells in untreated tumor

35

35

30

30

25

25

20

20

15

15

10

10

5

5

0

6

24 72 After treatment (hour)

Ki-67 positive cells in treated tumor

40

View

View

40

120

0

6

24 72 After treatment (hour)

120

Figure 9: Result of the quantitative evaluation of the Ki-67 staining. Ki-67 positive cell nuclei were counted in 10 randomly chosen area of the living part of the tumor samples. In a very interesting way the number of Ki-67 positive cells was significantly decreased in the living part of the treated tumor compared to the control tumors.

Conference Papers in Medicine Tumor tissue oxigenization

50 45 40 35 30 25 20 15 10 5 0

35

Tumor tissue oxigenization in average

30 pO2 level (mmHg)

O2 level (mmHg)

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25 20 15 10 5 0

1

2

3

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5 6 7 Rat number

8

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Before treatment

After treatment

Before treatment After treatment

Before treatment After treatment (a)

(b)

Figure 10: Result of the tumor tissue oxygenization level measurement in each animal (a) and in average (b). Tumor tissue pO2 level was significantly higher right after the oncothermia treatment compared the pO2 level measured right before the treatment in case of 10 out of the total 11 animals. The pO2 level was almost double after the treatment in average.

References [1] A. Szasz, “Hyperthermia, a modality in the wings,” Journal of Cancer Research and Therapeutics, vol. 3, no. 1, pp. 56–66, 2007. [2] A. Szasz, N. Szasz, and O. Szasz, Oncothermia. Principles and Practices, Springer, Dordrecht, The Netherlands, 2010, http://www.springer.com/biomed/cancer/book/978-90-4819497-1?changeHeader. [3] G. Andocs, O. Szasz, and A. Szasz, “Oncothermia treatment of cancer: from the laboratory to clinic,” Electromagnetic Biology and Medicine, vol. 28, no. 2, pp. 148–165, 2009. [4] G. Andocs, H. Renner, L. Balogh, L. Fonyad, C. Jakab, and A. Szasz, “Strong synergy of heat and modulated electromagnetic field in tumor cell killing,” Strahlentherapie und Onkologie, vol. 185, no. 2, pp. 120–126, 2009. [5] N. Meggyeshazi, “Programmed cell death induced by modulated electro-yperthermia,” in Proceedings of International Clinical Hyperthermia Society (ICHS ’12), 2012. [6] N. Meggyeshazi, “Early changes in protein expression releated to modulated electro-hyperthermia,” in Proceedings of International Clinical Hyperthermia Society (ICHS ’12), 2012.

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