Bystander effect of oncothermia

Andocs G, Meggyeshazi N, Okamoto Y, Balogh L, Szasz A (4) Department of Veterinary Clinical Medicine, School of Veterinary Medicine, Tottori University, Tottori, 4-101 Minami, Koyama-cho., tottori Pref., Japan (5) 1sr Department of Pathology and Experimental Cancer Research, Semmelweis University, 1085-Budapest, Ulloi ut 26., Hungary (6) “Frederic Joliot Curie” National Research Institute for Radiobiology and Radiohygiene 1221-Budapest, Anna u. 5., Hungary (7) Department of Biotechnics, Szent Istvan University, 2100-Godollo, Pater K. u. 1., Hungary Corresponding author: Gabor Andocs gandocs@gmail.com


Background
Oncothermia (OTM) is an electro-hyperthermia modality, a long time (since 1989) applied method, [1], used successfully in human oncology [2].OTM changes the paradigm of hyperthermia by targeted microscopic heat-liberation at the membrane of the malignant cells.This method creates inhomogeneous heating, and the microscopic temperature greatly differs far from the thermal equilibrium.The tumor destruction efficacy and the role of temperature independent effects of the OTM were proven earlier by a laboratory research, and were presented elsewhere [3], [4].Bystander effect (abscopal effect) means that a local tumor treatment can affect the behavior of the far distant metastases.It was first discovered by radio-oncologists and remained a highly controversial topic until recent years.[5], [6].Intensive research is being conducted to reveal the immunbiological basis [7], [8], [9] and the mechanism of the action of this effect [10] and to use the benefits in the regular oncological practice.Our objective is to show the newest results of oncothermia in research of the bystander effect.

Experimental setup and treatment
A single shot 30 min oncothermia treatment was done, reaching maximum 41-42oC intratumoral temperature, using the LabEHY system (Oncotherm Ltd.Germany), under precise tumor temperature control using fluoroptic temperature measurement system (Lumasense, Luxtron m3300).Digital microscopy analysis: All histological slides were digitalized using Panoramic Slide Scanner (3D HisTech) and a special software was used for imaging and evaluation.
HT29 human colorectal carcinoma cell line derived xenograft tumor model in nude mice.The use of the mice and the procedures used in this study were approved by the Animal Experiment Ethical Committee of National Research Institute for Radiobiology and Radiohygiene.

Figure 1 .
Figure 1.Process of the tumor induction of the experimental animals

Figure 3 .
Figure 3.All the oncothermia-treated experimental animals involved in this studyTumor sample processing: At the time of the sampling the single-treatment animals were sacrificed and both the control and treated tumors were removed and studied in pairs.

Figure 4 .
Figure 4. Method of the tumor sample processing Due to the extremely high number of the tumor samples, tissue microarray (TMA) technology was used to perform accurate immuno-histochemical reactions on many samples in one block.

Figure 5 .
Figure 5.The computer-controlled tissue-microarray device and the tissue sample multiblock were created by the TMA Master device (3DhisTech).One multiblock contains many small representative tumor tissue samples, so really identical and highly standardized immunohistochemical reaction can be performed in all the samples.This is the real advantage of this technology Immunohistochemistry (IHCH): The following reactions and IHCH analysis were performed on the TMA samples: TUNEL (Invitrogen); TRAIL-R2 (DR5) (Cell Signaling), HSP70 (Cell Signaling); Myeloperoxidase (Sigma); CD3 (Dako), CD4 (ABDSerotech).