# Bystander Effect of Oncothermia

Authors: G. Andocs; N. Meggyeshazi; Y. Okamoto; L. Balogh; O. Szasz
Journal: Conference Papers in Medicine
 (2013)

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## Abstract

Metastatic form of malignant tumor diseases is the most serious problem in oncology and the greatest challenge in tumor therapy. Conventional therapeutical approaches (surgery, irradiation, and chemotherapy) cannot manage this challenge in oncological practice. According to our theory, oncothermia treatment-induced immunogenic tumor cell death can be a very good basis for immunotherapy combination to make systemic tumor control from a local tumor destruction effect. We summarize the molecular basis of the oncothermia treatment-induced immunogenic cell death as a necessary basic condition to achieve the bystander effect.


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## Body

## 1. Background

Oncothermia (OTM) is an electrohyperthermia modality, a long-time (since 1989) applied method in oncology, [1] with great clinical success [2]. OTM changes the paradigm of hyperthermia by targeted microscopic heat liberation at the membrane of the malignant cells. This method creates inhomogeneous heating, microscopic temperature differences far from thermal equilibrium. The tumor destruction efficacy and the role of temperature independent effects of the OTM were proven earlier by laboratory research and 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 radiooncologists and remained a higly controversial topic until recent years [5, 6]. Intensive research is conducting to reveal the immunbiological basis [7–9] and mechanism of action of this effect [10] and using the benefits in the regular oncological practice.The objective is showing the newest results of oncothermia in research bystander effect.


## 2. Materials and Methods

### 2.1. Animal Model

HT29 human colorectal carcinoma cell line derived xenograft tumor model in nude mouse. See Figure1.Figure 1
Process of the tumor induction of the experimental animals.


### 2.2. Experimental Setup and Treatment

A single shot 30 min oncothermia treatment was done, reaching maximum 41-42°C intratumoral temperature, using the LabEHY system (Oncotherm Ltd.), under precise tumor temperature control using fluoroptic temperature measurement system (Lumasense, Luxtron m3300). See Figure2.Figure 2
Experimental setup of the oncothermia treatments in the laboratory.


### 2.3. Study Design

Time course study was performed. After a single shot treatment, sampling was made after 0, 1, 4, 8, 14, 24, 48, 72, 120, 168, and 216 hours. Three mice were sacrificed at each time point, keeping 5 sham-treated animals.  See Figure3.Figure 3
All the oncothermia-treated experimental animals involved in this study.


### 2.4. Tumor 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. See Figure4.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 immunohistochemical reactions on many samples in one block. See Figure5.Figure 5
The computer controlled tissue microarray device and the tissue sample multiblock created by the TMA Master device (3DHisTech). One multiblock contains many small representative tumor tissue samples, so really identical and higly standardized immunohistochemical reaction can be performed in all the samples. This is the real advantage of this technology.


### 2.5. Immunohistochemistry (IHCH)

The following reactions and IHCH analysis were performed on the TMA samples: TUNEL (Invitrogen); TRAIL (DR5), HSP70 (Cell Signaling); Myeloperoxidase (Sigma); CD3 (Dako), CD4 (ABDSerotech).


### 2.6. Digital Microscopy Analysis

All histological slides were digitalized using Panoramic Slide Scanner (3DHisTech) and special software was used for imaging and evaluation. See Figure6.Figure 6
The Panoramic slide scanner device and a screenshot from the Panoramic viewer software, dedicated for precise histomorphological analysis.


## 2.1. Animal Model

HT29 human colorectal carcinoma cell line derived xenograft tumor model in nude mouse. See Figure1.Figure 1
Process of the tumor induction of the experimental animals.


## 2.2. Experimental Setup and Treatment

A single shot 30 min oncothermia treatment was done, reaching maximum 41-42°C intratumoral temperature, using the LabEHY system (Oncotherm Ltd.), under precise tumor temperature control using fluoroptic temperature measurement system (Lumasense, Luxtron m3300). See Figure2.Figure 2
Experimental setup of the oncothermia treatments in the laboratory.


## 2.3. Study Design

Time course study was performed. After a single shot treatment, sampling was made after 0, 1, 4, 8, 14, 24, 48, 72, 120, 168, and 216 hours. Three mice were sacrificed at each time point, keeping 5 sham-treated animals.  See Figure3.Figure 3
All the oncothermia-treated experimental animals involved in this study.


## 2.4. Tumor 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. See Figure4.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 immunohistochemical reactions on many samples in one block. See Figure5.Figure 5
The computer controlled tissue microarray device and the tissue sample multiblock created by the TMA Master device (3DHisTech). One multiblock contains many small representative tumor tissue samples, so really identical and higly standardized immunohistochemical reaction can be performed in all the samples. This is the real advantage of this technology.


## 2.5. Immunohistochemistry (IHCH)

The following reactions and IHCH analysis were performed on the TMA samples: TUNEL (Invitrogen); TRAIL (DR5), HSP70 (Cell Signaling); Myeloperoxidase (Sigma); CD3 (Dako), CD4 (ABDSerotech).


## 2.6. Digital Microscopy Analysis

All histological slides were digitalized using Panoramic Slide Scanner (3DHisTech) and special software was used for imaging and evaluation. See Figure6.Figure 6
The Panoramic slide scanner device and a screenshot from the Panoramic viewer software, dedicated for precise histomorphological analysis.


## 3. Results

### 3.1.  Histomorphological Changes

See Figure7.Figure 7
All the processed and HE stained tumor samples in this study. Morphologically the first significant sign of cell destruction was seen 8 h after the treatment. Drastic and selective tumor destruction was detected 24 h after OTM which became more emphasized after 48 h. 72 hours after the treatment a significant leucocyte infiltration (marked with red arrows) appeared around the destructed tumor tissue and reached its maximum 168 hours after the treatment.


### 3.2. Appearance of the Hallmarks of Immunogenic Cancer Cell Death

#### 3.2.1. Apoptotic Body Formation

See Figure8.Figure 8
HE and TUNEL stained whole cross-section tumor samples 48 h after the treatment. Oncothermia treatment induce apoptotic cell death, and this process is really very emphasized 48 h after a single shot treatment. Almost all the cell nuclei of the killed tumor cells are TUNEL positive. In the process of this programmed cell death a huge number of apoptotic body was formed (marked with red arrows).


#### 3.2.2. TRAIL (DR5) Expression

See Figure9.Figure 9
TRAIL detection IHCH from TMA multiblock. TRAIL (DR5) is a higly immunogenic cell surface receptor. Expression was increased in the treated side 8 h after the treatment and became more emphasized after 14 h.


#### 3.2.3. HSP70 Expression Changes and Molecular Dynamics

See Figure10.Figure 10
HSP70 detection IHCH from TMA multiblock. Definite increase of the HSP70 expression was observed 14 hours after the treatment. After 24 hours, unusual molecular dynamic changes of the increased amount of HSP70 can be visible: intracellular condensation (marked with green rectangle) and relocalization to cell membrane. After 72 hours the membrane relocalization of the HSP70 became more emphasized, especially in the region of the leukocyte invasion (marked with yellow rectangle).


### 3.3. Strong Local Immune Reaction

#### 3.3.1. Myeloperoxidase (MPO) Detection

See Figure11.Figure 11
Myeloperoxidase (MPO) detection from TMA multiblock. MPO is a marker of neutrophyle granulocytes. The leukocyte invasion ring what appears at 72 h and became very characteristic at 168 h around the destructed tumor area, containing high number of MPO positve cells ( neutrophiles).


#### 3.3.2. CD3 and CD4 Detection

See Figure12.Figure 12
The 168 h tumor tissue sample area, marked with green rectangle in Figure11. was analyzed by CD3 IHCH staining and CD3/CD4 dual fluorescent IHCH staining. The analysis showed that the invasion ring, beside the neutrophiles, also contains large amount of CD3+ T cells and CD4+ cells, probably dendritic cells.


## 3.1.  Histomorphological Changes

See Figure7.Figure 7
All the processed and HE stained tumor samples in this study. Morphologically the first significant sign of cell destruction was seen 8 h after the treatment. Drastic and selective tumor destruction was detected 24 h after OTM which became more emphasized after 48 h. 72 hours after the treatment a significant leucocyte infiltration (marked with red arrows) appeared around the destructed tumor tissue and reached its maximum 168 hours after the treatment.


## 3.2. Appearance of the Hallmarks of Immunogenic Cancer Cell Death

### 3.2.1. Apoptotic Body Formation

See Figure8.Figure 8
HE and TUNEL stained whole cross-section tumor samples 48 h after the treatment. Oncothermia treatment induce apoptotic cell death, and this process is really very emphasized 48 h after a single shot treatment. Almost all the cell nuclei of the killed tumor cells are TUNEL positive. In the process of this programmed cell death a huge number of apoptotic body was formed (marked with red arrows).


### 3.2.2. TRAIL (DR5) Expression

See Figure9.Figure 9
TRAIL detection IHCH from TMA multiblock. TRAIL (DR5) is a higly immunogenic cell surface receptor. Expression was increased in the treated side 8 h after the treatment and became more emphasized after 14 h.


### 3.2.3. HSP70 Expression Changes and Molecular Dynamics

See Figure10.Figure 10
HSP70 detection IHCH from TMA multiblock. Definite increase of the HSP70 expression was observed 14 hours after the treatment. After 24 hours, unusual molecular dynamic changes of the increased amount of HSP70 can be visible: intracellular condensation (marked with green rectangle) and relocalization to cell membrane. After 72 hours the membrane relocalization of the HSP70 became more emphasized, especially in the region of the leukocyte invasion (marked with yellow rectangle).


## 3.2.1. Apoptotic Body Formation

See Figure8.Figure 8
HE and TUNEL stained whole cross-section tumor samples 48 h after the treatment. Oncothermia treatment induce apoptotic cell death, and this process is really very emphasized 48 h after a single shot treatment. Almost all the cell nuclei of the killed tumor cells are TUNEL positive. In the process of this programmed cell death a huge number of apoptotic body was formed (marked with red arrows).


## 3.2.2. TRAIL (DR5) Expression

See Figure9.Figure 9
TRAIL detection IHCH from TMA multiblock. TRAIL (DR5) is a higly immunogenic cell surface receptor. Expression was increased in the treated side 8 h after the treatment and became more emphasized after 14 h.


## 3.2.3. HSP70 Expression Changes and Molecular Dynamics

See Figure10.Figure 10
HSP70 detection IHCH from TMA multiblock. Definite increase of the HSP70 expression was observed 14 hours after the treatment. After 24 hours, unusual molecular dynamic changes of the increased amount of HSP70 can be visible: intracellular condensation (marked with green rectangle) and relocalization to cell membrane. After 72 hours the membrane relocalization of the HSP70 became more emphasized, especially in the region of the leukocyte invasion (marked with yellow rectangle).


## 3.3. Strong Local Immune Reaction

### 3.3.1. Myeloperoxidase (MPO) Detection

See Figure11.Figure 11
Myeloperoxidase (MPO) detection from TMA multiblock. MPO is a marker of neutrophyle granulocytes. The leukocyte invasion ring what appears at 72 h and became very characteristic at 168 h around the destructed tumor area, containing high number of MPO positve cells ( neutrophiles).


### 3.3.2. CD3 and CD4 Detection

See Figure12.Figure 12
The 168 h tumor tissue sample area, marked with green rectangle in Figure11. was analyzed by CD3 IHCH staining and CD3/CD4 dual fluorescent IHCH staining. The analysis showed that the invasion ring, beside the neutrophiles, also contains large amount of CD3+ T cells and CD4+ cells, probably dendritic cells.


## 3.3.1. Myeloperoxidase (MPO) Detection

See Figure11.Figure 11
Myeloperoxidase (MPO) detection from TMA multiblock. MPO is a marker of neutrophyle granulocytes. The leukocyte invasion ring what appears at 72 h and became very characteristic at 168 h around the destructed tumor area, containing high number of MPO positve cells ( neutrophiles).


## 3.3.2. CD3 and CD4 Detection

See Figure12.Figure 12
The 168 h tumor tissue sample area, marked with green rectangle in Figure11. was analyzed by CD3 IHCH staining and CD3/CD4 dual fluorescent IHCH staining. The analysis showed that the invasion ring, beside the neutrophiles, also contains large amount of CD3+ T cells and CD4+ cells, probably dendritic cells.


## 4. Conclusions

(1)
Oncothermia treatment can induce programmed cell death in the tumors which create many apoptotic bodies. Presence of apoptotic bodies in a destructed tumor tissue is essential to induce immunogenic reactions.
(2)
Oncothermia treatment-induced cell death is higly immunogenic, showing all the key molecular pattern dynamic changes what is characteristic of immunogenic tumor cell death.
(3)
Oncothermia treatment can induce strong and very unusual local immune reaction at the site of the treatment, long time after the electromagnetic intervention.
(4)
The local antitumor immune reaction might be systemic if the host has an intact immune system and proper immunstimulating agent is administered. This process can control the distant metastases by bystander effect, making possible the systemic control of the malignant disease with local treatment.Ongoing intensive research is in progress on immunocompetent tumor models, to investigate  and reveal the mechanism of action of this controlled bystander effect.



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*Source: 953482-2013-08-07.xml*