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.

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 [79] 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 Figure 1.

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 Figure 2.

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 Figure 3.

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 Figure 4.

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 Figure 5.

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 Figure 6.

3. Results

3.1. Histomorphological Changes

See Figure 7.

3.2. Appearance of the Hallmarks of Immunogenic Cancer Cell Death
3.2.1. Apoptotic Body Formation

See Figure 8.

3.2.2. TRAIL (DR5) Expression

See Figure 9.

3.2.3. HSP70 Expression Changes and Molecular Dynamics

See Figure 10.

3.3. Strong Local Immune Reaction
3.3.1. Myeloperoxidase (MPO) Detection

See Figure 11.

3.3.2. CD3 and CD4 Detection

See Figure 12.

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.