The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3+ and CD8+ cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3+ and CD8+ cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.
The abscopal effect is a phenomenon noted in the treatment of metastatic cancer where localized irradiation of a tumor causes shrinking not only of the immediate target, but also of tumors located far from the irradiated area. This phenomenon is extremely rare, but when it does occur, its anticancer effects can be dramatic, leading to the disappearance of malignant growths throughout the body. Such success has been described for a variety of malignancies, including melanoma, lymphoma, and lung metastases of hepatocellular carcinoma (
The modulated electro-hyperthermia (mEHT) system is a fast-developing complementary treatment method that is effective against different types of tumors (
Hyperthermia is usually applied as an adjunct to an already established treatment modality, such as radiotherapy and chemotherapy, in order to achieve tumor temperatures of 40–46°C. Hyperthermia was originally controlled through modulating temperature alone, whereas the more recently developed mEHT (also described as ‘oncothermia’) is based on energy dose control, replacing the single temperature concept (
Although hyperthermia has been extensively studied, the effects of mEHT combined with dendritic cell (DC) treatment on squamous cell carcinoma have not been addressed. The goal of this study was to evaluate the antitumor effects of combined direct intratumoral injection of DCs and mEHT treatment using a mouse squamous cell carcinoma (SCCVII) cancer model. We showed that this combined treatment causes both local and distant shrinkage of SCCVII-derived tumors, and thereby offers hope to patients with esophageal squamous cell carcinoma, particularly those who have recurrent disease and are unable to undergo further radiotherapy. Thus, this newly developed technique could represent a new strategy for the treatment of squamous cell carcinoma, as well as other cancers.
Mouse SCCVII cells, which have been previously characterized (
DCs were generated using the method established by Lutz
We determined whether the cells generated could be used as DCs by performing flow cytometry analysis. Surface markers such as MHC class II, CD11c, CD80 and CD86 were highly expressed by 70.6, 35.2, 63.4, and 72.0% of the cells, respectively, indicating that they were indeed DCs.
Syngeneic 6- to 10-week-old female C3H/He mice, purchased from Japan SLC (Shizuoka, Japan), were maintained in our facility under specific pathogen-free conditions. SCCVII cancer cells at a dose of 5×105 per mouse were inoculated subcutaneously into the left leg in order to seed the primary treatment tumor, and the same number of SCCVII cells was simultaneously inoculated subcutaneously into the chest wall to seed a distant, non-treatment tumor. Mice were examined every two or three days, and only those of a similar size were selected for treatment. Animals that had developed palpable tumors 9 days after inoculation prepared for treatment were divided into independent experimental groups, each consisting of at least 4 mice. Animal care was provided in accordance with the guidelines of Chiba University. All animal experiments were conducted to the Guidelines for the Welfare and Use of Animals in Cancer Research (
We used the LAB-EHY device (Oncotherm Ltd. Hungary) to induce mEHT treatment on the left leg tumors of the mice. The RF power level was regulated using the fluoroptic temperature measurement system (Luxtron m3300; Lumasense, Santa Clara, CA, USA). Tumors on the left legs of the mice in the mEHT-only treatment group and in the DC combined with mEHT treatment group were treated on day 9, 11 and 13 after SCCVII cell inoculation. In the DC-only treatment group and DC combined with mEHT treatment group, 1×106 DCs for each mouse were directly administered only into the tumor on the left leg of the mice after the mEHT treatment on day 9, 11 and 13.
After the mice were sacrificed on day 39 after SCCVII cell inoculation, the tumor draining lymph nodes (TDLNs) were collected and washed with PBS. After red blood cell reduction, the cells were treated with mouse BD Fc Block (2.4G2; Pharmingen™, BD) and then stained with antibodies conjugated with fluorescent agents. For the investigation of cytotoxic T lymphocytes (CTLs) in the TDLNs, CD3-PE (2134-0034; Biogenesis Inc., Kingston, NH, USA) and CD8-FITC (2134-0083; Biogenesis Inc.) were used. Cell counting was performed with a Coulter Epics XL cytometer (Beckman Coulter, Miami, FL, USA), and cell populations were evaluated with gating software, FlowJo for Windows (Tree Star Inc., Ashland, OR, USA).
After the mice were sacrificed on day 39 after SCCVII cell inoculation, the tumor tissues on the chest were harvested. The tissues were stained with anti-mouse CD8 (250596; ABBIOTEC), S100 (GEX48819; Gene Tex), Foxp3 (NBP1-18319; Novus Biologicals) and a fluorescein-conjugated secondary antibody (k4003; Dako). The expression of CD8, S100 and Foxp3 was observed under a microscope (Carl Zeiss, Oberkochen, Germany). According to the method developed by Xavie
The RNA was extracted from tumor tissues on the chest using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and 1 μg of RNA was transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The resulting 1 μg of cDNA was amplified via RT-PCR. RT-PCR was conducted using SsoFast™ EvaGreen® Supermix (Bio-Rad, Hercules, CA, USA) with the following primers (Operon, Biotechnology, Tokyo, Japan): GP96, 5′-ACACGGC TTGCTAAACTTCT-3′ and 5′-ACTACAGTCTGCGGTCC AAA-3′. β-actin was used as an internal control and the sequences used were 5′-TCATGAAGATCCTCACCGAG-3′ and 5′-TTGCCAATGGTGATTGACCTG-3′. The PCR was performed using MyiQ2 thermal cycler (Bio-Rad). The PCR conditions consisted of 40 cycles of 95°C for 30 sec, 95°C for 5 sec and 60°C for 10 sec. This assay was performed in triplicate.
Results are expressed as the mean ± SE, and statistical comparisons were performed using ANOVA. A P-value <0.05 was considered to indicate a statistically significant result. All data were analyzed by the SPSS software package (SPSS version 19.0; SPSS Inc., USA).
By using the LAB-EHY device, it was possible to increase the temperature within the tumor to 42°C within 10 min. By controlling the energy input, we were able to maintain the temperature within a range of 42–43°C. At the same time of treatment, we found that the temperature on the surface of the tumor was ~40°C, and the rectal temperature was maintained close to 35–37°C (
We examined the antitumor effect by the combination of DCs and mEHT treatment against the local tumor. The tumor on the left leg of the mice in the control group grew to a mean volume of 1,709.25±2,557 4 mm3 by day 39. The mean volume of the tumor on the left leg in the group treated with a combination of DCs and mEHT was 359.92±62.01 mm3. The tumor growth rate was obviously slow in the DC and mEHT treatment group compared to that in the control group (P<0.05). The mean tumor volume in the DC-only treatment group and the mEHT-only treatment group was smaller compared to the control group (DC treatment group: 902.78±109.74 mm3, P<0.05; mEHT treatment group: 607.19±87.28 mm3, P<0.05) (
We examined the tumors on the chest wall of the mice to investigate whether the combination treatment of DCs and mEHT could elicit a systemic antitumor effect. On day 39, the mean volume of the tumors on the chest wall of the mice in the control group was 852.43±270.78 mm3, whereas the mean volume of the tumors in the DC and the mEHT treatment group was only 99.84±9.21 mm3. The tumor growth rate was obviously slower in the DC and mEHT treatment group compared to the control group (P<0.05). The mean chest tumor volumes in the DC-only treatment group and the mEHT-only treatment group were 396.32±58.52 and 152.78±19.12 mm3, respectively, both of which were also significantly smaller (P<0.05) than the mean tumor volume in the control group (
We investigated whether the intratumoral injection of DCs in addition to mEHT can elicit a systemic antitumor effect
For the immunostaining, the tumor tissues were examined for CD8, S100 and Foxp3 protein expression. The IOD was calculated as the number of positive cells expressing the protein. We found that in the DC plus mEHT treatment group, a greater number of cells were positive for CD8 than the numbers in the other groups (
RT-PCR was used to analyze the cDNA reverse-transcribed RNA of the chest tumors from different groups. As shown in
Head and neck and esophageal cancer are still among the most deadly malignant neoplasms, particularly in the Asian populaton. These cancers have the tendency to metastasize easily to lymph nodes (
Hyperthermia is a type of cancer treatment in which tissue is exposed to a high temperature. Previous studies have shown that high temperatures can damage and kill cancer cells, usually with minimal injury to normal tissues. mEHT which is also described as ‘oncothermia’ in Europe is a personalized treatment using energy delivery to the targeted tumor (
In the present study, we found that treatment of the tumors on the leg of mice resulted in the concurrent growth inhibition of the distant tumors on the chest. This phenomenon is known as the abscopal effect and is defined as a reaction following irradiation but occurring outside the zone of actual radiation absorption. It was first described more than 60 years ago by Dr R.H. Mole, of Britain’s Medical Research Council (
The mechanism that underlies the abscopal effect remains unclear, although studies in mice suggest that it may depend upon activation of the immune system, and is mediated by T cells (
In the present research, we found that mEHT enhanced the expression of GP96
In a case study from the Memorial Sloan-Kettering Cancer Center in New York, changes in a metastatic melanoma patient’s immune system were measured over the course of treatment (
Our findings suggest that the combination of hyperthermia with an intratumoral injection of DCs can induce whole body antitumor effects. Both DC and mEHT therapy are non-invasive, and therefore, this treatment model can be safely adapted for treating patients. Based on our findings, the combination of direct intratumoral administration of DCs and mEHT can be a feasible future treatment, and warrants further studies, including human clinical trials.
This study was supported by the JSPS KAKENHI (grant no. 24501328).
The electric current of the alternating magnetic fields was controlled and the temperature of the thermoseeds was maintained within a range of 42–43°C. The rectal temperature was maintained at 35–37°C.
Tumor growth inhibition results. (A) The effectiveness of the combination treatment of dendritic cells (DCs) and modulated electro-hyperthermia (mEHT). The DC plus mEHT treatment completely suppressed the tumor growth on the left leg of the mice. (B) Tumor growth inhibition mediated by the systemic antitumor effect induced by the combination of DCs and mEHT on the tumors without treatment. In these groups, growth of the distant tumor on the chest wall of the mice without treatment was markedly suppressed, compared with mice treated with DCs and mEHT alone.
Flow cytometric analysis of CD3+ and CD8+ T cells in the tumor draining lymph nodes of mice treated with dendritic cells (DCs) and modulated electro-hyperthermia (mEHT), either alone or in combination. A significant increment in the population of CD3+ and CD8+ T cells was observed in the DC plus mEHT treatment group.
Changes in CD8, S100 and Foxp3 expression in the chest tumor tissues of the different groups (original magnification ×100). (A) Control group. (B) Dendritic cell (DC) treatment group. (C) Modulated electro-hyperthermia (mEHT) treatment group. (D) DC plus mEHT treatment group. (E) The intergrated optical density (IOD) values were examined between the different groups separately.
Analysis of GP96 expression in the different groups. Dendritic cell (DC) plus mEHT treatment upregulated the expression of GP96 compared to the other groups.