Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy

Abstract

Background: The treatment of intratumoral dentritic cells (DCs) commonly fails because it cannot evoke immunity in a poor tumor microenvironment (TME). Modulated electro-hyperthermia (mEHT, trade-name: oncothermia) represents a significant technological advancement in the hyperthermia field, allowing the autofocusing of electromagnetic power on a cell membrane to generate massive apoptosis. This approach turns local immunogenic cancer cell death (apoptosis) into a systemic anti-tumor immune response and may be implemented by treatment with intratumoral DCs.

Methods: The CT26 murine colorectal cancer model was used in this investigation. The inhibition of growth of the tumor and the systemic anti-tumor immune response were measured. The tumor was heated to a core temperature of 42 °C for 30 min. The matured synergetic DCs were intratumorally injected 24 h following mEHT was applied.

Results: mEHT induced significant apoptosis and enhanced the release of heat shock protein70 (Hsp70) in CT26 tumors. Treatment with mEHT-DCs significantly inhibited CT26 tumor growth, relative to DCs alone or mEHT alone. The secondary tumor protection effect upon rechallenging was observed in mice that were treated with mEHT-DCs. Immunohistochemical staining of CD45 and F4/80 revealed that mEHT-DC treatment increased the number of leukocytes and macrophages. Most interestingly, mEHT also induced infiltrations of eosinophil, which has recently been reported to be an orchestrator of a specific T cell response. Cytotoxic T cell assay and ELISpot assay revealed a tumor-specific T cell activity.

Conclusions: This study demonstrated that mEHT induces tumor cell apoptosis and enhances the release of Hsp70 from heated tumor cells, unlike conventional hyperthermia. mEHT can create a favorable tumor microenvironment for an immunological chain reaction that improves the success rate of intratumoral DC immunotherapy.

Figures

Fig. 1

Apoptosis in mEHT-treated CT26 cells. One and a half million CT26 cells were heated to 42 °C for 30 min using LabEHY or a water bath (control). The apoptosis of CT26 cells after 24 h of hyperthermia treatment was analyzed using Annexin-V assay. (*, p < 0.05; n = 3)

Fig. 2

Expression and release of Hsp70 following hyperthermia treatment. One and a half million CT26 cells were heated to 42 °C for 30 min using LabEHY or a water bath (control). a After 24 h of incubation, cells were harvested for western blot analysis of Hsp70 or HMGB1 protein in lysates of CT26 (control) (37 °C), a water bath (control) (42 °C) or mEHT. β-actin was used as an internal control. b After 24 h of incubation, supernatants were harvested and concentration of Hsp70 was measured using ELISA (n = 3). Data are presented as mean +/− SD from three independent experiments. * indicates p < 0.05

Fig. 3

Inhibition of tumor growth and rechallenge inoculation. a Mice in different groups were injected with 5 × 105 CT26 tumor cells (subcutaneously) in right femoral area on day zero and treated with mEHT on day 14, before receiving DC injection on day 15. Data obtained from each mouse after tumor-cell inoculation (n = 7) were plotted. b A secondary rechallenge with CT26 tumor cells was administered to mice 30 days after first injection with DC alone or following mEHT or mEHT-DC therapy. Contra-lateral flanks of mice in treated groups and untreated control BALB/c mice were inoculated subcutaneously (1 × 105 parental CT26 cells). Percentage of mice that developed tumors at contra-lateral site was obtained using Kaplan–Meier method. (n = 7 mice per group.)

Fig. 4

Areas of tumor infiltrated by immune cells after mEHT and DC treatment. Representative images of immunohistochemical straining revealed that quantities of CD45 (a) and F4/80 (b) were increased in tumors that were treated with DC and mEHT, either alone or in combination. Proportion of positive cells in one field that was randomly selected from ten fields, was calculated. c Amount of eosinophil increased significantly upon treatment of tumor; error bars represent standard errors. (***) P < 0.001 (t-test) relative to control. EH: electro-hyperethermia

Fig. 5

Tumor-specific CTLs. Mice in various groups were injected with 5 × 105 CT26 tumor cells subcutaneously on day zero and with mEHT on day 14, before being given DC injection on day 15. On day 30 following tumor injection, splenocytes were harvested for CTL assay. Cytotoxic activity of splenocytes was determined by LDH-release assay at various effector/target cells (E/T) ratios

Fig. 6

ELISpot assays. a Representative results of ELISpot assay of mice splenocytes that were pulsed with AH1. Top two rows, 2 × 105 splenocytes/well; bottom two rows, 105 splenocytes/well. PC: positive control, splenocytes treated with ConA (5 mg/ml) for 24 h. NC: negative control, splenocytes treated with BSA for 24 h. b Numbers of IFN-γ-secreting T-cells in DC-treated and mEHT + DC-treated mice significantly exceeded those in mice treated with mEHT alone and untreated control mice. Error bars represent standard errors. (***) P < 0.001 (t-test) relative to control. EH: electro-hyperethermia