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. 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. 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. 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.

The natural killer cell line, NK-92MI, is cytotoxic against various types of cancer. The aim of this study was to develop chimeric antigen receptor-modified (CAR) NK-92MI cells targeting carcinoembryonic antigen-expressing (CEA) tumours and increase killing efficacy by pharmacologically modifying CEA-expression. We generated anti-CEA-CAR NK-92MI cells by retroviral vector transduction. This genetically-modified cell line recognised and lysed high CEA-expressing tumour cell lines (LS174T) at 47.54 ± 12.60% and moderate CEA-expressing tumour cell lines (WiDr) at 31.14 ± 16.92% at a 5:1 effector: target (E/T) ratio. The cell line did not lyse low CEA-expressing tumour cells (HCT116) as they did their parental cells (NK-92MI cells). The histone deacetylase-inhibitor (HDAC) sodium butyrate (NaB) and the methylation-inhibitor 5-azacytidine (5-AZA), as epigenetic modifiers, induced CEA-expression in HCT116 and WiDr cells. Although the IC50 of 5 fluorouracil (5-FU) increased, both cell lines showed collateral sensitivity to anti-CEA-CAR NK-92MI cells. The cytolytic function of anti-CEA-CAR NK-92MI cells was increased from 22.99 ± 2.04% of lysis background to 69.20 ± 11.92% after NaB treatment, and 69.70 ± 9.93% after 5-AZA treatment, at a 10:1 E/T ratio in HCT116 cells. The WiDr cells showed similar trend, from 22.99 ± 4.01% of lysis background to 70.69 ± 10.19% after NaB treatment, and 59.44 ± 10.92% after 5-AZA treatment, at a 10:1 E/T ratio. This data indicates that the effector-ability of anti-CEA-CAR NK-92MI increased in a CEA-dependent manner. The combination of epigenetic-modifiers like HDAC-inhibitors, methylation-inhibitors, and adoptive-transfer of ex vivo-expanded allogeneic-NK cells may be clinically applicable to patients with in 5-FU resistant condition.

A cationic liposome-PEG-PEI complex (LPPC) was employed as a carrier for achieving targeted delivery of drug to human epidermal growth factor receptor-2 (HER2/neu)-expressing breast cancer cells. LPPC can be easily loaded with an anti-tumor drug and non-covalently associated with an anti-tumor antibody such as Herceptin that is clinically used to rapidly form immunoparticles within 1 h. Drug-loaded LPPC have an average size about 250 nm and a zeta potential of about 40 mV. Herceptin was complexed onto surface of the LPPC to form the drug/LPPC/Herceptin complexes. The size of curcumin/LPPC/Herceptin complexes were 280 nm and the zeta potentials were about 23 mV. Targeting ability of this delivery system was demonstrated through specific binding on surface of cells and IVIS images in vivo, which showed specific binding in HER2-positive SKBR3 cells as compared to HER2-negative Hs578T cells. Only the drug/LPPC/Herceptin complexes displayed dramatically increased the cytotoxic activity in cancer cells. Both in vitro and in vivo results indicated that Herceptin adsorbed on LPPC directed the immunocomplex towards HER2/neu-positive cells but not HER2/neu-negative cells. The complexes with either component (curcumin or doxorubicin) used in the LPPC-delivery system provided a better therapeutic efficacy compared to the drug treatment alone and other treatment groups, including clinical dosages of Herceptin and LipoDox, in a xenografted model. LPPC displays important clinical implications by easily introducing a specific targeting characteristic to drugs utilized for breast cancer therapy.

Background: Modulated electro-hyperthermia (mEHT) is a form of hyperthermia used in cancer treatment. mEHT has demonstrated the ability to activate host immunity by inducing the release of heat shock proteins, triggering apoptosis, and destroying the integrity of cell membranes to enhance cellular uptake of chemo-drugs in tumor cells. Both curcumin and resveratrol are phytochemicals that function as effective antioxidants, immune activators, and potential inhibitors of tumor development. However, poor bioavailability is a major obstacle for use in clinical cancer treatment. Methods: This purpose of this study was to investigate whether mEHT can increase anti-cancer efficacy of nanosized curcumin and resveratrol in in vitro and in vivo models. The in vitro study included cell proliferation assay, cell cycle, and apoptosis analysis. Serum concentration was analyzed for the absorption of curcumin and resveratrol in SD rat model. The in vivo CT26/BALB/c animal tumor model was used for validating the safety, tumor growth curve, and immune cell infiltration within tumor tissues after combined mEHT/curcumin/resveratrol treatment. Results: The results indicate co-treatment of mEHT with nano-curcumin and resveratrol significantly induced cell cycle arrest and apoptosis of CT26 cells. The serum concentrations of curcumin and resveratrol were significantly elevated when mEHT was applied. The combination also inhibited the growth of CT26 colon cancer by inducing apoptosis and HSP70 expression of tumor cells while recruiting CD3+ T-cells and F4/80+ macrophages. Conclusions: The results of this study have suggested that this natural, non-toxic compound can be an effective anti-tumor strategy for clinical cancer therapy. mEHT can enable cellular uptake of potential anti-tumor materials and create a favorable tumor microenvironment for an immunological chain reaction that improves the success of combined treatments of curcumin and resveratrol.

Purpose: Modulated electro-hyperthermia (mEHT) stands to be a significant technological advancement in the hyperthermia field, utilizing autofocusing electromagnetic power on the cell membrane to create massive apoptosis. Since mEHT possesses the unique ability to excite cell membranes, we hypothesized that mEHT could enhance the uptake of liposomal drugs by enhancing phagocytic activity. Materials and methods: Water bath control and mEHT were used to compare the enhancement of liposome-encapsulated doxorubicin (Lipodox^®) uptake by cancer cells. Cancer cells were made visible by doxorubicin fluorescence to investigate drug uptake. Viable cell yield was determined via the Trypan Blue exclusion method. Various substrates were used to investigate the mechanism of drug-uptake enhancement. The murine colon carcinoma model, CT26, was used to confirm the tissue infiltration of Lipodox^® and its therapeutic effect. Results: mEHT treatment showed a significant enhancement of Lipodox^® uptake of doxorubicin fluorescence compared with 37°C or 42°C water bath treatment. Tumor tissue sections also confirmed that mEHT treatment achieved the highest doxorubicin concentration in vivo (1.44±0.32 µg/g in mEHT group and 0.79±0.32 µg/g in 42°C water bath). Wortmannin was used to inhibit the macropinocytosis effect and 70 kDa dextran-FITC served as uptake substance. The uptake of dextran-FITC by cancer cells significantly increased after mEHT treatment whereas such enhancement was significantly inhibited by wortmannin. Conclusion: The result showed mEHT-induced particle-uptake through macropinocytosis. mEHT-enhanced uptake of Lipodox^® may amplify the therapeutic effect of liposomal drugs. This novel finding warrants further clinical investigation.