Objective: Modulated electro‐hyperthermia (mEHT), a noninvasive complementary treatment of human chemo‐ and radiotherapy, can generate selective ~42°C heat in cancer due to elevated glycolysis (Warburg‐effect) and electric conductivity in malignant tissues. Here we tested the molecular background of mEHT and its combination with doxorubicin chemotherapy using an in vitro model.Methods: C26 mouse colorectal adenocarcinoma cultures were mEHT treated at 42°C for 2 × 60 minutes (with 120 minutes interruption) either alone or in combination with 1 µmol/L doxorubicin (mEHT + Dox). Cell stress response, apoptosis, and cell cycle regulation related markers were detected using qPCR and immunocytochemistry supported with resazurin cell viability assay, cell death analysis using flow‐cytometry and clonogenic assay.Result: Cell‐stress by mEHT alone was indicated by the significant upregulation and release of hsp70 and calreticulin proteins 3 hours posttreatment. Between 3 and 9 hours after treatment significantly reduced anti‐apoptotic XIAP, BCL‐2, and BCL‐XL and elevated pro‐apoptotic BAX and PUMA, as well as the cyclin dependent kinase inhibitor p21^waf1 mRNA levels were detected. After 24 hours, major elevation and nuclear translocation of phospho‐p53(Ser15) protein levels and reduced phospho‐Akt(Ser473) levels were accompanied by a significant caspase‐3‐mediated programmed cell death response. While mEHT dominantly induced apoptosis, Dox administration primarily led to tumor cell necrosis, and both significantly reduced the number of tumor progenitor colonies 10 days post‐treatment. Furthermore, mEHT promoted the uptake of Dox by tumor cells and the combined treatment additively reduced tumor cell viability and augmented cell death near to synergy.Conclusion: In C26 colorectal adenocarcinoma mEHT‐induced irreversible cell stress can activate both caspase‐dependent apoptosis and p21^waf1 mediated growth arrest pathways, likely to be driven by the upregulated nuclear p53 protein. Elevated phospho‐p53(Ser15) might contribute to p53 escape from mdm2 control, which was further supported by reduced phospho‐Akt(Ser473) protein levels. In combinations, mEHT could promote the uptake and significantly potentiate the cytotoxic effect of doxorubicin.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.

Auranofin, an FDA-approved antirheumatic drug and thioredoxin reductase 1 (TXNRD1) inhibitor, has demonstrated anti-tumoral properties, but its immunological effects are not well characterized. Here, we report that auranofin unexpectedly promotes regulatory T cell (Treg) expansion. In a B16F10 melanoma model, auranofin treatment increased lung tumor coverage, IL-10 serum levels, and FOXP3⁺CD44⁺CD4⁺ T cell frequencies. It also altered the proportion of antigen-presenting cells (APCs), increasing B cells and reducing dendritic cells. To test whether Treg expansion occurs independently of tumor antigens, we stimulated T cells ex vivo in lymph node cultures from naïve mice using anti-CD3/CD28, with or without auranofin. Auranofin increased Treg frequency in these cultures, as well as in treated human PBMCs. Similar effects were observed with the TXNRD1 inhibitor TRi-1, suggesting a ROS-dependent mechanism. Using mice with conditional expression of neutrophil cytosolic factor 1 (NCF1), we found that both TXNRD1 inhibition and APC-specific NCF1-NOX2-ROS expression enhanced tumor burden and Treg expansion. Alternatively, sorted T cells from mice harboring conditional TXNRD1 knockouts showed reduced FOXP3 and GITR expression in the naïve state and reduced tumor burden when challenged with B16F10. These data suggest TXNRD1 inhibitors likely drive Treg expansion by elevating ROS levels in APCs during T cell priming and less by intrinsic Treg TXNRD1 blockade. Our findings reveal a paradoxical immunosuppressive effect of TXNRD1 inhibitors that may contribute to their limited efficacy in immunocompetent cancer models. This work provides mechanistic insight and underscores the need to consider Treg-mediated immune suppression when designing TXNRD1-targeted therapies.