Modulated Electro-Hyperthermia Accelerates Tumor Delivery and Improves Anticancer Activity of Doxorubicin Encapsulated in Lyso-Thermosensitive Liposomes in 4T1-Tumor-Bearing Mice

Abstract

Modulated electro-hyperthermia (mEHT) is an adjuvant cancer therapy that enables tumor-selective heating (+2.5 °C). In this study, we investigated whether mEHT accelerates the tumor-specific delivery of doxorubicin (DOX) from lyso-thermosensitive liposomal doxorubicin (LTLD) and improves its anticancer efficacy in mice bearing a triple-negative breast cancer cell line (4T1). The 4T1 cells were orthotopically injected into Balb/C mice, and mEHT was performed on days 9, 12, and 15 after the implantation. DOX, LTLD, or PEGylated liposomal DOX (PLD) were administered for comparison. The tumor size and DOX accumulation in the tumor were measured. The cleaved caspase-3 (cC3) and cell proliferation were evaluated by cC3 or Ki67 immunohistochemistry and Western blot. The LTLD+mEHT combination was more effective at inhibiting tumor growth than the free DOX and PLD, demonstrated by reductions in both the tumor volume and tumor weight. LTLD+mEHT resulted in the highest DOX accumulation in the tumor one hour after treatment. Tumor cell damage was associated with cC3 in the damaged area, and with a reduction in Ki67 in the living area. These changes were significantly the strongest in the LTLD+mEHT-treated tumors. The body weight loss was similar in all mice treated with any DOX formulation, suggesting no difference in toxicity. In conclusion, LTLD combined with mEHT represents a novel approach for DOX delivery into cancer tissue.

Ivermectin Synergizes with Modulated Electro-hyperthermia and Improves Its Anticancer Effects in a Triple-Negative Breast Cancer Mouse Model

“…Mice underwent four mEHT treatments, with 48 h intervals between the treatments (Figure ). mEHT treatment was done using a LabEHY 200 device (Oncotherm kft., Budaörs, Hungary) as described previously. , Briefly, mice under 5% isoflurane anesthesia were placed on a heating pad (i.e., the lower electrode), and the upper electrode was positioned on the tumor. mEHT was conducted at 0.7 ± 0.3 W for 30 min in a temperature-controlled manner, following a 3 min warm-up period.…”

“…FFPE tumors were cut into 2.5 μm sections, mounted on glass slides, and kept in a thermostat set to 65 °C for 1 h. Tumor sections were deparaffinized and rehydrated for hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC). H&E staining was performed as described in detail earlier . H&E-stained tumor sections were scanned and evaluated digitally using the Slide Viewer software (v.2.6, 3DHISTECH, Budapest, Hungary).…”

“…43 IHC for p-HSPB1, cC3, and Kiel-67 (Ki67) was conducted with a Polymer-Peroxidase system (Histols, Histopathology Ltd., Pećs, Hungary) as described in detail earlier. 40 The stained slides were scanned, and the positively stained area was analyzed using the QuantCenter module of Slide Viewer…”

“…H&E staining was performed as described in detail earlier. 40 H&Estained tumor sections were scanned and evaluated digitally using the Slide Viewer software (v.2.6, 3DHISTECH, Budapest, Hungary). The damaged tumor area was annotated from the viable tumor area under high magnification.…”