PhD Scientific Days 2023

Pathology - Posters D

Synergism between methotrexate and modulated electro-hyperthermia is mediated by H19 lnc-RNA

Text of the abstract

Introduction: Modulated electro-hyperthermia (mEHT) is a novel antitumor therapy, with strong tumor-progression inhibiting potential according to in vitro, in vivo experiments and clinical studies in monotherapy and as complementary therapy as well. Implantation of 4T1 cancer cells into immunocompetent mice serves as a reliable model for triple negative breast cancer (TNBC). H19 long non-coding RNA promotes breast cancer cell invasion, migration and metastasis.
Aims: Our aim was to investigate the effect of mEHT on H19 in TNBC mouse model
Material and method: In-vitro 4T1 spheroids were treated with mEHT for 30 minutes. In-vivo TNBC cells (4T1, 4T07) were inoculated orthotopically in female BALB/c mice. Tumor growth was monitored in vivo by digital caliper and ultrasound. Mice were randomized into groups treated with mEHT monotherapy or in combination with methotrexate (MTX) 2 or 3 times for 30 minutes. H19 expression was measured with RT-PCR from dissected tumors. Histological evaluation of tumor sections were performed by hematoxylin-eosin (H&E) and cleaved caspase-3 (cC3) immunohistochemistry stainings.
Results and discussion: H19 expression correlated with tumor aggressiveness (4T07:0.006±0.004 vs 4T1:0.4±0.07, p<0.0001). Single mEHT treatment of 4T1 spheroids reduced H19 expression vs normothermic control (Ctrl: 0.004±0.0004, mEHT: 0.0006±0.0002, p<0.0001). In vivo, MTX diminished metastatic burden (meta/lung area: Ctrl: 5.942±4.500 %, MTX: 1.723±1.138 %). mEHT and MTX significantly reduced tumor size and weight in monotherapy and combination of mEHT with MTX demonstrated synergistic effectiveness. mEHT but not MTX induced apoptosis (cC3+ area: sham: 18.8±11.4, mEHT: 50.9±26.2, MTX: 15.4±9.5, mEHT+MTX: 57.6±13.5%, p<0.0001). Tumor size reduction was accompanied by reduction of H19 expression (sham: 0.068±0.044, mEHT: 0.033±0.024, MTX: 0.104±0,038, mEHT+MTX: 0.056±0.025, p<0.01). H19 reduction peaked 12 hours after mEHT, recovering slowly by 48-72 hours. Furthermore, MTX induced H19 overexpression was reversed by cotreatment with mEHT (sham: 0.16±0.07, mEHT: 0.11±0.06, MTX: 0.21±0.08, mEHT+MTX: 0.11±0.05, p<0.05).
Conclusion: MTX and mEHT synergize each other’s tumor-killing effect, making their combination a potential therapeutic option. The observed synergism may be based on 1) different mechanisms of action: mEHT killing viable cells through apoptosis induction and MTX killing proliferating cells, and 2) inhibition of H19 lncRNA involved in both apoptosis and cancer cell proliferation.
Funding: NVKP_16-1-2016-0042; ÚNKP-22-4-I-SE-15; STIA-OTKA to PH; 2021-1.2.4-TÉT-2021-00060