PhD Scientific Days 2024

Poster Session K - Theoretical and Translational Medicine 2.

Potential Regulating Mechanisms behind Modulated Electro-hyperthermia and Antifolate Chemotherapy Synergism

Text of the abstract

Introduction: Triple Negative Breast Cancer (TNBC) has the worst prognosis among breast cancer types, due to the lack of hormone and HER2 targets. Modulated electro-hyperthermia (mEHT) is a local hyperthermia method, able to transfer heat to solid tumors even in deep regions. MEHT is used in the clinics mostly as complementary treatment for chemotherapy. H19 is Long Non-coding RNA (Lnc RNA), promoting various pro-tumoral cellular mechanisms via regulating pathways including Wnt-β-catenin pathway. Inoculation of 4T1/4T07 in the mammary area of Balb/c mice allows to study TNBC.
Aims: to investigate the potential therapeutic effects of MTX and mEHT combination
Method: mEHT treatment of TNB (4T1) spheroids was performed with in vitro applicator. 4T1/4T07 cells were inoculated into BALB/c mice in vivo. Digital caliper and ultrasound measurements were performed repeatedly for tumor growth follow-up. Sham and mEHT+/-Methotrexate (MTX) treated groups were randomized, based on tumor volume. H19 expression was measured with RT-PCR. Hematoxylin-eosin (H&E) and cleaved caspase-3 (cC3) immunohistochemistry stainings were made from the tumors. Mass Spectrometry (MS) was performed from tumor samples.
Results and discussion: 1x repeated mEHT treatment reduced H19 expression in spheroids (ctrl: 0.004±0.0004, mEHT: 0.0006±0.0002, p<0.0001). H19 expression correlated with tumor aggressiveness (4T07:0.006±0.004 vs 4T1:0.4±0.07, p<0.0001). MTX decreased metastatic area in the lungs and combination of mEHT and MTX inhibited tumor growth with synergistic effectiveness. mEHT induced caspase-mediated 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. H19 was overexpressed by MTX, and reversed by 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). β-catenin expression was significantly decreased by mEHT (MS: sham: 23,7 ± 0,3, mEHT: 21,9 ± 0,5 LFQ).
Conclusion: MTX treatment’s anti-tumoral effect is synergistically enhanced by mEHT in TNBC preclinical model. MTX-induced Lnc RNA H19 expression is reversed by mEHT. Decreased activation of Wnt-pathway by mEHT may explain the molecular mechanism of synergism between MTX and mEHT.
Funding: NVKP_16-1-2016-0042; ÚNKP-23-4-I-SE-22; OTKA_K 145998; HUN-REN Hungarian Research Network