Molecular Basis of Modulated Electro-Hyperthermia Combination with Radio- and Chemo-Therapies

1. ESHO Molecular basis of modulated electro-hyperthermia combination with radio- and chemo-therapies Oliver Szasz, Ph.D. • Possible conflict of interest and expertise: • CEO of company Oncotherm (1988) • Privatedocent (associate professor) atBiotechnicsDepartment of St.Istvan University

2. Outline • Cellular selection • Molecular selection • Complementary synergy

3. Effects of modulated electrohyperthermia (mEHT) Malignant cell High conductivity (extracellular) (Warburg, nano-effect) Membrane bonds ordering (nano-effect) Interfacial dispersion (Schwan effect, cross-membrane nano-effect Conductivity dependent Permittivity dependent Increased dielectric permittivity High ionic concentration β (δ)-dispersion Membrane rafts and ion-channels Cytoskaleton Due to high metabolic rate, the tumor ionic concentration is high consequently the tumor has conductivity selection  Stochastic resonant (ratchet , nano-effect) Due to missing cellular network, extracellular matrix of malignant cells have high dielectric permittivity selection  Time-fractal amplitude modulation E-cadherin+β-catenin –cytoskeleton complex Due to large number of clusters of transmembrane proteins the protein-lipid complex is attacked, frequency dispersion selection  Pattern recognition (Micro-effect) Produce damage associated molecular pattern (DAMP) and immunogenic cell death (ICD), synchronization selection 

4. Stochastic resonant “ratchet” Complex forming extrinsic signal + fractal modulation RF- carrier Modulated Original thermal energy to destroy (> 42.5 oC) activated complex Necessary electric & thermal energy to destroy Oncothermia “catalysis” advantage by “ratchet” Ea Hyperthermia Oncothermia complex1 complex2 Enzymes in action, Signal excitation, etc. • pushes the reaction into one direction • selects by the autocorrelation time-lag

5. Outline • Cellular selection • Molecular selection • Complementary synergy

6. Cellular selective, heterogeneous heating The three ways of apoptosis is realized: Casp-8 Casp-9 AIF Healthy tissue Targeted tumor mEHT INTRINSIC SMAC/ DIABOLO EXTRINSIC XIAP CASPASE Independent AIF Septin 4 It is thermal excitation, proven by Arrhenius plot Blocking the blocker

7. Three apoptotic pathways are verified Tae-Won Jeon, et al;; (2016 Int.J.Hyperthermia, http://dx.doi.org/10.1080/02656736.2016.1186290 INTRINSIC EXTRINSIC Septin 4 XIAP SMAC/ DIABOLO Andocs G, et al,. (2014) Cell Stress and Chaperones 20(1):37-46, AIF Meggyeshazi N, et al. (2014), StrahlentherOnkol, 190:815-822 CASPASE Independent Meggyeshazi N, et al. (2014), StrahlentherOnkol, 190:815-822 Meggyeshazi N, et al. (2014), StrahlentherOnkol, 190:815-822 Yang K-L, et.al. (2016), Oncotarget, doi: 10.18632/oncotarget.11444

8. Challenges in invasion/dissemination cell 1 cell 2 intracellular electrolyte intracellular electrolyte Inhomogeneous electric field turns the proteins in extracellular electrolyte β-catenin cell-membrane cell-membrane E-cadherin β-catenin α-catenin β-catenin E-cadherin α-catenin p120-catenin E-cadherin p120-catenin Modulated external field helps to reunite the broken connections extracellular electrolyte Malignant autonomy

9. Hyperthermia, 42 oC Hyperthermia, 42 oC Oncothermia, 42 oC Oncothermia 42 oC Block the invasion and dissemination E-cadherin β-catenin Control 37ᴼC Water-bath 42ᴼC Capacitive 42ᴼC Oncothermia 42ᴼC In vitro A431 + human fibroblastco-culture, E-cadherine and β-catenin; 24h afterthetreatments Yang K-L, Huang C-C, Chi M-S, Chiang H-C, Wang Y-S, Andocs G, et.al. (2016) In vitro comparison of conventional hyperthermia and modulated electro-hyperthermia, Oncotarget, oi: 10.18632/oncotarget.11444 Andocs G, Szasz O, Szasz A (2009) Oncothermia treatment of cancer: from the laboratory to clinic. ElectromagnBiol Med 28(2):148–165

10. Outline • Cellular selection • Molecular selection • Complementary synergy

11. Radiation with mEHT In vitro SCCVII (SCC7), a mouse head and neck carcinoma cell line In vivo oral-cancer (SAS) cell-line Matsumoto Y. et al. (2018), 35th Annual conference of Japanese Thermal Society, Fukui August 27-28 Airi OTA et al.(2017) Conf. JHA of Cancer X-rays Water-bath 41 °C Water-bath 42 °C mEHT 42 °C mEHT 41 °C In vivo lung tumor (A549 and HCI-H1299 cell-lines; Bibin Prasad, et al; (2019); Scientific reports 9:3942; www.nature.com/scientificreports

12. mEHT with doxorubicin chemotherapy (Poster P01) Vancsik T et al (2019) Modulated electro-hyperthermia induced p53 driven apoptosis and cell cycle arrest additively support doxorubicin chemotherapy of colorectal cancer in vitro,

13. mEHT + DC therapycombination Treated femoral region Tumor induced on femoral region (treated by mEHT) sham control Tumor induced on chest region like model of metastasis, not treated by mEHT Clear abscopal effect on chest Wei Quin et.al; Modulated electro-hyperthermia enhances dendritic cell therapy through an abscopal effect in mice; Oncology Reports 32: 2373-2379, 2014

14. Thank you very much Oliver Szasz, PhD, associateprofessor biotech@gek.szie.hu This work was supported bytheHungarianCompetitiveness and Excellence Programme grant (NVKP_16-1-2016-0042).

15. Outline • Cellular selection • Molecular selection • Verification • Applications

16. mEHT heats differently (mRNA-based info) (42⁰C) Human lymphoma U937 cell (in-vitro) cell death-related genes, such as EGR1, JUN, and CDKN1A 1. Control (37⁰C) [CTRL] . 2. Water-bath (42⁰C) [WHT] (42⁰C) 1 2 3 (42⁰C) mEHT (42⁰C) cytoprotective gene network (HSPs) 3 . (42⁰C) Upregulated gene-group (42⁰C) (42⁰C) ERK activation and the relationship of JUN and ERK Andocs G, et al. (2016) Cell Death Discovery (Nature Publishing Group), 2, 16039

17. mEHT with mitomycin-C 66.1 70 60 50 Efficacy of cell killing (active – control)(%) 40 30 20 7.7 10 0 Hyperthermia 42oC Oncothermia 42oC Aim of the study: Investigating the difference of the effects of hyperthermia and oncothermia combined both method with mitomycin-c (MMC) single dose chemotherapy in vivo at tissue and cellular level using histological examinations. Animals: HT29 human colorectal carcinoma cell line derived xenograft tumor model in nude mouse. As a result of oncothermia treatment the dead part of the tumor grew 66% compared to the non-treated tumor pair, while this rate was hardly 8% in case of hyperthermia. Oncothermia drastically enhances the antitumor effect of the MMC!

18. Randomized study (n=6+6) for pharmacokinetics mEHT (with Nefopam) Lee SY, Kim M-G (2015); Int J Hyp, 31:869; 2015 mEHT effect Despite the mild overall heating mEHT significantly increases the maximum of the kinetic curve (Cmax ) and also significantly decreases the time at maximum (Tmax) parameters. The area under the peak (AUC) was stable, indicating the unchanged systemic adverse effects, despite the increase of the absorption of the drug. Conventional hyperthermia (with Mitomycin-C) Sørensen O, et al. (2014) Impact of Hyperthermia on Pharmacokinetics of Intraperitoneal Mitomycin C in Rats …, Journal of Surgical Oncology;109:521–526 This effect depends on the permeability of the vessel-wall, which is increased by the applied electric field. MMC concentration Time (min)

19. Prospective study (n=20) for blood-flow changes in cervix Biopsy proven cervical carcinoma by mEHT therapy. Peri-tumor temperature: internal organ temperature probe. Tumor blood flow: by determining the peak systolic velocity/end-diastolic velocity ratio (S/D ratio) and the resistance index (RI) within blood vessels. (3D color Doppler ultrasound) Lee S-Y, Kim JH, Han YH, Cho DH; Int. J. Hyperthermia, Jan 3:1-24, (2018) 38.5ᴼC 60 min (S/D and RI are displayed in negative for guiding of the eye) 60→150W 6 min steps up 400→1600W 5 min steps up 39.2ᴼC 1. 0. 3. 2. D. Fatehi, Technical Quality of Deep Hyperthermia Using the BSD-2000, Uitgeverij Box Press, Oisterwijk, The Netherlands, 2007, PhD.

20. mEHT with radiotherapy – hypoxia investigation FSaII tumors three days after treatment. Radiation: Tumors were irradiated with 15 Gy. Oncothermia: Tumors were heated once at 41 °C for 30 min. Radiation+Oncothermia: Tumors were irradiated with 15 Gy and immediately heated once at 41 ° C for 30 min Five fields in each of the three tissue sections per tumor were studied and the % positive area calculated. Means of five tumors ±1 SE are shown. _indicates p<0.05. Wonwoo Kim, Mi-Sook Kim, Hee-jong Kim, Eunjin Lee, Jae-hoon Jeong, Inhwan Park, YounKyoungJeong & Won Il Jang (2017): Role of HIF-1α in response of tumors to a combination of hyperthermia and radiation in vivo, International Journal of Hyperthermia, DOI: 10.1080/02656736.2017.1335440; https://doi.org/10.1080/02656736.2017.1335440

21. Oncothermia with radiotherapy (FSaII tumors) Radiation: Tumors were irradiated with 15 Gy. Oncothermia: Tumors were heated once at 41°C for 30 min. Radiation+Oncothermia: Tumors were irradiated with 15 Gy and immediately heated once at 41°C for 30 min Three days after treatment HIF-1a → hypoxia inducible factor CA9 → is a cellular biomarkers of hypoxia VEGF → Vascular endothelial growth factor Ten images were captured for each of the three tissue sections per tumor and % positive areas calculated. Means of 4–6 tumors ±1 SE are shown. *indicates p<0.05. Wonwoo Kim, et al. (2017) Int.J.Hyp. DOI: 10.1080/02656736.2017.1335440; tps://doi.org/10.1080/02656736.2017.1335440