PhD Scientific Days 2023

Translational Medicine II.

Digoxin mediated inhibition of potential hypoxia related angiogenic repair in murine triple-negative breast cancer tumors treated with modulated electro-hyperthermia (mEHT)

Text of the abstract

Introduction
Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer type with no targeted therapy, hence limited treatment options. Modulated electro-hyperthermia (mEHT) is a novel complementary therapy where a 13.56 MHz radiofrequency current targets cancer cells selectively, inducing tumor damage by thermal and electromagnetic effects. Complementary mEHT treatment can therefore improve the efficacy of available TNBC treatments. However, the astounding ability of tumor cells to adapt and evade therapy is a constant challenge, thus a better understanding of the therapeutic effects of mEHT has great translational potential. We observed angiogenic alterations in mEHT treated tumors and aimed to investigate the effects of mEHT on angiogenesis in our TNBC mouse model.
Aims
We aim to inhibit this hypoxia-mediated repair by using digoxin; a cardiac glycoside drug, as a Hypoxia inducible factor (HIF1-a) inhibitor and investigate the effect of this inhibition on angiogenic repair and tumor growth
Methods
TNBC murine isografts were treated three to five times with LabEHY 200 at 0.7 ± 0.3 W for 30 min every 48 hours and daily intraperitoneal injection of digoxin. Tumor growth was monitored with ultrasound and digital calipers. Tumor destruction histology, blood capillary damage, and molecular changes were detected using immunohistochemistry, next generation sequencing, and Nanostring technology.
Results
mEHT induced capillary damage detected as free red blood cells in the interstitium of TNBC isografts on HE stained slides, 24h after 3 mEHT treatments in a time-dependent manner starting 4h post-treatment. The bleeding peaked at 12h and interstitial red blood cell count was significantly reduced by 24h. Similar bleeding was observed after five mEHT treatments and the number of viable blood vessels was significantly reduced as assessed by CD31 immunostaining.
Significant upregulation of stress-related genes in response to mEHT treatment was observed in NGS corroborated by nanostring and MS, which may be partially due to capillary damage-mediated hypoxia. Furthermore, angiogenic repair in response to mEHT was detected by a reduction and subsequent continuous upregulation of tumor vasculature marker CD105. Additionally, tumor angiogenic marker CD-31 staining was reduced at 12h followed by an upregulation after 24 hours to sham level.
Treatment with digoxin, a cardiac glycoside also known to reduce expression of Hypoxia Inducible Factor (HIF-1a), in combination with mEHT demonstrated a reduction in tumor volume, tumor weight, and tissue hypoxia. We further aim to identify, if this reduction in tumor hypoxia is translated to reduced tumor angiogenesis and enhanced tumor damage.
Conclusion
Our data suggest that mEHT induces blood capillary damage and triggers a stress response that repairs the tumor vasculature 24 hours after the last mEHT treatment. Combining mEHT treatment with digoxin can reduce tumor tissue hypoxia and can potentially reduce this angiogenic repair in tumor cells and hence can enhance the effectivity of mEHT and potentially other forms of cancer treatment.
Funding Funding: EFOP-3.6.3-VEKOP-16-2017-00009