# Targeting DAMPs by Aspirin Inhibits Head and Neck Cancer Stem Cells and Stimulates Radio-Sensitization to Proton Therapy

## TL;DR

Aspirin and proton therapy together can target cancer stem cells in head and neck cancer by inhibiting harmful signals.

## Contribution

A new combination therapy using aspirin, poly (I:C), and proton irradiation to target resistant cancer stem cells is proposed.

## Key findings

- TLR3 activation promotes stemness and cancer cell migration in head and neck cancer.
- Aspirin and metformin inhibit DAMP secretion and reverse TLR3-induced migration.
- Proton therapy combined with aspirin and poly (I:C) effectively eliminates cancer stem cells.

## Abstract

This article investigates the pro-tumorigenic role of Toll-like receptor 3 (TLR3) and its influence on cancer stem cells (CSCs) in head and neck cancer. We demonstrate that TLR3 activation promotes stemness in head and neck cancer cell lines. Furthermore, it triggers the secretion of damage-associated molecular patterns (DAMPs), increasing cancer cell migration—an effect that is reversible with common drugs serving as DAMP inhibitors like aspirin and metformin. Importantly, this study identifies a potential therapeutic strategy targeting CSCs, which are often resistant to conventional radio- and chemotherapy, using a combination of aspirin, poly (I:C), and proton irradiation.

Background: Cancer stem cells (CSCs) are a subpopulation of cancer cells known for their self-renewal capacity, tumorigenicity, and resistance to treatment. Toll-like receptor 3 (TLR3) plays a complex role in cancer, exhibiting both pro-apoptotic and pro-tumorigenic effects. This study investigates the pro-tumorigenic role of TLR3, specifically its impact on CSCs in head and neck cancer. Methods: We have investigated Detroit 562, FaDu and SQ20B cell lines, the latter being stably transfected with a plasmid containing inducible shRNA for TLR3, by cultivating them to form tumor spheres in order to study CSCs. Results: Our findings demonstrate that TLR3 activation promotes stemness in head and neck cancer cell lines. This is evidenced by increased tumor sphere formation, promotion of epithelial-to-mesenchymal transition (EMT), upregulated stemness gene expression, and elevated aldehyde dehydrogenase (ALDH) activity. Conditional TLR3 knockdown abolished tumor sphere formation, confirming its important role. Furthermore, TLR3 activation triggers the secretion of damage-associated molecular patterns (DAMPs) into the tumor microenvironment, leading to increased cancer cell migration. This was inhibited by DAMP inhibitors. In patient tissue samples, we observed co-localization of TLR3 with stemness markers CD133 and ALDH1, as well as with heat shock protein 70 (HSP70) and receptor for advanced glycation end products (RAGE). We then explored potential CSC-targeted therapies, initially combining the apoptosis inducer poly (I:C) with DAMP inhibitors and γ-irradiation. While this combination proved effective in adherent cells, it failed to eliminate tumor spheres. Nevertheless, we discovered that proton radiotherapy, particularly when combined with aspirin (HMGB1 inhibitor) and poly (I:C), effectively eliminates CSCs. Conclusions: This novel combination holds promise for the development of new therapeutic strategies for head and neck cancers, particularly given the promising results of proton therapy in treating this disease.