Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.

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Background Tumor Treating Fields (TTFields), alternating electric fields with intermediate field intensity, have shown clinical efficacy in several tumor entities, leading to clinical approval for glioblastoma and mesothelioma treatment. As mesenchymal stromal cells (MSCs) belong to the tumor microenvironment and exhibit - depending on the tumor type and microenvironment - either pro-tumorigenic or anti-tumorigenic abilities, we aimed to study the effects of TTFields on human MSCs. Methods Human MSCs were isolated from the bone marrow of three different healthy donors and were exposed to TTFields for 72 hours. MSCs’ clonogenic survival, proliferation and viability was analyzed after different TTFields frequencies. Boyden chamber assays were carried out to investigate the influence of TTFields on the migratory potential of MSCs. Flow cytometry analyses of caspase-3 and annexin V were conducted to determine MSCs’ apoptosis rates after TTFields treatment. TTFields-induced senescence was quantified using beta-galactosidase stainings. MSCs’ characteristic surface marker expression (CD73, CD90 and CD105) was studied after TTFields treatment. Result TTFields led to significantly reduced clonogenic survival (70-90% reduction compared to controls) and cellular proliferation (approximately 50% reduction) in all analyzed MSC specimens. However, MSCs’ metabolic viability was largely unaffected after TTFields exposure. Within a range between 100 and 500 kHz, there was no frequency dependence of TTFields’ anti-proliferative effect on human MSCs. As determined by Bodyen Chamber assays, TTFields resulted in a considerable reduction of MSCs’ migratory potential. TTFields increased both cellular apoptosis and senescence in all MSC samples, whereas there was no alteration of MSCs’ characteristic surface marker pattern. Conclusion As TTFields considerably reduce MSCs’ survival and migratory potential, TTFields could attenuate the pro-tumorigenic effects of tumor-associated MSCs that were recently described in glioblastoma patients. Co-culture experiments of MSCs and tumor cells after TTFields treatment as well as in vivo experiments are planned in the future. Citation Format: Alexander Rühle, Maren Strack, Anca-Ligia Grosu, Nils H. Nicolay. Tumor Treating Fields reduce cellular survival of human mesenchymal stromal cells via apoptosis and senescence induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3156.