Effects of tumor treating fields (TTFields) on human mesenchymal stromal cells.

PURPOSE: Mesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs. METHODS: Single-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC' characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs' migratory potential was evaluated with Boyden chamber assays. RESULTS: Single-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs' migratory capacity. CONCLUSION: We could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields.

Human Mesenchymal Stromal Cells Do Not Cause Radioprotection of Head-and-Neck Squamous Cell Carcinoma.

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.

Human mesenchymal stromal cells maintain their stem cell traits after high-LET particle irradiation - Potential implications for particle radiotherapy and manned space missions.

The influence of high-linear energy transfer (LET) particle radiation on the functionalities of mesenchymal stromal cells (MSCs) is largely unknown. Here, we analyzed the effects of proton (1H), helium (4He), carbon (12C) and oxygen (16O) ions on human bone marrow-MSCs. Cell cycle distribution and apoptosis induction were examined by flow cytometry, and DNA damage was quantified using γH2AX immunofluorescence and Western blots. Relative biological effectiveness values of MSCs amounted to 1.0-1.1 for 1H, 1.7-2.3 for 4He, 2.9-3.4 for 12C and 2.6-3.3 for 16O. Particle radiation did not alter the MSCs' characteristic surface marker pattern, and MSCs maintained their multi-lineage differentiation capabilities. Apoptosis rates ranged low for all radiation modalities. At 24 h after irradiation, particle radiation-induced ATM and CHK2 phosphorylation as well as γH2AX foci numbers returned to baseline levels. The resistance of human MSCs to high-LET irradiation suggests that MSCs remain functional after exposure to moderate doses of particle radiation as seen in normal tissues after particle radiotherapy or during manned space flights. In the future, in vivo models focusing on long-term consequences of particle irradiation on the bone marrow niche and MSCs are needed.