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.

The value of plasma hypoxia markers for predicting imaging-based hypoxia in patients with head-and-neck cancers undergoing definitive chemoradiation.

BACKGROUND: Tumor hypoxia worsens the prognosis of head-and-neck squamous cell carcinoma (HNSCC) patients, and plasma hypoxia markers may be used as biomarkers for radiotherapy personalization. We therefore investigated the role of the hypoxia-associated plasma proteins osteopontin, galectin-3, vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) as surrogate markers for imaging-based tumor hypoxia. METHODS: Serial blood samples of HNSCC patients receiving chemoradiation within a prospective trial were analyzed for osteopontin, galectin-3, VEGF and CTGF concentrations. Tumor hypoxia was quantified in treatment weeks 0, 2 and 5 using [18F]FMISO PET/CT. The association between PET-defined hypoxia and the plasma markers was determined using Pearson's correlation analyses. Receiver-operating characteristic analyses were conducted to reveal the diagnostic value of the hypoxia markers. RESULTS: Baseline osteopontin (r = 0.579, p < 0.01) and galectin-3 (r = 0.429, p < 0.05) correlated with the hypoxic subvolume (HSV) prior to radiotherapy, whereas VEGF (r = 0.196, p = 0.36) and CTGF (r = 0.314, p = 0.12) showed no association. Patients with an HSV > 1 mL in week 2 exhibited increased VEGF (p < 0.05) and CTGF (p < 0.05) levels in week 5. Pretherapeutic osteopontin levels were higher in patients exhibiting residual hypoxia at the end of treatment (104.7 vs. 60.8 ng/mL, p < 0.05) and could therefore predict residual hypoxia (AUC = 0.821, 95% CI 0.604-1.000, p < 0.05). CONCLUSION: In this exploratory analysis, osteopontin correlated with the initial HSV and with residual tumor hypoxia; therefore, there may be a rationale to study hypoxic modification based on osteopontin levels. However, as plasma hypoxia markers do not correspond to any spatial information of tumor hypoxia, they have limitations regarding the replacement of [18F]FMISO PET-based focal treatments. The results need to be validated in larger patient cohorts to draw definitive conclusions.

Multipotent mesenchymal stromal cells are sensitive to thermic stress - potential implications for therapeutic hyperthermia.

Purpose: Hyperthermia demonstrated clinical efficacy in multimodal cancer treatment. Multipotent mesenchymal stromal cells (MSCs) as part of the tumor-supporting stroma modulate tumor response and tissue regeneration after hyperthermia. We aimed to investigate the effects of hyperthermia on the survival, stem cell characteristics and heat shock expression of human MSCs.Materials and methods: Human MSCs and normal human dermal fibroblasts (NHDFs) were exposed to temperatures between 37 °C and 44 °C for 60 min, and hyperthermic sensitivity was examined by clonogenicity, proliferation and viability assays. The influence of 42 °C hyperthermia on the MSCs' adhesion potential, migratory capacity, surface marker expression and multi-lineage differentiation capability was investigated. Cell cycle distribution, apoptosis and senescence after 42 °C hyperthermia were determined by flow cytometry and ß-galactosidase staining. Heat shock protein expression was determined by Western Blots.Results: MSCs exhibited decreased clonogenic survival after 40 °C and 42 °C hyperthermia compared to NHDFs, while proliferative activity and viability were comparable after hyperthermia up to 44 °C. MSC adhesion was reduced after 42 °C hyperthermia, while the characteristic surface marker expression and the migratory ability remained unaffected in 42 °C hyperthermia-exposed MSCs. 42 °C hyperthermia diminished the adipogenic differential potential of all tested MSC samples. A pronounced G2/M arrest was found after 42 °C hyperthermia and was associated with increased apoptosis and senescence levels in MSCs. MSCs exhibited slightly lower heat shock protein levels compared to NHDFs.Conclusion: Human MSCs exhibit a thermosensitive phenotype which reduced the multipotent cells' regenerative abilities, resulting in impaired tissue regeneration after hyperthermia treatment or thermal injuries. On the other hand, tumor-associated MSCs may be efficiently targeted by hyperthermia treatment.