Importance of the senescence-associated secretory phenotype (SASP) for the radio-resistance of HNSCC cell lines after particle irradiation

Photon-based radiotherapy is the major treatment option for locally advanced HPV-neg. head and neck squamous cell carcinoma (HNSCC). This treatment can be used as a precise and efficient tool to achieve tumor cell killing. However, despite the application of high doses above 70 Gy, therapeutic failure is common, which is attributed to the inherent and acquired radioresistance of tumor cells. This radioresistance is considered to be strongly associated with the senescence induced by ionizing irradiation. Senescent cells are able to secrete SASP (senescence-associated secretory phenotype) factors, which might enhance the cellular radioresistance and thereby diminish the effect of radiation on cell killing. The aim of this project was to better understand the cellular and molecular mechanisms of this senescence-induced radioresistance in HPV-neg. HNSCC cells. The experiments were performed with five different HPV-neg. cell lines (Cal27, Cal33, UPCISCC040, UPCISCC099, UPCISCC131) in order to cover a broad range of radiosensitivity, which is a typical characteristic of this entity. The mechanisms were studied for X-rays and for the first time also for 12C-ions, which is another high precision radiotherapy available at the Marburger Ionenstrahl-Therapiezentrum (MIT). This method uses particle irradiation to achieve efficient tumor cell killing. The following results were obtained: 1. Cellular radiosensitivity towards 12C-ions as determined via colony formation assay was clearly higher when compared with X-rays with an RBE10 of ~ 2-3, also confirming previous results. 2. An active DSB repair as examined by immunofluorescent visualization of γH2AX/53BP1-foci was seen in all cell lines and also after both, X- and 12C-irradiation. However, repair capacity was clearly less after 12C-irradiation. 3. Senescence was studied using a flow-cytometer based method to detect senescence-associated β-galactosidase activity after X-irradiation and for the first time also after 12C-irradiation. After both types of irradiation, senescence increases over time and dose. However, the final level approached was much stronger after 12C-irradiation. This level of senescence was well correlated with the respective cellular radioresistance. But this association was much steeper for 12C-irradiation showing that for X-irradiation, a moderate increase in senescence was associated with a strong increase in radioresistance, while for 12C-irradiation the opposite was seen with a strong increase of senescence being associated with a low increase in radioresistance. This finding shows for the first time, that impact of senescence on cellular radioresistance appeared to be less for 12C-ions when compared to X-rays. 4. Gene expression profile of SASP factors were measured via both, qRT-PCR and RNA-seq. A strong induction of SASP factors were seen after both radiation types with very similar dynamics. Using a principal component analysis (PCA) a clear discrimination was achieved for the five cell lines used, demonstrating that each cell is characterized by a specific profile of SASP factors. The data sets unveiled also a strong irradiation-induced induction of these factors. 5. For IL1α and IL1β it is shown for the first time that there is strong association with radioresistance as well as senescence after both X- and 12C-irradiation. The secretion of IL1β as measured in the supernatant of cell cultures using ELISA increases with time after irradiation and was shown to be stronger after 12C-irradiation. This secretion was clearly high in radioresistant cell lines. 6. A mechanistic study was performed for IL1α and β by using siRNA mediated knockdown. Most surprisingly this knockdown was found to have no effect on cellular radiosensitivity, DSB repair and senescence. And this was seen for both X- and 12C-irradiation and not only for a single but also for a combined knockdown of these two factors. 7. These data demonstrate for the first time that the two important SASP factors IL1α and β, although strongly associated with cellular radioresistance, DSB repair and senescence, are not functionally involved in any of these processes. Due to best knowledge, this is the first study examining the impact of the specific SASP factors IL1α and β associated with senescence after X- as well as after 12C-irradiation in HNSCC cell lines. Due to the data obtained, IL1α/β can now be excluded as exclusive drivers of irradiation-induced senescence and with that cellular radioresistance. Future research has to concentrate on other factors probably relevant for these biological end points.