With almost 900.000 new patients per year suffering from head and neck squamous cell carcinomas (HNSCC), who after radiotherapy treatments experience severe side effects, the focus is drawn to proton therapy. Proton therapy results to less side effects since healthy tissues surrounding the tumour receive less radiation dose. This results from the proton’s specific depth-dose profile with its Bragg peak.
To further reduce the effect on surrounding tissues, major improvements should be made in understanding the biologic response to proton therapy which will eventually lead to enlarging the therapeutic window. Here in vitro analysis with DNA damage repair inhibition or increasing proton dose rates can be performed to assess which DNA repair pathways repair radiation induced DNA damage and to assess the effect of proton dose rates on biological tissues. This will be investigated in vitro, while in vivo experiments are necessary as well. However, currently most mice are irradiated with protons in the plateau of the Bragg curve instead of the Bragg peak. Therefore, a dosimetric pipeline should be designed to allow for accurate placement of mouse tumours in the proton’s Bragg peak.
In this thesis, the osteosarcoma- and HNSCC-derived U2OS and FaDu cell lines were incubated with non-homologous end-joining or homologous recombination inhibitors during x-ray and proton irradiations. We have shown a radiosensitising effect of the non-homologous end joining inhibitor AZD7648 on FaDu cells during x-ray irradiation, but an effect during proton irradiation could not be proved nor neglected. The homologous recombination inhibitor B02 could not effectively inhibit homologous recombination and was shown to not affect clonogenic survival of FaDu during X-ray irradiations. The observed radiosensitising effect of the non-homologous recombination inhibitor AZD7648 can be exploited in patient selection based on already existing DNA damage repair deficiencies in the tumour. Furthermore, combination therapies could be used of photon irradiations with AZD7648 targeted to the tumour to artificially enlarge the therapeutic window.
Besides this, the HNSCC-derived FaDu cells were irradiated with varying dose rates to assess induction of a FLASH effect. This FLASH effect is usually observed after irradiations of >40 Gy/s resulting in a reduction of normal tissue complications while tumour control is maintained. In our analysis, small differences in low dose rates did not have any impact. The experimental set up for proton FLASH irradiations of cells was prepared and though we performed the first set of experiments no definite conclusion could be drawn due to the level of biological variation we observed.
Of course, differential effects between tumours and tissues like the FLASH effect should be investigated in vivo. Therefore, a set-up should be created to irradiate the mural tumour with Bragg peak protons. To reach this, two micro-CT scanners were calibrated to link Hounsfield units to stopping power ratios. The proton range in mice was determined and a 3D-printed mouse-like phantom was irradiated. In this thesis, determined stopping powers of all CIRS phantom inserts except for lung tissues were accurate with a maximum deviation of 2% or 6.5% after calibration with QuantumGx or VECTor micro-CT scanners. Larger deviations were observed for CIRS lung inserts or Gammex inserts. The irradiated gafchromic films inserted in the murinemorphic phantom showed dose distributions visualising the mouse’s anatomy, yet the dose was too low due to irradiation in the distal edge of the Bragg peak. The latter was confirmed with Monte Carlo simulations. This dosimetric set-up to place mice tumours in the proton Bragg peak should thus be slightly improved and can then be of great value for in vivo experiments of proton therapy. This enables execution of many new experiments with DNA damage repair inhibition and FLASH irradiations, potentially leading to an increase of the therapeutic window in proton therapy and less side effects for HNSCC-patients.

Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.

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