To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

The definition of clinical target volume (CTV) margins around gross tumor volume (GTV) for radiotherapy of esophageal cancer (EC) and many solid tumors is still a challenge hence the currently available in-vivo imaging techniques still fail to detect areas of microscopic tumor extension (MTE). Many parameters of the tumor microenvironment (TME), e.g., tumor cell proliferation, cancer stem cells, hypoxia, kinases, immune architecture and patient-specific parameters are hypothesized as inducers of MTE in esophageal cancer and other tumors. The correlation of these TME biomarkers with MTE before, during or after radiochemotherapy (RCHT) is crucial in the era of image-guided, adaptive high-precision photon or particle therapy. In this thesis, two study cohorts were used to assess some selected TME biomarkers and their predictive value on MTE for an improved CTV definition. The first study used immunohistochemistry analysis for the assessment of TME marker namely HIF-1α, Ki67, p53, CXCR4 and PD1 in a cohort of retrospectively collected formalin-fixed paraffin-embedded (FFPE) blocks of EC patients treated with either neoadjuvant radiochemotherapy plus resection (NRCHT+R) or resection alone (R). The subsequent study employing a multiplex-immunofluorescence technique assessed the expression of various markers, i.e., FAK, ILK, CD44, HIF-1α and Ki67, in a cohort of prospectively prepared FFPE resection specimens of EC patients with implantable fiducial gold markers at the proximal and distal tumor borders illustrating the GTV prior to NRCHT+R and correlated those markers to the MTE. The findings from our first study showed upregulation of HIF-1α, Ki67, p53, CXCR4 and PD1, within squamous cell carcinoma (SCC) and adenocarcinoma (AC) patients treated with R compared to those having undergone NRCHT+R. In the second study higher expression of FAK+, CD44+, HIF-1+, and Ki67+ cells in tumor-nests than in tumor-stroma of both SCC and AC patients was found, although ILK+ cells were higher in tumor stroma. In addition, MTE reaching up to 31 mm beyond the fiducial markers was found in three patients (all cT3N1) with a stronger expression of FAK+, CD44+ and ILK+ cells in tumor-nests in between the fiducial markers (former GTV) and beyond those (former CTV), even after NRCHT. In conclusion, there is thus far no evidence that the TME influences the CTV margin on an individual patient basis, hence differences in the TME between patients with residual tumor cells in the original CTV compared to those without were not detected.

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