It is the intrinsic radiosensitivity of the tumor cell, which is primarily linked to its genetic background, but also the radiosensitivity of the tumor vasculature, that co-determine the treatment response to high-dose and fractionated irradiation (Begg, Stewart, Vens, 2011; Brown, Wilson, 2004; Garcia-Barros, Paris, Cordon-Cardo, Lyden, Rafii, Haimovitz-Friedman, Fuks, Kolesnick, 2003). However, we have only limited insights on the immediate and long-term stress responses of the tumor vasculature to ionizing radiation. Fractionated irradiation exploits the phenomenon of (iterative) reoxygenation, in which hypoxic cells become reoxygenated and subsequently more radiosensitive, i.e. a dose of ionizing radiation will preferentially kill the normoxic cell population, and the remaining cell population with a higher relative proportion of hypoxic cells will become reoxygenated (and more radiosensitive) to be killed by the next dose of a fractionated treatment schedule.
Reoxygenation might be linked to rapid recirculation through temporarily closed vessels, reduced respiration rates in damaged cells, ischemic and mitotic cell death of irradiated tumor cells and normalization of the stressed tumor vasculature. These different mechanisms might be the cause for differential time courses and levels of reoxygenation (Dewhirst, Cao, Moeller, 2008; Mortensen, Busk, Nordsmark, Jakobsen, Theil, Overgaard, Horsman, 2011; Overgaard, 2011). The concurrent chemotherapy given to head and neck cancers will most likely not affect the process of radiotherapy-dependent reoxygenation, which is the major focus of this research topic. We anticipate gaining detailed insights into this fundamental radiobiological concept of reoxygenation, with these novel non-invasive methods that allow serial determination of microenvironmental parameters during a fractionated treatment regimen of tumor xenografts.
The aim of the preclinical sub-project is to analyze (IR-induced) biological processes and the dynamics of tumor microenvironmental parameters during and in response to different RT-regimens (low dose fractionation, high dose hypofractionation, etc.) with already established techniques and the novel set of non-invasive techniques, which allow us to image oxygenation of tissue and organs with high spatial and temporal resolution. It will be of high interest to investigate these dynamic processes, which might even represent the better endpoint with regard to clinical outcome. Close interaction with the clinical part of this radiotherapy-oriented KFSP-subproject (see project Riesterer & Studer) will strengthen the translational aspect of the preclinical research.
In work package 1, several preclinical tumor models will be established to investigate the dynamics of IR-affected tumor oxygenation. These include genetically-defined tumor xenograft and spontaneous murine tumor models (lung carcinoma and head and neck squamous cell carcinoma). In these murine tumor models we will use NIRI and O-/Misonidazole PET (as soon as available and depending on the spatial resolution) to assess the dynamics of tumor oxygenation/hypoxia in response to treatment. Tumor cell lines will also be stably transfected with specific luminescence-based reporter constructs to determine dynamic parameters of tumor oxygenation (see also project Rudin and project Wenger & Borsig). These reporter systems also enable serial determination of tumor hypoxia during and in response to treatment.
We will first investigate - with high spatial and temporal resolution - immediate changes of tumor oxygenation and of the tumor vasculature to increasing doses of ionizing radiation. Taking the novel radiotherapeutical innovations with precise dose conformality into consideration - which will be implemented for many tumor entities in the near future - we will specifically focus on the treatment response to single and fractionated irradiation regimens using high-dose fractions of ionizing radiation (up to 20Gy/fraction). It will be important to arrange and determine the preclinical tumor models relevant for this KFSP among the multiple preclinical research groups and subprojects. Furthermore, it will be important to quantitatively and qualitatively cross-validate the different invasive and non-invasive techniques using defined tumor models, which have been previously characterized with regard to tumor hypoxia, and tumor clamping techniques.
In work package 2, biochemical and cell biological parameters in the tumor and the irradiated tumor bed will be analyzed. Classic immunohistochemical analysis on (tumor) tissue sections will be combined with bioimaging techniques to determine IR-induced de-/stabilization of the tumor vasculature, changes of tumor infiltrating cells, indicative for immunological responses and angiogenic switches, e.g. from angiogenesis to vasculogenesis, and secretion of angiogenic factors as part of tumor stress/rescue processes. Complementary mechanistic experiments will be perfomed in tumors derived from genetically-modified cells to determine the relevance of the identified stress/rescue mechanisms. Eventually, the identified parameters might represent relevant targets for combined treatment modalities xenografts, which have previously been characterized for their hypoxia-dependent treatment response. Furthermore, we will investigate an experimental model of impaired tumor angiogenesis in the irradiated tumor bed. This experimental model provokes tumor vasculogenesis in the tumor bed, mimicking the microenvironment of tumor persistence or tumor recurrence after radiotherapy. Preirradiation of the transplantation site will be performed to induce radiation damage to the supplying host tissues (tumor bed effect, TBE) as an experimental model of impaired tumor angiogenesis and to provoke tumor vasculogenesis at putative sites of local recurrences. The analysis IR-induced biological processes and the dynamics of tumor microenvironmental parameters during and in response to different RT-regimens (low dose fractionation, high dose hypofractionation, etc.) are the primary focus of this preclinical radiotherapy-oriented KFSP-subproject (in the tumor and in the tumor bed). However these experiments will be extended to other parameters investigated as part of this KFSP, but now in response to IR and other treatment modalities.
Qualitative and quantitative characterization of these preclinical (and clinical) situations with the respective high-resolution approaches will lead to an improved understanding of the relevance of tumor hypoxia and reoxygenation for radiation resistance on the individual level, and eventually to personalized radiotherapy protocols with the tumor microenvironment as a critical parameter.