We have made significant progress towards our objectives through a series of focused research activities:
1. Understanding Cell-Intrinsic Radiobiological Parameters:
We have successfully performed various survival and DNA damage assays to assess cell-intrinsic parameters, establishing patterns specific to beta and X-ray irradiation. Experiments for alpha irradiation will begin in 2025. Additionally, we have set up methods to detect senescence and cell death using flow cytometry, western blotting, and microscopy. We are currently defining specific timepoints and doses for each method.
2. Assessing Subcellular Localization of TRT:
We have performed uptake assays to assess the subcellular localizations of radiopharmaceuticals in different cell lines. The Cy5-DOTA-TATE compound is currently being investigated using molecular assays and for microscopy to identify subcellular localization in more detail. Live cell microscopy pilot experiments showed good resolution, and we will commence full experiments once the cell lines are finalized. DNA damage has been analysed in fixed cells over time, with results resembling previous data acquired in our laboratory. Cell lines for live cell imaging of DNA damage have been constructed, microscope settings have been optimized, and we have optimized our image analysis pipeline using deep learning methods. We have established foci tracking and have the first biological results as outcome. We see a clear distinction between beta and Xray in terms of number of foci at a specific time, however the duration of DNA repair is the same between both treatments.
3. Analysing Intra-Tumoral Localization of TRT:
This part of the project has not yet started. The first steps to acquire animal ethical approval have been finalized and we expect to start the first tests in Q1/Q2 2025.
4. Imaging In Vivo TRT Efficacy:
This objective will be pursued once objective 3 is completed.
5. Performing Realistic Dosimetric Simulations:
This work is ongoing, and we have optimized dosimetric models for in vitro (2D and 3D) neuroendocrine cell lines.
In addition to the research described above, we are developing novel in vitro and in vivo models for our studies. So far, we have successfully created and validated a 53BP1-mClover knock-in in U2OS cells. We are in the process of repeating this for the Bon1 cell line. The U2OS-53BP1_mClover cells have further been engineered to stably express SSTR2 or SNAP-SSTR2 for TRT imaging. Based on these and other outcomes, we have created a realistic dosimetric model for 2 neuro-endocrine tumor cell lines (focus of this project). We have shown that using Monte Carlo simulations of suspension, adherent and cluster forming cells, resemble the biological situation and used this to calculate the relative biological effectiveness of Xray vs beta radiation.
In addition to these objectives, we are creating new research models to better study TRT. We have successfully modified cancer cells with a marker (53BP1-mClover) to visualize DNA damage in live cells, and we are replicating this for the Bon1 cell line. These modified cells are now being used to optimize live cell imaging techniques and test the effects of radiotherapy.
Our research is paving the way for a more detailed and precise understanding of how TRT works at the cellular level. By improving our knowledge of these processes, we aim to enhance TRT's effectiveness and provide better outcomes for cancer patients.
