Deciphering the radiobiology of targeted radionuclide therapy: from subcellular to intra-tumoural analyses

Project Information

RADIOBIO

Grant agreement ID: 101042537

DOI

10.3030/101042537

EC signature date 28 January 2022

Start date 1 June 2022

End date 31 May 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 750 000,00

EU contribution

€ 1 750 000,00

Coordinated by

ERASMUS UNIVERSITAIR MEDISCH CENTRUM ROTTERDAM
Netherlands

Periodic Reporting for period 1 - RADIOBIO (Deciphering the radiobiology of targeted radionuclide therapy: from subcellular to intra-tumoural analyses)

Reporting period: 2022-06-01 to 2024-11-30

Summary of the context and overall objectives of the project

My ERC project aims to deepen our understanding of targeted radionuclide therapy (TRT) for treating tumours. TRT is a promising cancer treatment that delivers radioactive substances directly to cancer cells, minimizing damage to healthy tissues. However, the effects of TRT can vary significantly among different tumour cells. Understanding these variations is crucial for optimizing and improving TRT outcomes.

Key factors influencing TRT's effectiveness include the type of radionuclide used, the rate at which radiation is delivered, and the total radiation dose. These physical parameters play a vital role in determining how tumour cells respond to TRT. When exposed to TRT, tumour cells experience DNA damage, which can lead to different cellular responses such as DNA repair or cell death (apoptosis). These processes are not yet fully understood within the context of TRT.

The overall objective of my project is to identify and quantify the specific mechanisms by which TRT affects tumour cells, both in cultured cells (in vitro) and in mice (in vivo). By doing so, we aim to provide a more detailed radiobiological understanding of TRT, which could lead to more effective and personalized cancer treatments. The expected impact of our project includes enhancing the precision of TRT, improving patient outcomes, and contributing to the development of more accurate dosimetric models.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

My ERC project has achieved several breakthroughs in understanding and improving TRT for cancer treatment. These advances have the potential to significantly impact the field and pave the way for more effective cancer therapies.

• Key Achievements and Their Impact
1. Improved Understanding of Cellular Responses:
We have gained valuable insights into how different types of radiation affect tumour cells at the DNA level. By studying these effects, we can better predict how cells will respond to TRT, leading to more precise and effective treatments. We have developed new methods to detect when cells stop dividing or die, which helps us understand how and when TRT is most effective.

2. Enhanced Visualization Techniques:
Our work on tracking the subcellular localization of radioactive substances within tumour cells has provided clearer images and more detailed information about how TRT works at the microscopic level. We have optimized imaging techniques that allow us to observe DNA damage in live cells, providing real-time insights into the cellular processes affected by TRT.

3. Innovative Dosimetric Models:
We have created advanced models to simulate how TRT affects cells in both two-dimensional and three-dimensional settings. These models help us predict the outcomes of TRT more accurately and design better treatment plans.

• Ensuring Further Uptake and Success
To ensure that our findings lead to real-world improvements in cancer treatment, several steps are necessary:

1. Further Research and Demonstration:
Continued research is needed to validate our findings in larger and more diverse sets of tumour cells and to test these findings in animal models and clinical trials. Demonstrating the effectiveness of our new methods and models in real-world scenarios will be crucial for gaining wider acceptance and application.

2. Access to Markets and Finance:
Securing funding for further research and development is essential. This includes grants, investments, and partnerships with industry leaders. Access to markets where our innovations can be applied and tested will help bring our advancements to patients more quickly.

3. Commercialization and IPR Support:
Protecting our intellectual property through patents and other legal means will help ensure that our innovations can be developed and commercialized effectively. Developing a clear commercialization strategy, including potential partnerships with biotech companies, will be crucial for bringing our discoveries to market. We have full support from our technology transfer office for these activities.