OI Ljubljana

Cancer stem cell (CSC)-associated exosomes as regulators of tumor radiation response

Principal investigator at the Institute of Oncology Ljubljana: Maja Čemažar PhD

Radiation therapy (RT) is one of the main therapeutic approaches used in the breast cancer treatment. Nearly all breast cancer patients receive RT during the disease history either in curative or palliative settings. Despite the development of novel techniques and schedules of irradiation, the problems of primary and secondary radiation resistances are still not solved. It is currently accepted that an intratumoral subpopulation of cancer stem cells (CSCs) is responsible for radioresistance and disease progression in cancer patients after treatment. Therefore, it is logical to suggest that the malignant tumor (including breast cancer) could be cured when all CSCs will be destroyed and eliminated. CSCs with intrinsic radiation resistance can survive after irradiation of primary tumors and give rise to the tumor recurrences enriched for CSCs not only with intrinsic but also with acquired radiation resistances. Despite an intensive investigation of the molecular signaling underlying intrinsic and acquired CSC radiation insensitivity, it is still poorly understood how CSCs survive upon radiation exposure. The preliminary data have shown that radioresistant CSCs are characterized by affected metabolic activities, increased metastatic properties and affected DNA repair mechanisms. We have also found that secretomes obtained from CSC containing radioresistant breast carcinoma cells are enriched for different proteins affecting the radiation response in radiosensitive cells. Unfortunately, secretomes contain not only proteins secreted by carcinoma cells, but also molecules released by cells during their death. In order to avoid this limitation, cellular exosomes can be used as organelles containing proteins that are exlusively generated and released by cells for intercellular communications. It was observed that CSCs are characterized by increased number and volumes of microvesicles including exosomes. Therefore, it is suggested that CSC-specific exosomes can contain molecules contributing to cancer cell unlimited renewal, treatment resistance and metastatic spread. This research project will be focused on the role of CSC specific exosomes in the modulation of radiation response in different breast cancer cell subpopulations. This is a translational clinically relevant research project, because the preclinical data could help to develop exosome-related biomarkers and therapeutic targets to predict and/or combat an unfavourable disease progression due to radiation resistance in breast cancer patients.

Specific aims of the project: 1. To evaluate the protein patterns of exosomes obtained from different breast cancer cell subpopulations (CSCs expressing CD44+/CD24-/ALDHA1+ versus non-CSCs with CD44-/CD24+/ALDHA1+ or CD44- /CD24+/ALDHA1- or CD44-/CD24-/ALDHA1+ or CD44-/CD24-/ALDHA1-); 2. To study whether CSC-specific exosomes can change tumorigenic (mouse models will be used) and metastatic abilities of non-CSCs; 3. To determine whether CSC-related exosomes can affect a radiation sensitivity in non-CSCs (cell viability and clonogenic capacities, DNA damage response, metabolic activities, ROS production and scavenging will be determined); 4. Proteins of interest identified in the CSC-related exosomes will be studied for their role in radiation resistance of breast carcinoma cells. Methods: Following methods will be used: Cell viability assay (using ViCell Counter); clonogenic assay; Comet assay; migration assay using live cell imager CytoSMART and migration assay kits (Millipore AG); exosome isolation using commercial kits; scanning electron microscopy; fluorescence microscopy; FACS and MACSbased isolation of breast cancer cell subpopulations; evaluation of metabolic activities of breast carcinoma cells (oxygen consumption, acidification rate, proton production rate will be determined using Seahorse XFp instrument); Western blot analysis; ELISA, etc.



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