(174s) Analysis of Extracellular Vesicles and Particles for Investigating Intercellular Communication in the Irradiated Breast Microenvironment

Although breast tumors are managed through a combination of surgery, radiotherapy (RT), and chemotherapy, triple negative breast cancer (TNBC) patients experience high rates of local recurrence after treatment. RT is particularly critical for TNBC patients, as 60-70% of patients receive RT post-surgically. Recent studies show that local recurrence may be facilitated by the recruitment of circulating tumor cells to the irradiated tissue following RT. The role of intercellular communication in this process is not well understood. We hypothesized that cell-secreted nanoparticles function as key mediators of intercellular communication in response to RT. Specifically, extracellular vesicles (EVs) are secreted by cells in both homeostatic and pathological conditions and are understood to be capable of transmitting complex biological messages. In this study, the microenvironmental consequences of RT-induced intercellular communication were examined through the analysis of irradiated-cell derived EVs. This work represents a critical step toward determining how cell-cell crosstalk after RT may contribute to breast cancer recurrence.

Methods:

Primary human fibroblasts derived from reduction mammoplasty were utilized as a model of the post-surgical breast stroma. Fibroblasts were irradiated to a dose of 10 Gy and cultured for 48h. EVs were isolated from cell-culture conditioned media through differential ultracentrifugation. We used nanoparticle tracking analysis (NTA) to quantify EV number and size distribution. Moreover, we pursued tandem mass tag-based proteomics to comprehensively analyze the protein content of irradiated cell-derived EVs. Also, we analyzed EVs using super resolution microscopy to investigate the nature of key of cargo components. Finally, we treated bystander cells with irradiated cell derived EVs and evaluated changes in phenotype through western blot and fluorescence microscopy.

Results:

NTA results reveal that RT substantially enhances EV secretion but does not alter the size distribution of secreted EVs in fibroblasts. Through proteomics analysis, we reveal that irradiated fibroblasts derived EVs contain an enrichment of nucleosome proteins, including many canonical histone and histone linker variants. Furthermore, super resolution microscopy reveals an association of enriched cargo components with CD63-positive vesicles. Finally, EV treatment studies highlight the functionality of irradiated fibroblast EVs, with preliminary evidence that treatment with EVs induces a senescence-like phenotype.

Conclusions:

In this work, we evaluate changes in communication in the cellular microenvironment by analyzing secreted EVs. We show that, in fibroblasts, EV-mediated intercellular communication is altered post-RT. We begin to uncover how the breast microenvironment may develop post-RT, allowing us to gain deeper understanding of the mechanisms of local recurrence in TNBC patients. The techniques explored in this study, for instance, super resolution microscopy, can be further expanded upon to elucidate deeper aspects of intercellular communication such as the release and uptake of EVs. Ultimately, this work will further our understanding of EV-mediated communication after RT, with the goal of contributing to novel therapeutic strategies for preventing TNBC recurrence.