(28p) Aged and Young Breast Matrix Bound Vesicles in Breast Cancer

Extracellular vesicles (EVs) are small, lipid-bilayer membrane-derived particles released by cells into the extracellular environment to facilitate communication between cells and their surroundings. While most EV studies in cancer research focus on cancer cell secreted EVs, recently an unconventional, bioactive subgroup of EVs has been identified within the extracellular matrix (ECM) of tissues and have shown to provide tissue specific activities in multiple different tissues including urinary bladder, small intestine and heart. Given the recently recognized importance of tumor microenvironment, especially the ECM surrounding the tumor, matrix-bound EVs (MBVs) might have important roles in cancer initiation and progression. We have previously investigated the breast ECM alterations with older age, one of the biggest risk factors for breast cancer, but little is known on EVs in the aging breast microenvironment in general, especially the ECM-bound EVs.

Here, we showed the presence of MBVs in the breast tissue for the first time in literature. We isolated breast MBVs from young and aged mice, characterized and compared their physical, biochemical, and functional characteristics. Using novel fluorescence labeling technology, we visualized successful uptake of the breast MBVs from decellularized breast tissue sections and MBV-embedded ECM-mimicking 3D collagen gels by human mammary epithelial cells. We then studied the effects of MBVs on KTB21 and MDA-MB-231 cell motility and invasion and showed how MBVs could recapitulate the effects of the aged breast ECM on cellular behaviors. Finally, we performed full profiling of miRNA and cytokine contents of the MBVs and investigated the critical biomolecule components of the MBVs and their roles in these age-related influences. We identified three oncomiRs and a cytokine that can recapitulate the effect of MBVs on normal and cancer cell behavior. Investigating and comparing the characteristics and contents of MBVs and the influences of MBVs on breast cancer progression as a function of age will provide more detailed and thorough understanding of the breast microenvironment and pave way for developing new strategies to prevent and treat breast cancer.

EVs were isolated from the fourth mammary glands of young (2-6 months) and aged (20-23 months) mice and characterized with nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosomal markers were visualized with immunogold EM and quantified with ELISA. Normal (KTB21) and cancerous (MDA-MB-231) human mammary epithelial cells were used in this study. Cellular uptake of the MBVs directly from the native tissue sections and isolated MBVs embedded in 3D biomimetic collagen scaffolds were visualized with Exo-Glow exosome labeling technology. Cell motility and invasion were assessed by tracking cell movement microscopically with time-lapse imaging or quantifying invaded cells in transwell assays, respectively. Cytokine and miRNA contents of MBVs were profiled with Proteome cytokine profiling arrays and NanoString next generation sequencing technology. Specific targeted factors were selected for epithelial cell invasion assays to investigate whether these biomolecules could recapitulate the MBV-driven changes in cell behaviors.

NTA and TEM revealed the presence of round shaped, CD9 and CD63 positive vesicles in breast ECM with ~140 nm diameter (Fig. A and B). With fluorescence labels, MBV uptake from the decellularized tissue sections into epithelial cells were visualized (Fig. C). Interestingly, treatment with MBVs from aged mice (MBV-aged) significantly increased cell motility in both KTB21 (p=0.014) and MDA-MB-231 (p<0.0001) cells compared to untreated control, while treatment with MBVs from young mice (MBV-young) posed weaker influence on cell motility (Fig. D and E). Moreover, both MBV-aged and MBV-young treatment significantly increased the invasion of KTB21 (p=0.0043) and MDA-MB-231 (p<0.0001) cells, with higher impact of MBV-aged on the MDA-MB-231 cells than MBV-young (p=0.0002). Epithelial cells were also visualized to take up labeled MBVs embedded in ECM-mimicking 3D collagen gels and invade through the gels (Fig. F).

Cytokine profiling revealed a system of over 100 cytokines encapsulated in the MBVs, including significantly higher levels of adiponectin in the MBV-aged than MBV-young (p=0.0073). Adiponectin treatment significantly increased the invaded cell counts compared to non-treatment groups (p=0.0007 for KTB21 cells and p=0.0062 for MDA-MB-231 cells) (Fig. G). NanoString Quantitative RT-PCR showed higher levels of selected oncomiRs and lower levels of selected tumor-suppressive miRNA in the MBV-aged than MBV-young. Transfection of KTB21 (p<0.0001) and MDA-MB-231 (p=0.0014) cells with a cocktail of selected oncomiR mimics led to increased invasion (Fig. H), indicating that these miRNAs are involved in MBV-aged-driven increase in cell invasion.

In this study, we addressed a gap in the current understanding of how the aging breast microenvironment affects breast cancer progression and invasion. Although MBVs have been previously reported in a few other tissues, here, we for the first time visualized the cellular uptake of these MBVs from both native decellularized tissue sections and ECM-mimicking 3D hydrogel models and demonstrated the distinctively different biochemical contents and cellular effects of MBVs embedded in aged and young ECM.

In conclusion, we showed that as an integral component of the breast ECM that could interact with and be taken up by cells, these previous-unknown MBVs could play a pivotal role in how the breast tumor microenvironment alters epithelial cells toward cancer and metastasis as well as how the aging microenvironment further enhances these influences. Our results also show that the biomolecule cargos upregulated in MBV-aged could exert crucial influences in promoting the MBV-driven invasive cell behavior, indicating potential targets for future treatment development.