Poster S9 - Investigating the impact of fibroblast proximity to a tumor on fibroblast extracellular vesicle production

Introduction: Extracellular vesicles are an important method of intercellular communication. These are small particles with a bilipid membrane that are released from the cells [1,2]. Vesicles typically contain RNAs, microRNAs (miRNA), cytokines, and other molecules used for cell signaling. Different cells will produce EVs with different cargo. EVs have been proven to play an important role in breast cancer tumorigenesis, progression, and metastasis [3–5]. EVs have also been shown to contain important biomarkers for breast cancer [6,7]. This can detect breast cancer earlier and provide a more accurate prognosis based on content from the EVs. Fibroblasts within the tumor, termed cancer-associated fibroblasts (CAFs), contain more proinflammatory cytokines and miRNAs that promote cancer progression within their EVs as compared to normal fibroblasts [8–10].Many studies have been focused on EVs produced from biological fluids and cellular secretions. Recently, there is more interest in EVs that are within the ECM of tissues. EVs isolated from ECM are an important source to study as they may provide more insight to breast cancer initiation, progression, and metastasis. 3D engineered models can help to provide a more biologically relevant model than previous models. This study aims to investigate how the proximity of the tumor affects the EV production of the fibroblasts within stromal models.

Materials and

Methods: We created stromal models by 3D bioprinting two different fibroblasts, normal human mammary fibroblasts (hMFs) and normal tumor adjacent fibroblasts (NTAF), within a collagen gel. They were cultured for one week before isolating EVs from both the effluent media and the 3D stromal model. The resulting EVs were characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), ELISA, zeta potential, and cytokine array analysis of the cargo. After this, we used the EVs to treat breast cancer cells to investigate the effects the EVs have on the breast cancer cell behavior. Finally, we utilized a 3D breast tumor model to investigate the effects of the EVs on a tumor spheroid. To build the 3D breast tumor model, we first cultured MDA-MB-231 breast cancer cells within a round bottom ultra low attachment plate to form spheroids of 10,000 cells per spheroid. Then, we printed collagen gel into a well plate. The spheroids were then printed into the collagen gel and allowed to gelate before placing media and EV treatment onto the models.

Results, Conclusions, and Discussions: After 3D bioprinting the models, we characterized them through brightfield imaging and live/dead staining. TEM images show a consistent size and shape throughout the EV groups (Figure 1A) and NTA showed consistent size (Figure 1B). Cytokine array analysis revealed that the NTAF media group has consistent upregulation through most of the cytokines present in the array as compared to the rest of the groups (Figure 1C). The cytokine array results show 35 cancer promoting cytokines that have increased expression in the NTAF media group. After EV characterization, we then treated breast cancer cells. MDA-MB-231 breast cancer cells treated with NTAF groups had increased motility as compared to the hMF groups. NTAF media EVs caused the most significant increase in motility of 4.12 µm/hr (Figure 1D). Next, we utilized our tumor spheroids to create 3D breast tumor models. After model creation, these were treated with the EVs from the four EV groups. The breast tumor models were cultured for two weeks with images taken every three days to track spheroid diameter. The 3D models treated with the NTAF media EVs had the largest spheroid diameter increase of 1800 µm (Figure 1E). In this study, we show that the NTAF cells produce EVs that contain cytokines that promote breast cancer progression and influence the behavior of breast cancer cells. These cells have a distinct EV secretome different from normal human mammary fibroblasts, showing that the proximity to a tumor has an effect on the fibroblasts surrounding the tumor. The CAFs are a different population of fibroblasts that reside within the tumor that are directly influenced by the tumor. Both the tumor cells and the CAFs can impact the fibroblasts residing within the normal breast stroma near the tumor. Treatment with EVs from the NTAF groups caused an increase in 2D cell motility and 3D tumor spheroid diameter and subsequent local invasion. Overall, the NTAF EV groups promote breast cancer progression. This study can identify factors that are increased in areas surrounding the tumor which help the tumor progress and give researchers targets for treatment to slow this progression.

Acknowledgements (Optional): This study was funded by NIH award number 1R01CA275423-01A1.