EV Paper

Extracellular vesicles (EVs) are an important carrier of cellular communication that are secreted from the cell. Different cells will produce EVs with different cargo such as cytokines, RNAs, or microRNAs (miRNA). EVs have been proven to play an important role in breast cancer tumorigenesis, progression, and metastasis. Althought the role of cancer associated fibroblasts (CAFs), and EVs originated from them have been studied extensively, there is a lack in knowledge on the contribution of fibroblasts surrounding the tumor and their roles with respect to their proximity to the tumor. 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. Here we investigate how the proximity of the tumor affects the EV production of the fibroblasts. We created stromal models by 3D bioprinting the two different fibroblasts, normal human mammary fibroblasts (hMFs) and normal tumor adjacent fibroblasts (NTAF), within a collagen gel. After one week of culture, we isolated EVs from both the effluent media and the 3D stromal model, which were then characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), ELISA, zeta potential, and cytokine array analysis of the cargo. The EVs from each group were of consistent exosome size and displayed traditional exosome markers, however the EVs from different groups also displayed different cytokine profiles of their cargo, with the NTAF media group showing an upregulation of cytokines associated with breast cancer progression. After this, we used the EVs to treat breast cancer cells to investigate the effects the EVs from different tumor proximities have on the breast cancer cell behavior. The breast cancer cells treated with the NTAF groups had increased migration. Finally, we utilized a 3D breast tumor model to investigate the effects of the EVs on a tumor spheroid. Tumor spheroids treated with either NTAF EV groups showed increased proliferation, tumor radius, and local invasion. This study is the first to investigate the effect of proximity to a breast tumor on EV production and the first to utilize 3D bioprinting of stromal models specifically to obtain EVs. Overall, our results show that EVs from fibroblasts closer to a tumor produce EVs that promote breast cancer progression, regardless of the secretion location of the EVs. These cells have a distinct EV secretome different from normal human mammary fibroblasts, showing that the proximity to a tumor influences the fibroblasts surrounding the tumor.

细胞外囊泡（EVs）作为细胞通讯的重要载体，由细胞分泌而成。不同细胞会产生携带不同货物，如细胞因子、RNA或miRNA（miRNA）的EVs。研究证实，EVs在乳腺癌的肿瘤发生、进展和转移中发挥着重要作用。尽管癌症相关成纤维细胞（CAFs）及其来源的EVs的研究已相当广泛，但对于肿瘤周围成纤维细胞及其与肿瘤邻近度的作用尚缺乏深入了解。肿瘤内成纤维细胞，即癌症相关成纤维细胞（CAFs），相较于正常成纤维细胞，其EVs中含有更多促进癌症进展的促炎细胞因子和miRNA。本研究旨在探讨肿瘤邻近度如何影响成纤维细胞的EVs产生。我们通过3D生物打印技术，在胶原凝胶中构建了两种不同的成纤维细胞模型，即正常人类乳腺成纤维细胞（hMFs）和正常肿瘤邻近成纤维细胞（NTAF）。经过一周的培养，我们从流出介质和3D基质模型中分离EVs，并使用纳米颗粒追踪分析（NTA）、透射电子显微镜（TEM）、ELISA、ζ势和货物细胞因子阵列分析对其进行表征。每个组的EVs大小一致，并表现出传统的EVs标志物，然而，不同组的EVs也显示出其货物细胞因子谱的不同，其中NTAF介质组表现出与乳腺癌进展相关的细胞因子上调。随后，我们利用EVs处理乳腺癌细胞，以研究不同肿瘤邻近度的EVs对乳腺癌细胞行为的影响。接受NTAF组EVs处理的乳腺癌细胞迁移增加。最后，我们利用3D乳腺癌肿瘤模型研究EVs对肿瘤球体的作用。接受NTAF EV组处理的肿瘤球体显示出增殖、肿瘤半径和局部侵袭的增加。本研究首次探讨了乳腺癌肿瘤邻近度对EVs产生的影响，也是首次利用3D生物打印基质模型以获取EVs。总体而言，我们的结果表明，靠近肿瘤的成纤维细胞产生的EVs可促进乳腺癌进展，无论EVs的分泌位置如何。这些细胞具有与正常人类乳腺成纤维细胞不同的EVs分泌组，表明肿瘤邻近度会影响肿瘤周围的成纤维细胞。