This study evaluated the paracrine signaling between breast carcinoma-associated fibroblasts (CAFs) and breast cancer (BCa) cells. Resolving cell–cell communication in the BCa tumor microenvironment (TME) will aid the development of new therapeutics. Here, we utilized our patented TAME (tissue architecture and microenvironment engineering) 3D culture microphysiological system, which is a suitable pathomimetic avatar for the study of the BCa TME. We cultured in 3D BCa cells and CAFs either alone or together in cocultures and found that when cocultured, CAFs enhanced the invasive characteristics of tumor cells, as shown by increased proliferation and spread of tumor cells into the surrounding matrix. Secretome analysis from 3D cultures revealed a relatively high secretion of IL-6 by CAFs. A marked increase in the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) when carcinoma cells and CAFs were in coculture was also observed. We theorized that the CAF-secreted IL-6 functions in a paracrine manner to induce GM-CSF expression and secretion from carcinoma cells. This was confirmed by evaluating the activation of STAT3 and gene expression of GM-CSF in carcinoma cells exposed to CAF-conditioned media (CAF-CM). In addition, the treatment of CAFs with BCa cell-CM yielded a brief upregulation ofGM-CSFfollowed by a marked decrease, indicating a tightly regulated control ofGM-CSFin CAFs. Secretion of IL-6 from CAFs drives the activation of STAT3 in BCa cells, which in turn drives the expression and secretion of GM-CSF. As a result, CAFs exposed to BCa cell-secreted GM-CSF upregulate inflammation-associated genes such asIL-6,IL-6RandIL-8, thereby forming a positive feedback loop. We propose that the tight regulation ofGM-CSFin CAFs may be a novel regulatory pathway to target for disrupting the CAF:BCa cell symbiotic relationship. These data provide yet another piece of the cell–cell communication network governing the BCa TME.
本研究评估了乳腺癌相关成纤维细胞（CAFs）与乳腺癌细胞（BCa）之间的旁分泌信号传导。解析乳腺癌肿瘤微环境（TME）中的细胞间通讯将有助于新疗法的开发。本研究采用我们获得专利的TAME（组织结构与微环境工程）三维培养微生理系统，该系统是研究乳腺癌肿瘤微环境的理想病理模拟模型。我们分别对乳腺癌细胞和CAFs进行单独三维培养及共培养，发现共培养条件下CAFs能增强肿瘤细胞的侵袭特性，具体表现为肿瘤细胞增殖能力提升并向周围基质扩散。三维培养分泌组分析显示CAFs分泌较高水平的IL-6，同时观察到癌细胞与CAFs共培养时粒细胞-巨噬细胞集落刺激因子（GM-CSF）分泌显著增加。我们提出理论假设：CAFs分泌的IL-6通过旁分泌作用诱导癌细胞表达和分泌GM-CSF。通过评估暴露于CAF条件培养基（CAF-CM）的癌细胞中STAT3活化状态及GM-CSF基因表达，该假设得到验证。此外，使用BCa细胞条件培养基处理CAFs可短暂上调GM-CSF表达，随后出现显著下降，表明CAFs中GM-CSF受到严格调控。CAFs分泌的IL-6驱动BCa细胞中STAT3活化，进而促进GM-CSF的表达与分泌。由此，暴露于BCa细胞分泌的GM-CSF的CAFs会上调炎症相关基因（如IL-6、IL-6R和IL-8），从而形成正反馈循环。我们认为，CAFs中GM-CSF的严格调控可能成为靶向破坏CAF与BCa细胞共生关系的新型调控通路。这些数据为解析乳腺癌肿瘤微环境的细胞间通讯网络提供了新的重要依据。
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