Surface roughness modulates EGFR signaling and stemness of triple-negative breast cancer cells.
Surface roughness modulates EGFR signaling and stemness of triple-negative breast cancer cells.
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DOI：
10.3389/fcell.2023.1124250
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发表时间：
2023
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
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简介：癌症干细胞（CSC）是耐药表型和肿瘤复发的主要罪魁祸首，占TNBC细胞的不到2%，使得它们难以分离、研究，从而限制了我们对疾病发病机制的理解。目前用于CSC富集的方法，例如3D球状体培养、遗传修饰和干细胞调节，是耗时的、昂贵的，并且不适合于高通量测定。解决这些限制的一种方法是使用地形刺激来增强平面文化中的CSC群体。乳腺肿瘤微环境中的物理线索可以通过细胞外基质（ECM）的机械性质的变化来影响细胞行为。在这项研究中，我们使用聚苯乙烯薄膜上的地形线索，以研究它们对标准TNBC细胞系的蛋白质组和干性的影响。 方法：使用剃刀印刷和抛光方法生成基于聚苯乙烯的拓扑阵列。使用R软件分析蛋白质组数据并鉴定富集的生物过程。使用流式细胞术、免疫荧光、检测测定法测量CD 44、CD 24和ALDH标志物来评估干细胞，并使用乳腺球测定法进一步验证。采用酶联免疫吸附试验（ELISA）、免疫荧光和抗体膜阵列检测EGF/EGFR的表达和活性。进行剂量反应测定以进一步研究表面形貌对细胞对EGFR抑制剂的敏感性的影响。 结果：表面粗糙度富集CSC群体并调节TNBC细胞中的表皮生长因子受体（EGFR）信号传导活性。粗糙度下MDA-MB-468细胞的增殖增强与表皮生长因子（EGF）配体的上调相关，而与标准光滑培养表面相比，表皮生长因子（EGF）配体的上调又对应于EGFR表达的3倍增加和其磷酸化的42%增加。结果还表明，与地形（粗糙度）刺激相关的表型变化显著降低了对EGFR抑制剂吉非替尼的药物敏感性。此外，在MDA-MB-231和MDA-MB-468细胞系中，CD 44 +/CD 24 −/ALDH+的比例在表面粗糙度上增加。我们还证明了雅普/TAZ激活以粗糙度依赖性方式降低，证实了地形对细胞致癌活性的机械感应效应。 讨论内容：总体而言，本研究证明了表面粗糙度作为培养策略影响TNBC细胞中的致癌活性并在平面培养物中富集CSC群体的潜力。这样的培养策略可能有利于高通量筛选研究，以确定具有更广泛肿瘤疗效的化合物。
Introduction: Cancer stem cells (CSC), a major culprit of drug-resistant phenotypes and tumor relapse, represent less than 2 % of the bulk of TNBC cells, making them difficult to isolate, study, and thus, limiting our understanding of the pathogenesis of the disease. Current methods for CSC enrichment, such as 3D spheroid culture, genetic modification, and stem cell conditioning, are time consuming, expensive, and unsuitable for high-throughput assays. One way to address these limitations is to use topographical stimuli to enhance CSC populations in planar culture. Physical cues in the breast tumor microenvironment can influence cell behavior through changes in the mechanical properties of the extracellular matrix (ECM). In this study, we used topographical cues on polystyrene films to investigate their effect on the proteome and stemness of standard TNBC cell lines. Methods: The topographical polystyrene-based array was generated using razor printing and polishing methods. Proteome data were analyzed and enriched bioprocesses were identified using R software. Stemness was assessed measuring CD44, CD24 and ALDH markers using flow cytometry, immunofluorescence, detection assays, and further validated with mammosphere assay. EGF/EGFR expression and activity was evaluated using enzyme-linked immunosorbent assay (ELISA), immunofluorescence and antibody membrane array. A dose-response assay was performed to further investigate the effect of surface topography on the sensitivity of cells to the EGFR inhibitor. Results: Surface roughness enriched the CSC population and modulated epidermal growth factor receptor (EGFR) signaling activity in TNBC cells. Enhanced proliferation of MDA-MB-468 cells in roughness correlated with upregulation of the epidermal growth factor (EGF) ligand, which in turn corresponded with a 3-fold increase in the expression of EGFR and a 42% increase in its phosphorylation compared to standard smooth culture surfaces. The results also demonstrated that phenotypic changes associated with topographical (roughness) stimuli significantly decreased the drug sensitivity to the EGFR inhibitor gefitinib. In addition, the proportion of CD44+/CD24−/ALDH+ was enhanced on surface roughness in both MDA-MB-231 and MDA-MB-468 cell lines. We also demonstrated that YAP/TAZ activation decreased in a roughness-dependent manner, confirming the mechanosensing effect of the topographies on the oncogenic activity of the cells. Discussion: Overall, this study demonstrates the potential of surface roughness as a culture strategy to influence oncogenic activity in TNBC cells and enrich CSC populations in planar cultures. Such a culture strategy may benefit high-throughput screening studies seeking to identify compounds with broader tumor efficacy.
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影响因子：
4.6
作者：
Azimi I;Petersen RM;Thompson EW;Roberts-Thomson SJ;Monteith GR
通讯作者：
Monteith GR
影响因子：
7.5
作者：
Gao W;Wu D;Wang Y;Wang Z;Zou C;Dai Y;Ng CF;Teoh JY;Chan FL
通讯作者：
Chan FL
影响因子：
11.5
作者：
Culy, CR;Faulds, D
通讯作者：
Faulds, D
微胶状地面地形增强了小鼠骨髓衍生的间充质干细胞的细胞分裂和增殖。
影响因子：
3.7
作者：
Chaudhary JK;Rath PC
通讯作者：
Rath PC
影响因子：
4.6
作者：
Kawano M;Iwasaki T;Itonaga I;Kubota Y;Tanaka K;Tsumura H
通讯作者：
Tsumura H