PDF(Pubmed)
Abstract：
UNASSIGNED: Understanding the key role of the tumor microenvironment in specifying molecular markers of breast cancer subtypes is of a high importance in diagnosis and treatment. Therefore, the possibility of interconversion of luminal states and their specific markers alteration under the control of tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs) deserves to be further investigated.
UNASSIGNED: To activate normal human fibroblasts, liquid overlay technique or nemosis was used and α-SMA protein expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines were treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of both luminal A and B cells were evaluated by invert light microscopy and analyzed through the shape factor formula. Moreover, chemo-sensitivity, proliferation, and changes in ER-related and proliferative genes expression levels were assessed respectively via MTT assay, Ki67 expression Immunofluorescence assay, real time PCR and Annexin V-FITC techniques.
UNASSIGNED: Activated (spheroidal) fibroblasts, expressed αSMA marker two folds more than monolayer cultured fibroblasts. Our study indicated a significant increase in IC50 of both luminal A and B cell lines after being treated with conditioned medium particularly in treated group with spheroidal conditioned medium. Studying Morphological changes using shape factor formula demonstrated more aggressiveness with gaining mesenchymal features in both luminal A and B subtypes by increasing exposure time. Changes in the expression of Ki67 were observed following treatment with fibroblastic and spheroidal paracrine secretome. Driven Data from Ki67 assay supports the luminal A and B interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression analysis revealed that anti-apoptotic Bcl2 gene expression in both luminal types treated with condition medium has been increased though there has seen no interchange in expression of ER-related and proliferative genes between luminal A (MCF7) and luminal B (MDA-MB361) subtypes, the results of Annexin V-FITC flow cytometry test indicated a decrease in the population of both early and late apoptotic cells in groups treated with both fibroblastic and spheroidal condition medium compared to of control group.
UNASSIGNED: Under the paracrine influence of fibroblast cells, both luminal A (MCF7) and luminal B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated(spheroidal) fibroblasts conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and luminal B which share more similarities among breast cancer molecular subtypes.
UNASSIGNED: Understanding the key role of the tumor microenvironment in specifying molecular markers of breast cancer subtypes is of a high importance in diagnosis and treatment. Therefore, the possibility of interconversion of luminal states and their specific markers alteration under the control of tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs) deserves to be further investigated.
UNASSIGNED: To activate normal human fibroblasts, liquid overlay technique or nemosis was used and α-SMA protein expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines were treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of both luminal A and B cells were evaluated by invert light microscopy and analyzed through the shape factor formula. Moreover, chemo-sensitivity, proliferation, and changes in ER-related and proliferative genes expression levels were assessed respectively via MTT assay, Ki67 expression Immunofluorescence assay, real time PCR and Annexin V-FITC techniques.
UNASSIGNED: Activated (spheroidal) fibroblasts, expressed αSMA marker two folds more than monolayer cultured fibroblasts. Our study indicated a significant increase in IC50 of both luminal A and B cell lines after being treated with conditioned medium particularly in treated group with spheroidal conditioned medium. Studying Morphological changes using shape factor formula demonstrated more aggressiveness with gaining mesenchymal features in both luminal A and B subtypes by increasing exposure time. Changes in the expression of Ki67 were observed following treatment with fibroblastic and spheroidal paracrine secretome. Driven Data from Ki67 assay supports the luminal A and B interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression analysis revealed that anti-apoptotic Bcl2 gene expression in both luminal types treated with condition medium has been increased though there has seen no interchange in expression of ER-related and proliferative genes between luminal A (MCF7) and luminal B (MDA-MB361) subtypes, the results of Annexin V-FITC flow cytometry test indicated a decrease in the population of both early and late apoptotic cells in groups treated with both fibroblastic and spheroidal condition medium compared to of control group.
UNASSIGNED: Under the paracrine influence of fibroblast cells, both luminal A (MCF7) and luminal B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated(spheroidal) fibroblasts conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and luminal B which share more similarities among breast cancer molecular subtypes.
摘要：
■了解肿瘤微环境在确定乳腺癌亚型分子标志物中的关键作用在诊断和治疗中非常重要。因此,在肿瘤微环境（TME）的控制下，管腔状态的相互转换及其特异性标志物改变的可能性，特别是癌症相关成纤维细胞（CAFs）值得进一步研究。
■为了激活正常人成纤维细胞，使用液体覆盖技术或止血技术，并表达α-SMA蛋白，CAFs标记,在成纤维细胞球体中通过印迹测量。管腔A，MCF-7和腔B，将MDA-MB361细胞系用正常和球形/活化的成纤维细胞条件培养基处理48小时。通过倒置光学显微镜评估腔A和B细胞的形态变化，并通过形状因子公式进行分析。此外，化学敏感性，扩散,并通过MTT法分别评估ER相关基因和增殖基因表达水平的变化，Ki67表达免疫荧光测定,实时PCR和膜联蛋白V-FITC技术。
■激活（球形）成纤维细胞，表达αSMA标记比单层培养的成纤维细胞多两倍。我们的研究表明，用条件培养基处理后，腔A和B细胞系的IC50显着增加，特别是在用球形条件培养基处理的组中。使用形状因子公式研究形态变化表明，通过增加暴露时间，在腔A和B亚型中获得间充质特征具有更大的侵略性。用成纤维细胞和球形旁分泌体处理后，观察到Ki67表达的变化。来自Ki67测定的驱动数据通过管腔A中Ki67表达升高和管腔B中Ki67表达降低来支持管腔A和B的相互转换。基因表达分析显示，用条件培养基处理的两种管腔类型中的抗凋亡Bcl2基因表达已经增加，尽管在管腔A（CFM7）和管腔B（MDA-MB361）亚型之间的ER相关和增殖基因的表达没有互换，AnnexinV-FITC流式细胞术检测结果表明,与对照组相比,用成纤维细胞和球形条件培养基处理组的早期和晚期凋亡细胞数量均减少.
■在成纤维细胞的旁分泌作用下，乳腺癌的管腔A（MCF7）和管腔B（MDA-MB）亚型均获得侵袭性，抗凋亡，和化学抗性特征，其主要通过模拟CAF的活化（球状）成纤维细胞条件培养基增加。没有强有力的证据证明管腔A和管腔B在乳腺癌分子亚型之间具有更多的相似性。
■了解肿瘤微环境在确定乳腺癌亚型分子标志物中的关键作用在诊断和治疗中非常重要。因此,在肿瘤微环境（TME）的控制下，管腔状态的相互转换及其特异性标志物改变的可能性，特别是癌症相关成纤维细胞（CAFs）值得进一步研究。
■为了激活正常人成纤维细胞，使用液体覆盖技术或止血技术，并表达α-SMA蛋白，CAFs标记,在成纤维细胞球体中通过印迹测量。管腔A，MCF-7和腔B，将MDA-MB361细胞系用正常和球形/活化的成纤维细胞条件培养基处理48小时。通过倒置光学显微镜评估腔A和B细胞的形态变化，并通过形状因子公式进行分析。此外，化学敏感性，扩散,并通过MTT法分别评估ER相关基因和增殖基因表达水平的变化，Ki67表达免疫荧光测定,实时PCR和膜联蛋白V-FITC技术。
■激活（球形）成纤维细胞，表达αSMA标记比单层培养的成纤维细胞多两倍。我们的研究表明，用条件培养基处理后，腔A和B细胞系的IC50显着增加，特别是在用球形条件培养基处理的组中。使用形状因子公式研究形态变化表明，通过增加暴露时间，在腔A和B亚型中获得间充质特征具有更大的侵略性。用成纤维细胞和球形旁分泌体处理后，观察到Ki67表达的变化。来自Ki67测定的驱动数据通过管腔A中Ki67表达升高和管腔B中Ki67表达降低来支持管腔A和B的相互转换。基因表达分析显示，用条件培养基处理的两种管腔类型中的抗凋亡Bcl2基因表达已经增加，尽管在管腔A（CFM7）和管腔B（MDA-MB361）亚型之间的ER相关和增殖基因的表达没有互换，AnnexinV-FITC流式细胞术检测结果表明,与对照组相比,用成纤维细胞和球形条件培养基处理组的早期和晚期凋亡细胞数量均减少.
■在成纤维细胞的旁分泌作用下，乳腺癌的管腔A（MCF7）和管腔B（MDA-MB）亚型均获得侵袭性，抗凋亡，和化学抗性特征，其主要通过模拟CAF的活化（球状）成纤维细胞条件培养基增加。没有强有力的证据证明管腔A和管腔B在乳腺癌分子亚型之间具有更多的相似性。
