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Abstract：
BACKGROUND: Heterogeneous tumor cells are thought to be a significant factor in the failure of endocrine therapy in estrogen receptor-positive (ER+) cancers. Culturing patient-derived breast cancer cells (PDBCCs) provides an invaluable tool in pre-clinical and translational research for the heterogeneity of cancer cells. This study aimed to investigate the effects of different media components and culture methods on the BCSC-associated immunophenotypes and gene expression in ER + PDBCCs.
METHODS: Ten patients with ER + breast cancer were employed in this study, six of whom had neoadjuvant chemotherapy and four of whom did not. PDBCCs were isolated by enzymatic methods using collagen I and hyaluronidase. PDBCCs were grown as monolayers in mediums with different compositions and as multicellular spheroid in a suspended condition. Collagen I-coated plate and ultralow attachment plate coated with polymer-X were used for monolayer and spheroid culture. Flow cytometry, immunofluorescent staining, RT-PCR, and RNA-sequencing were employed to examine the immunophenotype and genetic profile of PDBCCs.
RESULTS: More than 95% of PDBCCs sustain EpCAM high/+/fibroblast marker- phenotypes in monolayer conditions by subculturing 3-4 times. A83-01 removal induced senescent cells with high β-galactosidase activity. PDBCCs grown as monolayers were characterized by the majority of cells having an EpCAM+/CD49f + phenotype. Compared to full media in monolayer culture, EGF removal increased EpCAM+/CD49f - phenotype (13.8-fold, p = 0.028), whereas R-spondin removal reduced it (0.8-fold, p = 0.02). A83-01 removal increased EpCAM+/CD24 + phenotype (1.82-fold, p = 0.023) and decreased EpCAM low/-/CD44+/CD24- phenotype (0.45-fold, p = 0.026). Compared to monolayer, spheroid resulted in a significant increase in the population with EpCAM-/CD49+ (14.6-fold, p = 0.006) and EpCAM low/-/CD44+/CD24- phenotypes (4.16-fold, p = 0.022) and ALDH high activity (9.66-fold, p = 0.037). ALDH1A and EMT-related genes were upregulated. In RNA-sequencing analysis between spheroids and monolayers, a total of 561 differentially expressed genes (2-fold change, p < 0.05) were enriched in 27 KEGG pathways including signaling pathways regulating pluripotency of stem cells. In a recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the up-and down-regulated genes identified in spheroids, 15 up-, and 14 down-regulated genes were associated with poor prognosis of breast cancer patients.
CONCLUSIONS: The media composition and spheroid culture method change in the BCSCs and EMT markers of PDBCCs, implying the importance of defining the media composition and culture method for studying PDBCCs in vitro.
METHODS: Ten patients with ER + breast cancer were employed in this study, six of whom had neoadjuvant chemotherapy and four of whom did not. PDBCCs were isolated by enzymatic methods using collagen I and hyaluronidase. PDBCCs were grown as monolayers in mediums with different compositions and as multicellular spheroid in a suspended condition. Collagen I-coated plate and ultralow attachment plate coated with polymer-X were used for monolayer and spheroid culture. Flow cytometry, immunofluorescent staining, RT-PCR, and RNA-sequencing were employed to examine the immunophenotype and genetic profile of PDBCCs.
RESULTS: More than 95% of PDBCCs sustain EpCAM high/+/fibroblast marker- phenotypes in monolayer conditions by subculturing 3-4 times. A83-01 removal induced senescent cells with high β-galactosidase activity. PDBCCs grown as monolayers were characterized by the majority of cells having an EpCAM+/CD49f + phenotype. Compared to full media in monolayer culture, EGF removal increased EpCAM+/CD49f - phenotype (13.8-fold, p = 0.028), whereas R-spondin removal reduced it (0.8-fold, p = 0.02). A83-01 removal increased EpCAM+/CD24 + phenotype (1.82-fold, p = 0.023) and decreased EpCAM low/-/CD44+/CD24- phenotype (0.45-fold, p = 0.026). Compared to monolayer, spheroid resulted in a significant increase in the population with EpCAM-/CD49+ (14.6-fold, p = 0.006) and EpCAM low/-/CD44+/CD24- phenotypes (4.16-fold, p = 0.022) and ALDH high activity (9.66-fold, p = 0.037). ALDH1A and EMT-related genes were upregulated. In RNA-sequencing analysis between spheroids and monolayers, a total of 561 differentially expressed genes (2-fold change, p < 0.05) were enriched in 27 KEGG pathways including signaling pathways regulating pluripotency of stem cells. In a recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the up-and down-regulated genes identified in spheroids, 15 up-, and 14 down-regulated genes were associated with poor prognosis of breast cancer patients.
CONCLUSIONS: The media composition and spheroid culture method change in the BCSCs and EMT markers of PDBCCs, implying the importance of defining the media composition and culture method for studying PDBCCs in vitro.
摘要：
背景：异质肿瘤细胞被认为是雌激素受体阳性（ER+）癌症内分泌治疗失败的重要因素。培养患者来源的乳腺癌细胞（PDBCC）在临床前和转化研究中为癌细胞的异质性提供了宝贵的工具。本研究旨在探讨不同培养基成分和培养方法对ER+PDBCC中BCSC相关免疫表型和基因表达的影响。
方法：本研究采用10例ER+乳腺癌患者，其中6人接受了新辅助化疗,4人未接受新辅助化疗.使用胶原蛋白I和透明质酸酶通过酶法分离PDBCC。PDBCC在具有不同组成的培养基中作为单层生长，并在悬浮条件下作为多细胞球体生长。胶原蛋白I包被板和涂有聚合物X的超低附着板用于单层和球体培养。流式细胞术,免疫荧光染色，RT-PCR,和RNA测序用于检测PDBCC的免疫表型和遗传谱。
结果:超过95%的PDBCC通过传代培养3-4次在单层条件下维持EpCAM高/+/成纤维细胞标记表型。A83-01去除诱导具有高β-半乳糖苷酶活性的衰老细胞。以单层生长的PDBCC的特征在于大多数细胞具有EpCAM+/CD49f+表型。与单层培养的全培养基相比，EGF去除增加了EpCAM+/CD49f-表型(13.8倍，p=0.028),而R-spondin去除减少了它(0.8倍，p=0.02)。A83-01去除增加了EpCAM+/CD24+表型（1.82倍，p=0.023）和降低的EpCAM低/-/CD44/CD24-表型（0.45倍，p=0.026)。与单层相比，球体导致EpCAM-/CD49+的种群显著增加（14.6倍，p=0.006)和EpCAM低/-/CD44+/CD24-表型(4.16倍，p=0.022）和ALDH高活性（9.66倍，p=0.037)。ALDH1A和EMT相关基因上调。在球体和单层之间的RNA测序分析中，总共561个差异表达基因(2倍变化，p<0.05）富集在27个KEGG途径中，包括调节干细胞多能性的信号传导途径。在基于Kaplan-Meier绘图仪数据库的无复发生存分析中，在球状体中鉴定出的上调和下调基因，15上-，14个下调基因与乳腺癌患者的不良预后相关。
结论：PDBCC的BCSC和EMT标志物的培养基组成和球体培养方法发生变化，这意味着定义体外研究PDBCC的培养基组成和培养方法的重要性。
方法：本研究采用10例ER+乳腺癌患者，其中6人接受了新辅助化疗,4人未接受新辅助化疗.使用胶原蛋白I和透明质酸酶通过酶法分离PDBCC。PDBCC在具有不同组成的培养基中作为单层生长，并在悬浮条件下作为多细胞球体生长。胶原蛋白I包被板和涂有聚合物X的超低附着板用于单层和球体培养。流式细胞术,免疫荧光染色，RT-PCR,和RNA测序用于检测PDBCC的免疫表型和遗传谱。
结果:超过95%的PDBCC通过传代培养3-4次在单层条件下维持EpCAM高/+/成纤维细胞标记表型。A83-01去除诱导具有高β-半乳糖苷酶活性的衰老细胞。以单层生长的PDBCC的特征在于大多数细胞具有EpCAM+/CD49f+表型。与单层培养的全培养基相比，EGF去除增加了EpCAM+/CD49f-表型(13.8倍，p=0.028),而R-spondin去除减少了它(0.8倍，p=0.02)。A83-01去除增加了EpCAM+/CD24+表型（1.82倍，p=0.023）和降低的EpCAM低/-/CD44/CD24-表型（0.45倍，p=0.026)。与单层相比，球体导致EpCAM-/CD49+的种群显著增加（14.6倍，p=0.006)和EpCAM低/-/CD44+/CD24-表型(4.16倍，p=0.022）和ALDH高活性（9.66倍，p=0.037)。ALDH1A和EMT相关基因上调。在球体和单层之间的RNA测序分析中，总共561个差异表达基因(2倍变化，p<0.05）富集在27个KEGG途径中，包括调节干细胞多能性的信号传导途径。在基于Kaplan-Meier绘图仪数据库的无复发生存分析中，在球状体中鉴定出的上调和下调基因，15上-，14个下调基因与乳腺癌患者的不良预后相关。
结论：PDBCC的BCSC和EMT标志物的培养基组成和球体培养方法发生变化，这意味着定义体外研究PDBCC的培养基组成和培养方法的重要性。
