异质性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显著因素。培养患者来源的乳腺癌细胞（PDBCCs）为癌细胞的异质性提供了一种宝贵的体外和转化研究工具。本研究旨在探究不同培养基成分和培养方法对ER+ PDBCCs中BCSC相关免疫表型和基因表达的影响。本研究纳入了10例ER+乳腺癌患者，其中6例进行了新辅助化疗，4例未进行。使用胶原I和透明质酸酶的酶法分离PDBCCs。将PDBCCs作为单层培养和悬浮状态下的多细胞球体培养。分别使用涂有胶原I的培养皿和涂有聚合物X的超低附着力培养皿进行单层培养和球体培养。采用流式细胞术、免疫荧光染色、RT-PCR和RNA测序方法检测PDBCCs的免疫表型和基因组信息。在经3-4次亚培养后，超过95%的PDBCCs在单层条件下保持EpCAM高/+/成纤维细胞标记-表型。A83-01的去除导致具有较高β-半乳糖苷酶活性的细胞衰老。单层培养的PDBCCs以大多数细胞具有EpCAM+/CD49f+表型为特征。与单层培养中的全培养基相比，去除EGF会增加EpCAM+/CD49f-表型（增加13.8倍，p=0.028），而去除R-spondin会减少其表型（减少0.8倍，p=0.02）。去除A83-01会增加EpCAM+/CD24+表型（增加1.82倍，p=0.023），减少EpCAM低/-/CD44+/CD24-表型（减少0.45倍，p=0.026）。与单层培养相比，球体培养会导致EpCAM-/CD49+（增加14.6倍，p=0.006），EpCAM低/-/CD44+/CD24-表型（增加4.16倍，p=0.022）和ALDH高活性（增加9.66倍，p=0.037）的细胞亚群增加。ALDH1A和EMT相关基因被上调。通过spheroid和monolayer的RNA测序分析，鉴定出了561个差异表达基因（2倍改变，p<0.05），富集在27条KEGG信号通路中，包括调节干细胞多能性的信号通路。根据Kaplan-Meier Plotter数据库中spheroid中鉴定的上调和下调基因对复发无病生存的分析结果，发现15个上调和14个下调基因与乳腺癌患者预后不良有关。媒体成分和球体培养方法的改变会改变PDBCCs的BCSCs和EMT标记物，这意味着在体外研究PDBCCs时定义媒体成分和培养方法的重要性。©2023. BioMed Central Ltd., Springer Nature成员.

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.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.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.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.© 2023. BioMed Central Ltd., part of Springer Nature.