Abstract：
One of the leading causes that complicate the treatment of some malignancies, including breast cancer, is tumor heterogeneity. In addition to inter-heterogeneity and intra-heterogeneity of tumors that reflect the differences between cancer cell characteristics, heterogeneity in the tumor microenvironment plays a critical role in tumor progression and could be considered an overlooked and a proper target for the effective selection of therapeutic approaches. Due to the difficulty of completely capturing tumor heterogeneity in conventional detection methods, Tumor-on-Chip (TOC) devices with culturing patient-derived spheroids could be an appropriate alternative. In this research, human-derived spheroids from breast cancer individuals were cultured for 6 days in microfluidic devices. To compare TOC data with conventional detection methods, immunohistochemistry (IHC) and ITRAQ data were employed, and various protein expressions were validated using the transcriptomic databases. The behavior of the spheroids in the collagen matrix and the cell viability were monitored over 6 days of culture. IHC and immunocytochemistry (ICC) results revealed that inter and intra-heterogeneity of tumor spheroids are associated with HER2/ER expression. HER2 expression levels revealed a more important biomarker associated with invasion in the 3D culturing of spheroids. The expression levels of CD163 (as a marker for Ma2 macrophages) and CD44 (a marker for cancer stem cells (CSCs)) were also evaluated. Interestingly, the levels of M2a macrophages and CSCs were higher in triple-negative specimens and samples that showed higher migration and invasion. Cell density and extracellular matrix (ECM) stiffness were also important factors affecting the migration and invasion of the spheroids through the matrix. Among these, rigid ECM revealed a more crucial role than cell density. To sum up, these research findings demonstrated that human-derived spheroids from breast cancer specimens in microfluidic devices provide a dynamic condition for predicting tumor heterogeneity in patients, which can help move the field forward for better and more accurate therapeutic strategies.
摘要：
导致某些恶性肿瘤治疗复杂化的主要原因之一，包括乳腺癌,是肿瘤异质性。除了反映癌细胞特征差异的肿瘤间异质性和内异质性外，肿瘤微环境中的异质性在肿瘤进展中起着至关重要的作用,可被认为是一个被忽视的,也是有效选择治疗方法的适当目标.由于常规检测方法难以完全捕获肿瘤异质性,具有培养患者来源的球状体的芯片上肿瘤(TOC)装置可能是合适的替代方案。在这项研究中,来自乳腺癌个体的人源球体在微流体装置中培养6天.为了将TOC数据与常规检测方法进行比较，采用免疫组织化学（IHC）和ITRAQ数据，使用转录组数据库验证了各种蛋白质表达。在6天的培养中监测胶原蛋白基质中球体的行为和细胞活力。IHC和免疫细胞化学（ICC）结果显示，肿瘤球体的内部和内部异质性与HER2/ER表达相关。HER2表达水平揭示了与球体3D培养中的侵袭相关的更重要的生物标志物。还评估了CD163(作为Ma2巨噬细胞的标志物)和CD44(癌症干细胞(CSC)的标志物)的表达水平。有趣的是,在三阴性标本和显示较高迁移和侵袭的样本中,M2a巨噬细胞和CSC的水平较高.细胞密度和细胞外基质（ECM）硬度也是影响球体通过基质迁移和侵袭的重要因素。其中,刚性ECM显示出比细胞密度更重要的作用。总而言之,这些研究结果表明，微流控设备中乳腺癌标本中的人源球体为预测患者肿瘤异质性提供了动态条件，这可以帮助推动该领域向前发展，以获得更好，更准确的治疗策略。
