Agüero Eduardo Imanol, Gómez López Silvia María, Peñaherrera-Pazmiño Ana Belén, Tellado Matías, Pérez Maximiliano Sebastián, Lerner Betiana, Belgorosky Denise, Eiján Ana María
Universidad de Buenos Aires, Facultad de Medicina, Instituto de Oncología Ángel H. Roffo, Área de Investigación, Buenos Aires C1417DTB, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires C1425FQD, Argentina.
Cancers (Basel). 2025 Jun 10;17(12):1922. doi: 10.3390/cancers17121922.
Cancer stem cells (CSCs) represent a minor yet critical subpopulation within tumors, endowed with self-renewal and differentiation capacities, and are implicated in tumor initiation, progression, metastasis, therapeutic resistance, and recurrence. Reliable in vitro functional assays to characterize CSCs are pivotal for the development of personalized oncology strategies. This study sought to establish and validate a microfluidic device (MD) platform for the enrichment, functional assessment, and therapeutic evaluation of CSC populations derived from experimental models and primary tumor samples. Murine (LM38LP) and human (BPR6) breast cancer cell lines were cultured within MDs to promote sphere formation. CSC enrichment was confirmed through the expression analysis of pluripotency-associated genes (Oct4, Sox2, Nanog, and CD44) by quantitative PCR (qPCR) and immunofluorescence. Sphere number, size, and gene expression profiles were quantitatively assessed before (control) and after chemotherapeutic exposure. To validate the MD platform against conventional scale, parallel experiments were performed in 12 well plates. To extend translational relevance, three primary canine tumor samples (solid thyroid carcinoma, simple tubular carcinoma, and reactive lymph node) were mechanically disaggregated and processed within MDs for CSC characterization. The MD platform enabled the consistent enrichment of CSC populations, showing significant modulation of sphere growth parameters and stemness marker expression following chemotherapeutic treatment. Beyond its comparability with conventional culture, the MD also supported immunofluorescence staining and allowed real-time monitoring of individual cell growth. Sphere formation efficiency (SFE) and CSC marker expression were similarly demonstrated in primary veterinary tumor cultures, highlighting the device's cross-species applicability. Microfluidic-based sphere assays represent a robust, reproducible, and scalable platform for the functional interrogation of CSC dynamics and therapeutic responses. This methodology holds great promise for advancing CSC-targeted therapies and supporting personalized oncology in both human and veterinary settings.
癌症干细胞（CSCs）是肿瘤内一个数量虽少但至关重要的亚群，具有自我更新和分化能力，与肿瘤的起始、进展、转移、治疗抵抗及复发相关。用于表征CSCs的可靠体外功能测定对于个性化肿瘤学策略的发展至关重要。本研究旨在建立并验证一个微流控装置（MD）平台，用于富集、功能评估及治疗评估源自实验模型和原发性肿瘤样本的CSC群体。将小鼠（LM38LP）和人（BPR6）乳腺癌细胞系培养在MDs中以促进球体形成。通过定量PCR（qPCR）和免疫荧光对多能性相关基因（Oct4、Sox2、Nanog和CD44）进行表达分析，确认CSC富集情况。在化疗暴露前后（对照），对球体数量、大小及基因表达谱进行定量评估。为了将MD平台与传统规模进行验证对比，在12孔板中进行了平行实验。为了拓展转化相关性，对三个犬原发性肿瘤样本（实性甲状腺癌、单纯管状癌和反应性淋巴结）进行机械解离，并在MDs中进行处理以表征CSCs。MD平台能够一致地富集CSC群体，显示出化疗处理后球体生长参数和干性标志物表达的显著调节。除了与传统培养具有可比性外，MD还支持免疫荧光染色，并允许对单个细胞生长进行实时监测。在原发性兽医肿瘤培养物中同样证实了球体形成效率（SFE）和CSC标志物表达，突出了该装置的跨物种适用性。基于微流控的球体测定代表了一个强大、可重复且可扩展的平台，用于对CSC动力学和治疗反应进行功能研究。这种方法在推进CSC靶向治疗及支持人类和兽医环境中的个性化肿瘤学方面具有巨大潜力。
