PO.TB04.05 · 肿瘤生物学
通过微环境应激调节商业化 OCSCC 球体培养中的癌症干性
Modulating cancer stemness in commercial OCSCC spheroid cultures through microenvironmental stress
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
口腔鳞状细胞癌（OCSCC）的复发与癌症干细胞（CSC）相关，后者尽管经过治疗仍持续存在。为实现可重复的 CSC 研究，我们建立了一个可扩展的系统，使用商业化的 ATCC SCC-9 细胞系，并施加物理和代谢应激梯度——标准单层培养、AggreWell 球体（中度应激）和超低吸附（ULA）球体（最大应激）——分别在营养丰富（RMk 培养基）和营养缺乏（CSC 培养基）条件下进行。
SCC-9 细胞在标准组织培养瓶中扩增，并以确定的浓度接种至五种实验条件中：(1) 标准贴壁单层培养，(2) RMk 培养基中的 24 孔 AggreWell 400 板，(3) CSC 培养基中的 24 孔 AggreWell 400 板，(4) RMk 培养基中的 ULA 板，(5) CSC 培养基中的 ULA 板。四天后，对球体进行成像，用 Accutase 解离，并通过流式细胞术分析 CSC 富集情况。CD44 表达通过中位荧光强度（MFI）定量，醛脱氢酶活性升高则使用 Aldefluor 检测法检测。CD44 的 MFI 随微环境应激逐步升高，从 18,340（单层）升至 42,924（CSC 培养基中的 ULA 板），增加了 2.3 倍。双阳性 CSC 样群体（CD44high/Aldefluor+）在低应激单层培养和中度应激 AggreWell 培养中几乎不存在，但在 ULA RMk 中增加至 0.12%，在 ULA CSC 培养基中增加三倍至 0.42%。这些结果表明，CSC 的比例和标志物强度均可通过调节物理和代谢应激而系统性地进行调控。
该方案使研究人员能够使用广泛可得的 OCSCC 细胞系逐步增强 CSC 样富集。该方法学支持药物测试、分化研究和免疫相互作用检测等应用，利用商业化的板和试剂实现跨实验室的可重复性。CSC 诱导的动态范围为研究 OCSCC 复发和治疗耐药提供了实用工具。
查看英文原文 English abstract
Oral cavity squamous cell cancer (OCSCC) recurrence is linked to cancer stem cells (CSCs), which persist despite treatment. To enable reproducible CSC studies, we established a scalable system using the commercially available ATCC SCC-9 cell line subjected to physical and metabolic stress gradients-standard monolayer, AggreWell spheroids (moderate stress), and ultra-low attachment (ULA) spheroids (maximal stress)-in both nutrient-rich (RMk media) and nutrient-deficient (CSC media) conditions.
SCC-9 cells were expanded in standard tissue culture flasks and seeded at defined concentrations into five experimental conditions: (1) standard attachment monolayer, (2) 24-well AggreWell 400 plates in RMk media, (3) 24-well AggreWell 400 plates in CSC media, (4) ULA plates in RMk media, and (5) ULA plates in CSC media. After four days, spheroids were imaged, dissociated with Accutase, and analyzed for CSC enrichment by flow cytometry. CD44 expression was quantified by median fluorescence intensity (MFI), and increased aldehyde dehydrogenase activity was detected using the Aldefluor assay. MFI for CD44 rose progressively with microenvironmental stress, from 18,340 (monolayer) to 42,924 (ULA plate in CSC media), representing a 2.3-fold increase. The double-positive CSC-like population (CD44high/Aldefluor+) was nearly absent in low-stress monolayer and moderate-stress AggreWell cultures, but increased to 0.12% in ULA RMk and tripled to 0.42% in ULA CSC media. These results demonstrate that both CSC prevalence and marker intensity can be systematically tuned by adjusting physical and metabolic stress.
This protocol allows researchers to enhance CSC-like enrichment in a stepwise manner using a widely available OCSCC cell line. The methodology supports applications in drug testing, differentiation studies, and immune interaction assays, leveraging commercial plates and reagents for reproducibility across laboratories. The dynamic range of CSC induction provides a practical tool for studying OCSCC recurrence and therapeutic resistance.
利益披露 Disclosure
A. Elapavaluru, None..
S. Keysar, None..
H. Li, None..
A. Jimeno, None..
M. Kim, None..
F. N. Chowdhury, None.