Lopez Arbil, Holbrook Joseph H, Hummon Amanda B
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States.
Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States.
Anal Chem. 2025 Jun 3;97(21):11239-11248. doi: 10.1021/acs.analchem.5c01309. Epub 2025 May 22.
Microphysiological systems, such as multicellular spheroids, hold great promise for drug screening experiments. Spheroids may be dosed statically, where the drug is introduced to the growing chamber at one time point, or dynamically, where the drug is introduced via a fluidic component. Dynamic dosing can generate pharmacokinetic curves that more closely represent those seen in vivo than static dosing. In this work, we demonstrate the dynamic dosing of colorectal cancer spheroids in a 3D printed fluidic device with liposomal doxorubicin. Spheroids are valuable models to evaluate dynamic dosing, as they recapitulate the nutrient, oxygen, and pH gradients of solid tumors. Spheroids feature distinct cellular populations with a necrotic core, quiescent middle layer, and proliferative outer layer. Drug and liposome penetration are tracked with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and fluorescence imaging, showing that liposomal doxorubicin is stable to fluidic dosing and penetrates spheroids after 48 h. To provide a comprehensive pharmacodynamic profile of the distinct cellular regions within spheroids, we employ spatially stable isotopic labeling by amino acids in cell culture (spatial SILAC) proteomics to isotopically label the core and outer layers. Proteomic analysis reveals 714 upregulated proteins in the core upon treatment and 30 in the outer layers, as well as 103 downregulated proteins in the core and 1276 in the outer layers. Spatial SILAC uncovers the differential regulation of proteins associated with glycolysis, the TCA cycle, and lipid synthesis upon drug treatment between the spheroid core and outer layers. Using MALDI MSI and spatial SILAC proteomics, we interrogate the effects of dynamic dosing with liposomal doxorubicin on spheroid regions that would be overlooked by bulk analysis.
微生理系统，如多细胞球体，在药物筛选实验中具有巨大潜力。球体可以进行静态给药，即在一个时间点将药物引入生长室，也可以进行动态给药，即通过流体组件引入药物。动态给药所产生的药代动力学曲线比静态给药更能准确反映体内情况。在这项研究中，我们展示了在3D打印的流体装置中使用脂质体阿霉素对结直肠癌球体进行动态给药。球体是评估动态给药的有价值模型，因为它们概括了实体瘤的营养、氧气和pH梯度。球体具有不同的细胞群体，包括坏死核心、静止中间层和增殖外层。通过基质辅助激光解吸/电离质谱成像（MALDI MSI）和荧光成像跟踪药物和脂质体的渗透情况，结果表明脂质体阿霉素在流体给药后保持稳定，并在48小时后穿透球体。为了全面了解球体中不同细胞区域的药效学特征，我们采用细胞培养中氨基酸的空间稳定同位素标记（空间SILAC）蛋白质组学对核心层和外层进行同位素标记。蛋白质组学分析显示，处理后核心层有714种上调蛋白，外层有30种；核心层有103种下调蛋白，外层有1276种。空间SILAC揭示了药物处理后球体核心层和外层之间与糖酵解、三羧酸循环和脂质合成相关蛋白质的差异调节。通过MALDI MSI和空间SILAC蛋白质组学，我们研究了脂质体阿霉素动态给药对整体分析可能会忽略的球体区域的影响。
