Department of Pathology, Leiden University Medical Center, PO Box 9600, 2300, RC, Leiden, The Netherlands.
Centre for Mass Spectrometry Imaging, Biomolecular Sciences Research Centre, Sheffield Hallam University, Howard Street, Sheffield, S1 1WB, UK.
J Mass Spectrom. 2020 Apr;55(4):e4461. doi: 10.1002/jms.4461. Epub 2019 Dec 2.
Three-dimensional (3D) cell culture is a rapidly emerging field, which mimics some of the physiological conditions of human tissues. In cancer biology, it is considered a useful tool in predicting in vivo chemotherapy responses, compared with conventional two-dimensional (2D) cell culture. We have developed a novel 3D cell culture model of osteosarcoma composed of aggregated proliferative tumour spheroids, which shows regions of tumour heterogeneity formed by aggregated spheroids of polyclonal tumour cells. Aggregated spheroids show local necrotic and apoptotic regions and have sizes suitable for the study of spatial distribution of metabolites by mass spectrometry imaging (MSI). We have used this model to perform a proof-of-principle study showing a heterogeneous distribution of endogenous metabolites that colocalise with the necrotic core and apoptotic regions in this model. Cytotoxic chemotherapy (doxorubicin) responses were significantly attenuated in our 3D cell culture model compared with those of standard cell culture, as determined by resazurin assay, despite sufficient doxorubicin diffusion demonstrated by localisation throughout the 3D constructs. Finally, changes to the distribution of endogenous metabolites in response to doxorubicin were readily detected by MSI. Principal component analysis identified 50 metabolites which differed most in their abundance between treatment groups, and of these, 10 were identified by both in-software t test and mixed-effects analysis of variance (ANOVA). Subsequent independent MSIs of identified species were consistent with principle component analysis findings. This proof-of-principle study shows for the first time that chemotherapy-induced changes in metabolite abundance and distribution may be determined in 3D cell culture by MSI, highlighting this method as a potentially useful tool in the elucidation of chemotherapy responses as an alternative to in vivo testing.
三维（3D）细胞培养是一个迅速发展的领域，它模拟了一些人体组织的生理条件。在癌症生物学中，与传统的二维（2D）细胞培养相比，它被认为是预测体内化疗反应的有用工具。我们开发了一种新型的骨肉瘤 3D 细胞培养模型，由聚集的增殖性肿瘤球体组成，这些球体显示出由多克隆肿瘤细胞聚集球体形成的肿瘤异质性区域。聚集的球体显示出局部坏死和凋亡区域，并且大小适合通过质谱成像（MSI）研究代谢物的空间分布。我们已经使用这种模型进行了一项原理验证研究，显示出内源性代谢物的异质分布，这些代谢物与该模型中的坏死核心和凋亡区域共定位。通过 Resazurin 测定法，与标准细胞培养相比，我们的 3D 细胞培养模型中的细胞毒性化疗（阿霉素）反应明显减弱，尽管通过局部扩散到整个 3D 结构中证明了足够的阿霉素扩散。最后，通过 MSI 可以很容易地检测到内源性代谢物对阿霉素的反应的分布变化。主成分分析确定了 50 种代谢物，它们在处理组之间的丰度差异最大，其中 10 种代谢物通过软件中的 t 检验和混合效应方差分析（ANOVA）都得到了鉴定。随后对鉴定出的物种的独立 MSI 与主成分分析结果一致。这项原理验证研究首次表明，通过 MSI 可以在 3D 细胞培养中确定化疗引起的代谢物丰度和分布的变化，突出了这种方法作为替代体内测试的化疗反应阐明的潜在有用工具。
