A method for reproducible high-resolution imaging of 3D cancer cell spheroids.
A method for reproducible high-resolution imaging of 3D cancer cell spheroids.
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一种对 3D 癌细胞球体进行可重复高分辨率成像的方法。
DOI：
10.1111/jmi.13169
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发表时间：
2023
影响因子：
2
通讯作者：
Phillips TA
中科院分区：
文献类型：
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作者：
Phillips TA
嵌入三维（3D）水凝胶或细胞外基质（ECM）中的多细胞肿瘤细胞球体被广泛用作研究癌症生长和侵袭的模型。将球体嵌入 3D 矩阵中的标准方法会导致在空间中随机放置，这限制了倒置荧光显微镜技术的使用，从而限制了在完整球体中对分子细节进行成像所能达到的分辨率。在这里，我们利用紫外光刻技术来微加工 PDMS（聚二甲基硅氧烷）印模，从而可以在多个不同的成像室中生成高含量、可重复的井状结构。将多细胞肿瘤球体添加到冲压胶原结构中可以在 3D 空间中精确定位球体，从而实现可重复的高分辨率/超分辨率成像。嵌入式球体可以实时成像或固定成像，并且适合免疫染色，从而使实验方法具有更大的灵活性。我们描述了使用这些球体成像室通过各种常用的倒置显微镜平台来分析细胞侵袭、细胞-ECM 相互作用、ECM 排列、力依赖性细胞内蛋白质动力学以及基于肌动蛋白的精细突起的延伸。该方法能够对多个肿瘤球体进行可重复的、高分辨率/超分辨率的实时成像，并有可能扩展到类器官和其他更复杂的 3D 体外系统的可视化。
Multicellular tumour cell spheroids embedded within three‐dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high‐content, reproducible well‐like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high‐/super‐resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell–ECM interaction, ECM alignment, force‐dependent intracellular protein dynamics and extension of fine actin‐based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high‐/super‐resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.