Quantitative image analysis of intracellular protein translocation in 3-dimensional tissues for pharmacodynamic studies of immunogenic cell death

免疫染色 流式细胞术 细胞生物学 内质网 细胞内 细胞 生物 细胞外 免疫原性细胞死亡 癌细胞 病理 程序性细胞死亡 细胞凋亡 癌症 分子生物学 免疫组织化学 免疫学 医学 生物化学 遗传学
作者
Yajing Sun,Ze Lu,John A. Taylor,Jessie L.‐S. Au
出处
期刊:Journal of Controlled Release [Elsevier BV]
卷期号:365: 89-100
标识
DOI:10.1016/j.jconrel.2023.11.023
摘要

A recent development in cancer chemotherapy is to use cytotoxics to induce tumor-specific immune response through immunogenic cell death (ICD). In ICD, calreticulin is translocated from endoplasmic reticulum to cell membrane (ecto-CRT) which serves as the 'eat-me-signal' to antigen-presenting cells. Ecto-CRT measurements, e.g., by ecto-CRT immunostaining plus flow cytometry, can be used to study the pharmacodynamics of ICD in single cells, whereas ICD studies in intact 3-dimensional tissues such as human tumors require different approaches. The present study described a method that used (a) immunostaining with fluorescent antibodies followed by confocal microscopy to obtain the spatial locations of two molecules-of-interest (CRT and a marker protein WGA), and (b) machine-learning (trainable WEKA segmentation) and additional image processing tools to locate the target molecules, remove the interfering signals in the nucleus, cytosol and extracellular space, enable the distinction of the inner and outer edges of the cell membrane and thereby identify the cells with ecto-CRT. This method, when applied to 3-dimensional human bladder cancer cell spheroids, yielded drug-induced ecto-CRT measurements that were qualitatively comparable to the flow cytometry results obtained with single cells disaggregated from spheroids. This new method was applied to study drug-induced ICD in short-term cultures of surgical specimens of human patient bladder tumors.
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