A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies
A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies
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DOI：
10.1186/s40478-020-00918-5
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发表时间：
2020-04-17
影响因子：
7.1
通讯作者：
Phadke, Rahul
中科院分区：
文献类型：
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作者：
Scaglioni, Dominic;Ellis, Matthew;Phadke, Rahul
许多杜氏肌营养不良症（DMD）临床试验的主要分子终点是横纹肌中肌营养不良蛋白的诱导或产量增加。为了进行准确的终点分析，必须有可靠、稳健和客观的定量方法，能够检测肌营养不良蛋白表达的细微变化。在这项工作中，我们提出了进一步的发展和优化的自动化，数字化，高通量脚本的定量分析的多路复用免疫荧光（IF）全载玻片图像（WSI）的肌营养不良蛋白，肌营养不良蛋白相关蛋白（DAP）和再生肌纤维（胎儿/发育肌球蛋白阳性）的DMD，贝克肌营养不良症（BMD）和控制骨骼肌活检的横切面。该脚本能够广泛自动评估肌纤维形态学，通过荧光强度和肌膜周长覆盖率进行蛋白质定量，肌营养不良蛋白和DAP的共定位数据以及单个肌纤维和整个切片水平的再生。分析显示，在不同DMD和BMD样本之间，肌营养不良蛋白强度、覆盖率和DAP量存在显著差异。通过一种新的背景减除方法准确鉴定肌营养不良蛋白，允许与肌营养不良蛋白阴性肌膜区域相比，肌营养不良蛋白阳性区域内DAP荧光强度的差异评估。这使得能够替代量化肌营养不良蛋白在DAP复合物组装中的分子功能。总的来说，数字脚本能够对肌纤维再生和肌营养不良蛋白的标记物与关键DAP进行多参数和无偏分析，并能够更好地表征BMD和DMD中观察到的肌营养不良蛋白表达模式的异质性，以及肌营养不良蛋白分子功能的替代评估。这两个方面都将与正在进行和未来的DMD和其他肌营养不良症临床试验具有重要意义，以帮助基准治疗效果。
The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, high-throughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres (fetal/developmental myosin-positive) in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns seen in BMD and DMD alongside the surrogate assessment of molecular functionality of dystrophin. Both these aspects will be of significant relevance to ongoing and future DMD and other muscular dystrophies clinical trials to help benchmark therapeutic efficacy.