DataSheet_1_Spatiotemporal quantitative microRNA-155 imaging reports immune-mediated changes in a triple-negative breast cancer model.pdf

IntroductionMicroRNAs are small non-coding RNAs and represent key players in physiology and disease. Aberrant microRNA expression is central to the development and progression of cancer, with various microRNAs proposed as potential cancer biomarkers and drug targets. There is a need to better understand dynamic microRNA expression changes as cancers progress and their tumor microenvironments evolve. Therefore, spatiotemporal and non-invasive in vivo microRNA quantification in tumor models would be highly beneficial.MethodsWe developed an in vivo microRNA detector platform in which the obtained signals are positively correlated to microRNA presence, and which permitted stable expression in cancer cells as needed for long-term experimentation in tumor biology. It exploits a radionuclide-fluorescence dual-reporter for quantitative in vivo imaging of a microRNA of choice by radionuclide tomography and fluorescence-based downstream ex vivo tissue analyses. We generated and characterized breast cancer cells stably expressing various microRNA detectors and validated them in vitro.ResultsWe found the microRNA detector platform to report on microRNA presence in cells specifically and accurately, which was independently confirmed by real-time PCR and through microRNA modulation. Moreover, we established various breast tumor models in animals with different levels of residual immune systems and observed microRNA detector read-outs by imaging. Applying the detector platform to the progression of a triple-negative breast cancer model, we found that miR-155 upregulation in corresponding tumors was dependent on macrophage presence in tumors, revealing immune-mediated phenotypic changes in these tumors as they progressed.ConclusionWhile applied to immunooncology in this work, this multimodal in vivo microRNA detector platform will be useful whenever non-invasive quantification of spatiotemporal microRNA changes in living animals is of interest.

引言
miRNA（微小RNA）作为一种重要的非编码RNA，在生理和疾病过程中扮演着关键角色。miRNA表达异常是癌症发生和发展过程中的核心因素，多种miRNA被提议作为潜在的癌症生物标志物和药物靶点。深入理解癌症进展过程中及其肿瘤微环境演变中的miRNA表达动态变化，显得尤为迫切。因此，在肿瘤模型中对miRNA进行时空和微创的体内定量分析将极为有益。
方法
我们开发了一种体内miRNA检测平台，该平台获取的信号与miRNA的存在呈正相关，并允许在癌细胞中稳定表达，这对于长期肿瘤生物学实验至关重要。该平台利用放射性核素-荧光双报告系统，通过放射性核素断层扫描和基于荧光的离体组织分析，对所选定的miRNA进行定量体内成像。我们生成并表征了稳定表达各种miRNA检测器的乳腺癌细胞，并在体外进行了验证。
结果
我们发现miRNA检测平台能够特异性、准确地报告细胞中的miRNA存在情况，这一点通过实时PCR和miRNA调控得到了独立验证。此外，我们在具有不同残留免疫系统水平的动物体内建立了各种乳腺癌肿瘤模型，并通过成像观察了miRNA检测器的读数。将检测平台应用于三阴性乳腺癌模型的发展过程，我们发现相应肿瘤中miR-155的上调依赖于肿瘤中的巨噬细胞存在，揭示了这些肿瘤在进展过程中免疫介导的表型变化。
结论
尽管本研究应用于免疫肿瘤学，但这一多模态体内miRNA检测平台在需要非侵入性定量检测活体动物体内miRNA时空变化的情况下，将具有广泛的应用价值。