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Introduction
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Immunology and Infection

冷冻组织部分多光谱荧光成像的快速方法

Published: March 30, 2020
doi:
10.3791/60806
, , ,
1Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine,Northwestern University, 2Department of Pathology,University of Illinois at Chicago, 3Department of Microbiology and Immunology, Feinberg School of Medicine,Northwestern University

Summary

我们描述了一种快速染色方法，用于对冷冻组织进行多光谱成像。

Abstract

在正式固定石蜡嵌入 （FFPE） 组织中进行多光谱荧光成像，能够检测单个组织样本中的多个标记，从而提供有关标记的抗原共表达和空间分布的信息。然而，缺乏适合正式固定组织的抗体可能会限制可检测到的标记的性质。此外，染色方法非常耗时。在这里，我们描述了一种在冷冻组织中执行多光谱荧光成像的快速方法。该方法包括所使用的荧光波组合、小鼠和人类冷冻组织染色的详细步骤，以及扫描、采集和分析程序。对于染色分析，使用了商用半自动多光谱荧光成像系统。通过这种方法，在单个冷冻组织部分中，多达六个不同的标记被染色和检测。机器学习分析软件可以表型细胞，可用于定量分析。此处描述的冷冻组织方法可用于检测在 FFPE 组织中无法检测到的标记或 FFPE 组织不能针对的抗体。

Introduction

最近显微成像技术的进步大大增进了我们对生物过程和疾病状态的了解和理解。通过致色免疫组织化学（IHC）对组织中的蛋白质进行原位检测，在病理学中进行。然而，使用色谱IHC染色检测多个标记是具有挑战性的1，使用多路免疫荧光（mIF）染色方法的较新的方法，其中多个生物标记标记在单个组织样本上，正在开发中。多个生物标记的检测是有用的，因为有关组织结构、细胞空间分布和抗原共表达的信息都捕获在单个组织样本2中。多光谱荧光成像技术的使用使得检测多种生物标记成为可能。在这项技术中，使用特定的光学元件可以分离或”不混合”每个荧光波的荧光光谱，从而在没有任何光谱串扰3的情况下检测多个标记。多光谱荧光成像正在成为细胞生物学、临床前药物开发、临床病理学和肿瘤免疫功能44、5、65,6的关键方法。重要的是，免疫细胞（特别是CD8 T细胞）的分量可以作为现有肿瘤患者的预后因子7。

已经开发了多种荧光染色的各种方法，可以同时或按顺序执行。在同时染色的方法中，所有抗体在一个步骤中作为鸡尾酒一起加入，以标记组织。UltraPlex 技术使用触觉结合的初级抗体的鸡尾酒，然后采用氟磷结合抗触觉二次抗体的鸡尾酒。InSituPlex 技术8使用独特的 DNA 结合原抗体的混合物，这些抗体同时添加到组织中，然后进行放大步骤，最后是荧光结合探，这些探针与原抗体上每个独特的 DNA 序列相辅相成。这两种技术都能够检测四个标记和4’，6-2-苯胺烯酰胺（DAPI）的核染色。同时进行多路复用染色的另外两种方法是基于二次肌体质谱9。Hyperion 成像系统使用成像质量细胞学10来检测多达 37 个标记。该技术使用金属结合抗体的混合物来染色组织，组织的特定区域被激光清除，并转移到检测到金属离子的量量细胞计中。另一个类似的技术是IONPath，它使用多路复用的ion光束成像技术11。该技术使用经过改进的质谱仪和氧心源代替激光来清除金属偶联抗体。虽然所有这些同时多重染色方法能够检测多个标记，但无法低估将DNA、触觉或金属结合到抗体中所涉及的成本、消融造成的组织损失以及大量用于解混合的图像处理。此外，套件和染色协议目前仅适用于 FFPE 组织，开发定制面板需要额外的时间和支出。

相反，顺序多路复用染色方法包括将带有抗体的组织标记到一个标记，剥离以去除抗体，然后连续重复此过程以标记多个标记12。酪氨酸信号放大 （TSA） 是最常用的顺序多路复用方法。另外两种复用技术采用同时和顺序染色方法的组合。CODEX 平台13采用与独特的 DNA 寡核苷酸序列结合的抗体鸡尾酒，最终使用指数聚合步骤标记荧光磷，然后成像、剥离和重复这一过程，以检测多达 50 个标记。MultiOmyx 多路复用染色方法14是染色的迭代，其混合物由 3 到 4 个荧光素偶联抗体、成像、淬火荧光波和重复此循环来检测单个部分多达 60 个标记。与同时多重染色方法类似，虽然可以检测出广泛的标记，但染色、图像采集、处理和分析的时间也很大。剥离/淬火步骤涉及加热和/或漂白组织样本，因此，顺序多路复用染色方法通常对 FFPE 组织执行，在加热或漂白时保持组织完整性。

正式固定和随后的石蜡嵌入很容易在临床环境中进行，组织块易于存储，并提供多种多路染色方案。然而，FFPE组织的处理、嵌入和去压化，以及抗原检索15，抗体可以更好地获得表皮的过程，是费时的。此外，FFPE组织中的处理有助于自荧光16和口罩目标表皮，导致变异和缺乏抗体克隆可用于检测FFPE组织17，18，19的抗原。17,18,19一个例子是人类白细胞抗原（HLA）类I等位基因20。相反，组织的快速冻结不涉及修复前后的广泛处理步骤，无需对抗原检索21、22,22进行检测，有利于检测更广泛的靶点。因此，使用冷冻组织进行多光谱荧光成像对于检测临床前和临床研究的目标很有价值。

鉴于上述使用FFPE组织的限制，我们询问是否可以对冷冻组织进行多光谱荧光成像。为了解决这个问题，我们测试了一种同时使用荧光素结合抗体面板的多路复用染色方法，以检测多种抗原，并使用半自动多光谱成像系统对染色进行了分析。我们能够在90分钟内同时在单个组织部分中染色多达六个标记。

Protocol

小鼠脾脏和HLF16小鼠肿瘤组织23是从我们的实验室获得的。人扁桃体组织是从一家商业供应商那里购买的。详情载于资料表。 1. 组织嵌入 将新鲜组织嵌入 OCT（最佳切割温度）溶液中，并使用干冰或液氮进行卡扣冻结。 将组织储存在-80°C。 2. 冷冻切除 在低温器中切割 8 μm 部分，温度设定为 -25 °C?…

Representative Results

检测冷冻脾脏部分的单染色标记由于半自动成像系统使用液晶可调滤波器（LCTF）系统，允许更广泛的波长检测25，并且由于这里没有执行信号放大步骤，我们首先优化了显微镜上每个标记的初级结合抗体的检测。如图 1所示，其中每个单染色标记均为伪彩色红色。此处使用的Alexa Fluor偶联抗体已通过公司验证为免疫荧光和流细胞测定。但是，…

Discussion

冷冻组织已广泛用于mIF成像，传统上使用直接和间接方法32检测组织上的3到4个标记31。在直接方法中，抗体与荧光染料或量子点33结合以标记组织，而在间接方法中，非结合原抗体用于标记组织，然后由专门识别原抗体的氟磷结合二次抗体标记。前面讨论的一些最近同时使用的多路复用染色方法也可用于染色冷冻组织和检测四个以上的标记。?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

成像和分析指导由研究资源中心提供——芝加哥伊利诺伊大学研究组织学和组织成像核心，该中心由研究副校长办公室提供支持。这项工作得到了NIH/NCI RO1CA191317对中电的支持，国家卫生研究院/NIAMS（SBDRC向A.Paller博士授予1P30AR075049-01），以及罗伯特·卢里综合癌症中心对西北大学免疫治疗评估核心的支持。

Materials

Acetone (histological grade) Fisher Scientific A16F-1GAL Fixing tissues
Alexa Fluor 488 anti-mouse CD3 BioLegend 100212 Clone – 17A2; primary conjugated antibody
Alexa Fluor 488, eBioscience anti-human CD20 ThermoFisher Scientific 53-0202-82 Clone – L26; primary conjugated antibody
Alexa Fluor 555 Mouse anti-Ki-67 BD Biosciences 558617 Primary conjugated antibody
Alexa Fluor 594 anti-human CD3 BioLegend 300446 Clone – UCHT1; primary conjugated antibody
Alexa Fluor 594 anti-mouse CD8a BioLegend 100758 Clone – 53-6.7; primary conjugated antibody
Alexa Fluor 647 anti-human CD8a BioLegend 372906 Clone – C8/144B; primary conjugated antibody
Alexa Fluor 647 anti-mouse CD206 (MMR) BioLegend 141711 Clone – C068C2; primary conjugated antibody
Alexa Fluor 647 anti-mouse CD4 Antibody BioLegend 100426 Clone – GK1.5; primary conjugated antibody
C57BL/6 Mouse Charles River Laboratories 27 Mouse frozen tissues used for multispectral training
Coplin Jar Sigma Aldrich S6016-6EA Rehydrating and washing slides
DAPI Solution BD Biosciences 564907 Nucleic Acid stain
Diamond White Glass Charged Slides DOT Scientific DW7590W Adhering tissue sections
Dulbecco's Phosphate Buffered Saline 1x (without Ca and Mg) Fisher Scientific MT21031CV Washing and diluent
Gold Seal Cover Slips ThermoFisher Scientific 3306 Protecting stained tissues
Human Normal Tonsil OCT frozen tissue block AMSBio AMS6023 Human frozen tissue used for multispectral staining
Human Serum 1X Gemini Bio-Products 100-512 Blocking and diluent for human tissues
inForm Akoya Biosciences Version 2.4.1 Machine learning software
PerCP/Cyanine5.5 anti-human CD4 BioLegend 300529 Clone – RPA-T4; primary conjugated antibody
PerCP-Cy 5.5 Rat Anti-CD11b BD Biosciences 550993 Clone – M1/70; primary conjugated antibody
Phenochart Akoya Biosciences Version 1.0.8 Whole slide scan software
ProLong Diamond Antifade Mountant ThermoFisher Scientific P36965 Mounting medium
Research Cryostat Leica Biosystems CM3050 S Sectioning tissues
Superblock 1X ThermoFisher Scientific 37515 Blocking mouse tissues
Tissue-Tek O.C.T Solution Sakura Finetek 4583 Embedding tissues
Vectra 3.0 Automated Quantitative Pathology Imaging System, 6 Slide Akoya Biosciences CLS142568 Semi-automated multispectral imaging system
Vectra Software Akoya Biosciences Version 3.0.5 Software to operate microscope
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References

  1. van der Loos, C. M. Chromogens in Multiple Immunohistochemical Staining Used for Visual Assessment and Spectral Imaging: The Colorful Future. Journal of Histotechnology. 33 (1), 31-40 (2010).
  2. Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 70 (1), 46-58 (2014).
  3. Bian, L., et al. Multispectral imaging using a single bucket detector. Scientific Reports. 6, 24752 (2016).
  4. Zhou, L., El-Deiry, W. S. Multispectral fluorescence imaging. Journal of Nuclear Medicine. 50 (10), 1563-1566 (2009).
  5. Parra, E. R., et al. Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues. Scientific Reports. 7 (1), 13380 (2017).
  6. Wickenhauser, C., Pico de Coaña, Y., et al. . Immune Checkpoint Blockade: Methods and Protocols. , 13-31 (2019).
  7. Feng, Z., et al. Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma. Journal for ImmunoTherapy of Cancer. 3 (1), 47 (2015).
  8. Manesse, M., Patel, K. K., Bobrow, M., Downing, S. R. The InSituPlex((R)) Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples. Methods in Molecular Biology. 2055, 585-592 (2020).
  9. Gamble, L. J., Anderton, C. R. Secondary Ion Mass Spectrometry Imaging of Tissues, Cells, and Microbial Systems. Microscopy Today. 24 (2), 24-31 (2016).
  10. Giesen, C., et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nature Methods. 11 (4), 417-422 (2014).
  11. Angelo, M., et al. Multiplexed ion beam imaging of human breast tumors. Nature Medicine. 20 (4), 436-442 (2014).
  12. Tsujikawa, T., et al. Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis. Cell Reports. 19 (1), 203-217 (2017).
  13. Goltsev, Y., et al. Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging. Cell. 174 (4), 968-981 (2018).
  14. Gerdes, M. J., et al. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proceedings of the National Academy of Sciences of the United States of America. 110 (29), 11982-11987 (2013).
  15. Shi, S. R., Key, M. E., Kalra, K. L. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. Journal of Histochemistry & Cytochemistry. 39 (6), 741-748 (1991).
  16. Viegas, M. S., Martins, T. C., Seco, F., do Carmo, A. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. European Journal of Histochemistry. 51 (1), 59-66 (2007).
  17. Sorensen, I. V., et al. Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue. Journal of Histochemistry & Cytochemistry. 54 (10), 1075-1086 (2006).
  18. Parra, E. R., Villalobos, P., Mino, B., Rodriguez-Canales, J. Comparison of Different Antibody Clones for Immunohistochemistry Detection of Programmed Cell Death Ligand 1 (PD-L1) on Non-Small Cell Lung Carcinoma. Applied Immunohistochemistry & Molecular Morphology. 26 (2), 83-93 (2018).
  19. Boger, C., Kalthoff, H., Goodman, S. L., Rocken, C. Validation and comparison of anti-alphavbeta3 and anti-alphavbeta5 rabbit monoclonal versus murine monoclonal antibodies in four different tumor entities. Applied Immunohistochemistry & Molecular Morphology. 21 (6), 553-560 (2013).
  20. Torigoe, T., et al. Establishment of a monoclonal anti-pan HLA class I antibody suitable for immunostaining of formalin-fixed tissue: unusually high frequency of down-regulation in breast cancer tissues. Pathology International. 62 (5), 303-308 (2012).
  21. Dapson, R. W. Macromolecular changes caused by formalin fixation and antigen retrieval. Biotechnic & Histochemistry. 82 (3), 133-140 (2007).
  22. Sompuram, S. R., Vani, K., Hafer, L. J., Bogen, S. A. Antibodies Immunoreactive With Formalin-Fixed Tissue Antigens Recognize Linear Protein Epitopes. American Journal of Clinical Pathology. 125 (1), 82-90 (2006).
  23. Cassetti, M. C., et al. Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes. Vaccine. 22 (3-4), 520-527 (2004).
  24. Kiernan, J. A. Histological and Histochemical Methods: Theory and Practice, 3rd Edition. Shock. 12 (6), 479 (1999).
  25. Favreau, P., et al. Thin-film tunable filters for hyperspectral fluorescence microscopy. Journal of Biomedical Optics. 19 (1), 011017 (2014).
  26. van Kempen, M. J., Rijkers, G. T., Van Cauwenberge, P. B. The immune response in adenoids and tonsils. International Archives of Allergy and Immunology. 122 (1), 8-19 (2000).
  27. Klein, U., Dalla-Favera, R. Germinal centres: role in B-cell physiology and malignancy. Nature Reviews Immunology. 8 (1), 22-33 (2008).
  28. Eiben, G. L., et al. Establishment of an HLA-A*0201 Human Papillomavirus Type 16 Tumor Model to Determine the Efficacy of Vaccination Strategies in HLA-A*0201 Transgenic Mice. Cancer Research. 62, 5792-5799 (2002).
  29. Gonzalez, H., Hagerling, C., Werb, Z. Roles of the immune system in cancer: from tumor initiation to metastatic progression. Genes & Development. 32 (19-20), 1267-1284 (2018).
  30. Sica, A., Schioppa, T., Mantovani, A., Allavena, P. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. European Jorunal of Cancer. 42 (6), 717-727 (2006).
  31. Au-Granier, C., et al. Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells. Journal of Visualized Experiments. (132), e56606 (2018).
  32. Odell, I. D., Cook, D. Immunofluorescence Techniques. Journal of Investigative Dermatology. 133 (1), 1-4 (2013).
  33. Xing, Y., et al. Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nature Protocols. 2 (5), 1152-1165 (2007).
  34. Schubert, W., et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nature Biotechnology. 24 (10), 1270-1278 (2006).
  35. de Vries, N. L., et al. High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity. Gut. , 03 (2019).
  36. Scalia, C. R., et al. Antigen Masking During Fixation and Embedding, Dissected. The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. 65 (1), 5-20 (2017).
  37. Sorrelle, N., et al. Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues. Journal of Immunology. 202 (1), 292-299 (2019).
  38. Ackerman, L. V., Ramirez, G. A. The indications for and limitations of frozen section diagnosis; a review of 1269 consecutive frozen section diagnoses. British Journal of Surgery. 46 (198), 336-350 (1959).
  39. Mezheyeuski, A., et al. Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients. The Journal of Pathology. 244 (4), 421-431 (2018).
  40. Feng, Z., et al. Multiparametric immune profiling in HPV- oral squamous cell cancer. JCI insight. 2 (14), 93652 (2017).
  41. Yang, L., Liu, Z., Tan, J., Dong, H., Zhang, X. Multispectral imaging reveals hyper active TGF-β signaling in colorectal cancer. Cancer Biology & Therapy. 19 (2), 105-112 (2018).
  42. Blom, S., et al. Systems pathology by multiplexed immunohistochemistry and whole-slide digital image analysis. Scientific Reports. 7 (1), 15580-15580 (2017).
  43. Roy, S., Axelrod, H. D., Valkenburg, K. C., Amend, S., Pienta, K. J. Optimization of prostate cancer cell detection using multiplex tyramide signal amplification. Journal of Cellular Biochemistry. 120 (4), 4804-4812 (2019).
  44. Vickovic, S., et al. High-density spatial transcriptomics arrays for in situ tissue profiling. bioRxiv. , 563338 (2019).

Tags

Multispectral Fluorescence ImagingBiomarker DetectionCo-localizationRapid Staining MethodSpectral LibraryMachine LearningImage Analysis

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Jaishankar, D., Cosgrove, C., Deaton, R. J., Le Poole, I. C. A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections. J. Vis. Exp. (157), e60806, doi:10.3791/60806 (2020).
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