一种用于乳腺肿瘤微环境活体内成像的无标记分割方法
Summary
本文描述的活体成像方法利用来自代谢辅助因子NAD（P）H的胶原二次谐波和内源性荧光，将未标记的肿瘤微环境非侵入性地分割成肿瘤，基质和血管室，以深入分析4D活体内图像。
Abstract
可视化活肿瘤微环境中多种细胞类型与细胞外基质（ECM）之间复杂和动态生理相互作用的能力是了解调节肿瘤进展机制的重要一步。虽然这可以通过当前的活体成像技术来实现，但由于组织的异质性以及实验观察中对空间背景的需求，它仍然具有挑战性。为此，我们开发了一种活体内成像工作流程，将胶原蛋白二次谐波生成成像，来自代谢共因子NAD（P）H的内源性荧光和荧光寿命成像显微镜（FLIM）配对，作为将肿瘤微环境非侵入性地划分为肿瘤巢，周围基质或ECM以及脉管系统的基本结构域的手段。这种非侵入性方案详细介绍了从采集乳腺肿瘤模型的延时图像到后处理分析和图像分割的分步过程。该工作流程的主要优点是，它利用代谢特征来将动态变化的活肿瘤微环境置于情境中，而无需使用外源性荧光标记，这使其有利于人类患者来源的异种移植物（PDX）模型和未来的临床使用，其中外在荧光团不易适用。
Introduction
已知肿瘤微环境中的细胞外基质（ECM）被多种细胞类型动态沉积和重塑，以进一步促进疾病进展1，2，3。这些基质改变提供了改变细胞行为的机械和生物学线索，并且通常导致基质重塑的持续循环4。对肿瘤细胞和细胞外基质之间动态，相互相互作用的研究通常使用三维（3D）体外培养或微流体系统进行。虽然这些自下而上的方法已经证明了ECM重塑5，6，7，增殖增加8，上皮到间充质转变9，10，11，12以及肿瘤细胞迁移和侵袭的机制7，13，14，15，16，他们的重点主要集中在均匀的3D基质中的几种细胞类型（例如，肿瘤细胞或成纤维细胞）上，与生理组织内存在的相互作用的多样性和异质性相比。除了体外系统外，离体肿瘤组织学还可以提供一些关于这些细胞-细胞和细胞-ECM相互作用的见解17。免疫组织化学的优点是能够分析与ECM的空间异质组成和结构相关的多种细胞类型，但是固定组织的静态终点不能捕获细胞与微环境之间相互作用的动态性质。活体成像为在原生肿瘤微环境的生理环境中询问各种动态相互作用打开了大门。
活体肿瘤成像的能力正在迅速提高。影像学窗口设计的改进和植入视窗的手术技术使得在各种解剖位置（即原发性肿瘤，淋巴结，转移部位18，19，20）进行长期纵向肿瘤成像。此外，光学仪器在多个维度（即光谱、空间荧光强度和寿命）以及高分辨率和高速（视频速率）下可视化和收集数据的能力正在变得广泛应用。改进的技术为探索生理环境中细胞信号传导和表型动力学的快速变化提供了机会。最后，光遗传学工具的扩增和广泛的遗传荧光构建体允许标记特定细胞类型以捕获肿瘤微环境中的细胞迁移或发育或疾病进展期间的细胞谱系示踪21，22。将这些工具与CRISPR / Cas9技术结合使用，为研究人员提供了及时生成独特动物模型的机会。
虽然所有这些进展使活体成像成为探索动态和生理细胞相互作用的日益强大的方法，但仍然需要制定在组织水平上为这些生物相互作用提供空间，时间和结构背景的策略。目前，许多活体成像研究通过将荧光染料注射到脉管系统中或采用外源性表达荧光蛋白以描绘物理特征的小鼠模型来弥补视觉特征（例如血管）的缺乏。可注射染料和底物如荧光葡聚糖被广泛用于成功标记活体内的脉管系统19， 23， 24。但是，这种方法并非没有限制。首先，它需要额外的鼠标操作，其效用仅限于短期实验。对于纵向研究，荧光葡聚糖可能会有问题，因为我们观察到葡聚糖在吞噬细胞中的积累或随着时间的推移扩散到周围组织中25。外源性荧光蛋白掺入小鼠模型已被提出为荧光葡聚糖的替代品，但存在其自身的局限性。小鼠模型中外源性荧光团的可用性和多样性仍然有限且创建成本高昂。此外，在特定的模型中，例如PDX模型，遗传操作是不可取的或不可能的。还已经表明，细胞内荧光或生物发光蛋白的存在被识别为小鼠内的外来物，并且在免疫功能正常的小鼠模型中，这减少了由于宿主免疫系统的反应而引起的转移量26，27。最后，用于空间背景或分割后续数据的外源荧光蛋白或荧光染料通常占据光谱的素数范围，否则可用于研究感兴趣的生理相互作用。
使用来自ECM的内在信号或来自组织内细胞的内源性荧光代表了一种潜在的通用无标记方法，用于分割活体内数据以进行更深入的细胞和空间分析。二次谐波产生（SHG）长期以来一直用于可视化ECM28。随着随后开发有助于表征纤维组织29，30，31的重要工具，可以表征相对于局部ECM结构的细胞行为。此外，来自内源性代谢物NAD（P）H的自发荧光提供了另一种无标记的工具，用于划分 体内肿瘤微环境。NAD（P）H在肿瘤细胞中发出明亮的荧光，可用于区分生长中的肿瘤巢的边界与其周围的基质21，32。最后，脉管系统是肿瘤微环境中的重要生理结构，也是关键细胞类型特异性相互作用的位点33，34，35。红细胞（RBC）或血浆的激发已被用于可视化肿瘤脉管系统，并且使用双光子或三光子激发（2P; 3P）的血流速率的测量已被证明是可能的36。然而，虽然较大的血管很容易通过其内源性荧光特征来识别，但识别细微的，可变的和荧光较少的小血管需要更多的专业知识。这些固有的困难阻碍了最佳图像分割。幸运的是，这些内源性荧光源（即红细胞和血浆）也可以通过荧光寿命成像37来测量，其利用了脉管系统独特的光物理特性，并代表了对不断增长的活体内工具箱的有益补充。
在该协议中，描述了从采集到分析的四维（4D）活体内成像的工作流程，明确使用内源性荧光和SHG等内在信号进行分割。该协议对于通过乳腺成像窗口进行的纵向研究特别相关，其中外源性荧光可能不实用或不可能，就像PDX模型一样。然而，这里概述的分割原理广泛适用于研究肿瘤生物学，组织发育甚至正常组织生理学的活体内用户。所报告的分析方法套件将允许用户区分排列或随机胶原纤维配置区域之间的细胞行为，比较驻留在肿瘤微环境特定区域的细胞的数量或行为，并仅使用无标记或内在信号将脉管系统映射到肿瘤微环境。这些方法共同创建了一个操作框架，用于最大化从乳腺4D活体内成像中获得的信息深度，同时最大限度地减少对其他外源性标记的需求。
Protocol
Representative Results
Discussion
4D活体成像是研究天然肿瘤微环境的空间和时间背景下动态生理相互作用的强大工具。该手稿提供了一个非常基本且适应性强的操作框架，仅使用来自二次谐波产生或NAD（P）H自发荧光的内源性信号来划分肿瘤肿块内，相邻基质或靠近血管网络的动态细胞相互作用。该协议提供了一种全面的分步方法，从植入成像窗口到图像采集，分析和分割。我们相信这种技术将有助于建立一个必要的分析框架，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢NCI R01 CA216248，CA206458和CA179556资助这项工作。我们还要感谢Kevin Eliceiri博士及其成像小组在早期开发我们活体内计划方面的技术专长。我们还感谢Ben Cox博士和Morgridge研究所Eliceiri制造小组的其他成员，他们在MIW的早期阶段进行了必要的技术设计。Ellen Dobson博士协助进行了有关ImageJ可训练WEKA分割工具的有用对话。此外，我们要感谢Melissa Skala博士和Alexa Barres-Heaton博士及时使用他们的显微镜。最后，我们要感谢 Brigitte Raabe 博士，D.V.M.，感谢他就我们的鼠标处理和护理所做的所有深思熟虑的讨论和建议。
Materials
| #1.5 12mm round cover glass | Warner Instruments | # 64-0712 | MIW construction |
| 1.0 mL syringe for SQ injection | BD | 309659 | Syringe |
| 20x objective | Zeiss | 421452-988 | Water immersion |
| 27G needle for SQ injection | Covidien | 1188827012 | Needle |
| 40x objective | Nikon | MRD77410 | Water immersion |
| 5-0 silk braided suture | Ethicon | K870 | Suture for MIW implantation |
| Artificial tears gel | Akorn | NDC 59399-162-35 | Eye gel |
| Betadine solution, 5% | Fisher Scientific | NC1558063 | Surgery antiseptic |
| cotton-tipped applicator | Fisher Scientific | 23-400-101 | |
| Cyanoacrylate adhesive | Loctite | 1365882 | MIW construction |
| fluorescent dextran | Sigma | T1287-50mg | intravenous labelling of vasculature |
| forceps | Mckesson.com | Miltex #18-782 | stainless, 4 inch, curved |
| GaAsP photomultiplier tube | Hamamatsu | ||
| heating blanket | CARA 72 heating pad | 038056000729 | Temperature selectable |
| heating chamber | home built | ||
| Fluorescent lifetime handbook | Becker and Hickl | https://www.becker-hickl.com/literature/handbooks | |
| inverted microscope base | Nikon | ||
| Isoflurane | Akorn | NDC 59399-106-01 | Anesthesia |
| Liqui-Nox | Fisher Scientific | 16-000-125 | MIW cleaning |
| Meloxicam | Norbrook | NDC 55529-040-10 | Analgesic |
| Micro Hose | Scientific Commodities INC. | BB31695-PE/1 | |
| multiphoton scan head | Bruker Ultima II | Multiphoton scanhead and imaging platform | |
| NADH FLIM filter | Chroma | 284994 | ET 440/80 m-2P |
| Nair | CVS | 339826 | Depilatory cream |
| objective heater | Tokai Hit | STRG-WELSX-SET | |
| SHG/FAD filter | Chroma | 320740 | ET450/40m-2P |
| Sparkle glass cleaner | Amazon.com | B00814ME24 | Glass Cleaner for implanted MIW |
| SPC-150 photon counting board | Becker and Hickl | ||
| surgical light | FAJ | B06XV1VQVZ | Magnetic LED gooseneck light |
| surgical micro-scissors | Excelta | 366 | stainless, 3 inch |
| Triple antibiotic ointment | Actavis Pharma | NDC 0472-0179-34 | Antibiotic |
| TV catheter | Custom | BD 30G needle: 305106 | Catheter for TV injection |
| Two photon filter | Chroma | 320282 | ET585/65m-2P |
| two-photon laser | Coherent charmeleon | Tunable multiphoton laser | |
| ultrasound gel | Parker | PKR-03-02 | Water immersion gel |
| Urea crystals | Sigma | U5128-5G | Optional: FLIM IRF |
References
- Eble, J. A., Niland, S. The extracellular matrix in tumor progression and metastasis. Clinical & Experimental Metastasis. 36 (3), 171-198 (2019).
- Afik, R., et al. Tumor macrophages are pivotal constructors of tumor collagenous matrix. The Journal of Experimental Medicine. 213 (11), 2315-2331 (2016).
- Varol, C., Sagi, I. Phagocyte-extracellular matrix crosstalk empowers tumor development and dissemination. The FEBS Journal. 285 (4), 734-751 (2018).
- Winkler, J., Abisoye-Ogunniyan, A., Metcalf, K. J., Werb, Z. Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nature Communications. 11 (1), 5120 (2020).
- Han, W., et al. Oriented collagen fibers direct tumor cell intravasation. Proceedings of the National Academy of Sciences of the United States of America. 113 (40), 11208-11213 (2016).
- Malandrino, A., Mak, M., Kamm, R. D., Moeendarbary, E. Complex mechanics of the heterogeneous extracellular matrix in cancer. Extreme Mechanics Letters. 21, 25-34 (2018).
- Lugo-Cintrón, K. M., et al. Breast Fibroblasts and ECM Components Modulate Breast Cancer Cell Migration Through the Secretion of MMPs in a 3D Microfluidic Co-Culture Model. Cancers. 12 (5), 1173 (2020).
- Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J., Keely, P. J. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. The Journal of Cell Biology. 163 (3), 583-595 (2003).
- Zhang, K., et al. The collagen receptor discoidin domain receptor 2 stabilizes SNAIL1 to facilitate breast cancer metastasis. Nature Cell Biology. 15 (6), 677-687 (2013).
- Malik, G., et al. Plasma fibronectin promotes lung metastasis by contributions to fibrin clots and tumor cell invasion. 암 연구학. 70 (11), 4327-4334 (2010).
- Bae, Y. K., Choi, J. E., Kang, S. H., Lee, S. J. Epithelial-mesenchymal transition phenotype is associated with clinicopathological factors that indicate aggressive biological behavior and poor clinical outcomes in invasive breast cancer. Journal of Breast Cancer. 18 (3), 256-263 (2015).
- Wei, S. C., et al. Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway. Nature Cell Biology. 17 (5), 678-688 (2015).
- Riching, K. M., et al. 3D collagen alignment limits protrusions to enhance breast cancer cell persistence. Biophysical Journal. 107 (11), 2546-2558 (2014).
- Carey, S. P., et al. Local extracellular matrix alignment directs cellular protrusion dynamics and migration through Rac1 and FAK. Integrative Biology: Quantitative Biosciences from Nano to Macro. 8 (8), 821-835 (2016).
- Ray, A., Morford, R. K., Ghaderi, N., Odde, D. J., Provenzano, P. P. Dynamics of 3D carcinoma cell invasion into aligned collagen. Integrative Biology: Quantitative Biosciences from Nano to Macro. 10 (2), 100-112 (2018).
- Szulczewski, J. M., et al. Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers. Acta Biomaterialia. 129, 96-109 (2021).
- Esbona, K., et al. The Presence of Cyclooxygenase 2, Tumor-Associated Macrophages, and Collagen Alignment as Prognostic Markers for Invasive Breast Carcinoma Patients. The American Journal of Pathology. 188 (3), 559-573 (2018).
- Entenberg, D., et al. A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nature Methods. 15 (1), 73-80 (2018).
- Kedrin, D., et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nature Methods. 5 (12), 1019-1021 (2008).
- Jacquemin, G., et al. Longitudinal high-resolution imaging through a flexible intravital imaging window. Science Advances. 7 (25), (2021).
- Boone, P. G., et al. A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion. Nature Communications. 10 (1), 5490 (2019).
- Dawson, C. A., Mueller, S. N., Lindeman, G. J., Rios, A. C., Visvader, J. E. Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue. Nature Protocols. 16 (4), 1907-1935 (2021).
- Leung, E., et al. Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway. Oncogene. 36 (19), 2680-2692 (2017).
- Jain, R. K. Normalizing tumor microenvironment to treat cancer: Bench to bedside to biomarkers. Journal of Clinical Oncology: Official Journal of The American Society of Clinical Oncology. 31 (17), 2205-2218 (2013).
- Wyckoff, J. B., et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. 암 연구학. 67 (6), 2649-2656 (2007).
- Baklaushev, V. P., et al. Modeling and integral X-ray, optical, and MRI visualization of multiorgan metastases of orthotopic 4T1 breast carcinoma in BALB/c Mice. Bulletin of Experimental Biology and Medicine. 158 (4), 581-588 (2015).
- Baklaushev, V. P., et al. Luciferase Expression Allows Bioluminescence Imaging But Imposes Limitations on the Orthotopic Mouse (4T1) Model of Breast Cancer. Scientific Reports. 7 (1), 7715 (2017).
- Campagnola, P. J., Loew, L. M. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nature Biotechnology. 21 (11), 1356-1360 (2003).
- Bredfeldt, J. S., et al. Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer. Journal of Biomedical Optics. 19 (1), 16007 (2014).
- Liu, Y., et al. Fibrillar Collagen Quantification With Curvelet Transform Based Computational Methods. Frontiers in Bioengineering and Biotechnology. 8, 198 (2020).
- Püspöki, Z., Storath, M., Sage, D., Unser, M. Transforms and Operators for Directional Bioimage Analysis: A Survey. Advances in Anatomy, Embryology, and Cell Biology. 219, 69-93 (2016).
- Saytashev, I., et al. Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood. Biomedical Optics Express. 7 (9), 3449-3460 (2016).
- Harney, A. S., et al. Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discovery. 5 (9), 932-943 (2015).
- von Au, A., et al. Circulating fibronectin controls tumor growth. Neoplasia. 15 (8), 925-938 (2013).
- Murgai, M., et al. KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis. Nature Medicine. 23 (10), 1176-1190 (2017).
- You, S., et al. Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy. Nature Communications. 9 (1), 2125 (2018).
- Yakimov, B. P., et al. Label-free characterization of white blood cells using fluorescence lifetime imaging and flow-cytometry: molecular heterogeneity and erythrophagocytosis. Biomedical Optics Express. 10 (8), 4220-4236 (2019).
- Arganda-Carreras, I., et al. Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics. 33 (15), 2424-2426 (2017).
- Guy, C. T., Cardiff, R. D., Muller, W. J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Molecular and Cellular Biology. 12 (3), 954-961 (1992).
- Provenzano, P. P., et al. Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Medicine. 4 (1), 38 (2006).
- Conklin, M. W., et al. Aligned collagen is a prognostic signature for survival in human breast carcinoma. The American Journal of Pathology. 178 (3), 1221-1232 (2011).
- Szulczewski, J. M., et al. In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging. Scientific Reports. 6, 25086 (2016).
- Hoffmann, E. J., Ponik, S. M. Biomechanical Contributions to Macrophage Activation in the Tumor Microenvironment. Frontiers in Oncology. 10, 787 (2020).
- Pakshir, P., et al. Dynamic fibroblast contractions attract remote macrophages in fibrillar collagen matrix. Nature Communications. 10 (1), 1850 (2019).
- Dobrolecki, L. E., et al. Patient-derived xenograft (PDX) models in basic and translational breast cancer research. Cancer and Metastasis Reviews. 35 (4), 547-573 (2016).
- Shirshin, E. A., et al. Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization. Scientific Reports. 7 (1), 1171 (2017).
- Weigert, M., et al. Content-aware image restoration: pushing the limits of fluorescence microscopy. Nature Methods. 15 (12), 1090-1097 (2018).
