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In vivo Imaging of Tumor and Immune Cell Interactions in the Lung
肺内肿瘤细胞与免疫细胞反应的体内成像   

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Abstract

Immunotherapy has demonstrated great therapeutic potential by activating the immune system to fight cancer. However, little is known about the specific dynamics of interactions that occur between tumor and immune cells. In this protocol we describe a novel method to visualize the interaction of tumor and immune cells in the lung of live mice, which can be applied to other organs. In this protocol fluorescent-labeled tumor cells are transferred to recipient mice expressing fluorescently tagged immune cells. Tumor-immune cell interactions in the lung are then imaged by confocal or two photon microscopy. Analysis of tumor interactions with immune cells using this protocol should aid in a better understanding of the importance of these interactions and their role in developing immunotherapies.

Keywords: In vivo imaging(活体成像), Cancer(癌症), Immunology(免疫学), Lung(肺), Phagocytosis(吞噬)

Background

A number of immunotherapies have demonstrated great promise in treating cancer. Understanding the spatial temporal resolution of how these tumor-immune interactions occur is important for enhancing and developing new immunotherapies. In this protocol we describe a novel method to directly visualize tumor-immune cell interactions in vivo in mouse lung. This protocol is initially described in our work examining the interactions of patrolling monocytes and tumor cells in the mouse lung (Hanna et al., 2015). This fluorescent microscopy protocol uses the vacuum imaging ring to stabilize and image the lung, which was initially described by Looney and colleagues (Thornton et al., 2012). In this protocol fluorescent-labeled tumor cells are transferred to recipient mice expressing fluorescently tagged immune cells. Tumor-immune cell interactions in the lung are then imaged by confocal or two photon fluorescent microscopy using the vacuum imaging ring. This protocol allows for the addition of other immune cell markers by intravenous (IV) injection of fluorescently labeled antibodies, and is adaptable to image tumor-immune cell interactions in other organs. Quantitative information such as the localization, engulfment of tumor material, timing, speed and frequency of these immune cell interactions can be collected using this protocol. This protocol should aid in helping to better understand the specific immune-tumor cell interactions that are important to developing better immunotherapies in the future.

Materials and Reagents

  1. Cell culture flask
  2. 30 gauge insulin syringe (BD, catalog number: 328431 ) Nr4a1-GFP (The Jackson Laboratory, catalog number: 018974 ), CX3CR1-GFP (The Jackson Laboratory, catalog number: 005582 ) or other fluorescent reporter mouse for visualizing immune cells.
  3. ½ micro-cover glass (12 mm diameter) (Electron Microscope Sciences, catalog number: 72230-01 )
  4. PE-90 tubing (BD, IntramedicTM, catalog number: 427420 )
  5. Mice
  6. Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP) or other fluorescent-tagged tumor cell line (AntiCancer.com)
  7. TrypLETM Express enzyme(Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 )
  8. Dulbecco's phosphate-buffered saline (DPBS) (GE Healthcare, HycloneTM, catalog number: SH30038.02 )
  9. Ketamine hydrochloride
  10. Xylazine hydrochloride
  11. Vetbond glue (3M, catalog number: 1469SB )
  12. Oxygen
  13. Ethanol
  14. Dow Corning® high vacuum grease (Sigma-Aldrich, catalog number: Z273554 )

Equipment

  1. Centrifuge
  2. Mechanical mouse ventilator (Harvard Apparatus, model: 845 )
  3. Fine dissecting tweezers and scissors (Fine Scientific Tools)
  4. Suction ring for imaging (Mekilect, catalog number: Suction Ring )
  5. Vacuum line with pressure regulator and pressure gauge
  6. Upright confocal or 2photon microscope with resonance scanner (we use a Leica SP5), heated stage and long distance 20-25x water immersion objective suitable for live imaging.

Software

  1. Imaris software (version 7.1.1 x 64) (Bitplane, http://www.bitplane.com/download/manuals/ReferenceManual6_1_0.pdf)
  2. Prism software (GraphPad Software)

Procedure

  1. Tumor injection
    1. Collect LLC-RFP tumor cells at about 70-80% confluency grown in a cell culture flask. Wash with DPBS two times and then gently trypsinize cells for approximately 3-5 min. We find that the greatest amount of reproducibility with tumors that are 70-80% confluent and highly viable (> 95%) when collected for injection.
      Note: If there is any question of tumor cell viability, test for viability by trypan blue staining or other method after tumor cell collection.
    2. Resuspend cells in DPBS and centrifuge for 5 min at 300 x g. Repeat DPBS wash.
    3. Count cells and confirm high viability (> 95%), then resuspend cells in DPBS at 3 x 106 cells/ml.
    4. Intravenously inject (in tail vein) 3 x 105 Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP) or other fluorescent-tagged tumor cell line resuspended in 100 μl of DPBS into the tail vein using a 30 gauge syringe.

  2. Mouse imaging
    1. Inject mouse intraperitoneally with an initial dose of 90 mg/kg ketamine hydrochloride and 15 mg/kg xylazine hydrochloride in 0.5 ml of PBS to anesthetize mouse. Maintain anesthesia during image acquisition with one-half dose subcutaneously boosting every 45 min for up to 3 h.
    2. Placed mouse in the supine position on a heated microscope stage to achieve a rectal temperature of 37 °C.
    3. Once adequate anesthesia is induced (tested by paw pinch), expose the trachea and separate from the surrounding connective tissue.
    4. Cut a small opening in the anterior trachea parallel to the cartilaginous rings, and insert PE-90 tubing trimmed at a 45 degree angle into the trachea toward the lungs. Secure the tubing and close the skin around the trachea with Vetbond glue and connect to the mechanical ventilator. Apply a tidal volume of 8 to 10 μl/g body weight (room air or higher fractions of oxygen) with respiratory rates of approximately 120 breaths per min. This is the approximate normal breathing rate of an average size adult mouse (25-30 g).
    5. Reassess depth of anesthesia with a paw pinch.
    6. Dab mouse lightly with 70% ethanol around left rib cage and remove skin to expose ribs.
    7. Gently insert scissors between the two lowest ribs and cut a 0.5 cm hole into the thoracic cavity.
    8. Pull up second rib above hole with tweezers and then pipette approximately 300 μl of PBS into hole to help drop left lobe of the lung from the rib cage.
    9. Continue applying upward pressure on the ribcage with tweezers in order to keep the lung away from the ribs and prevent lung damage while removing ribs. Carefully remove approximately 1.5 cm of the three anterior ribs overlying the left lung lobe as shown in Figure 1.


      Figure 1. Images of exposure of left lobe of lung as described in step B9

    10. Apply a thin layer of high vacuum grease around the inner edge of the vacuum ring and then apply a 12-mm glass coverslip sealed with high vacuum grease to the vacuum suction ring. Apply 20 to 25 mmHg vacuum suction to the ring using a dedicated vacuum line attached to pressure gauge and regulator to adjust pressure. 
    11. Carefully lower thoracic suction window onto the flat surface of the left lung lobe, which will then enter the thoracic window and be stabilized for imaging as shown in Figure 2.


      Figure 2. Image of suction window on left lobe of the lung

    12. Add ~200 μl DPBS to slideglass, engage microscope lens on imaging ring as shown in Figure 3, and acquire imaging as necessary. Confirm normal blood flow in lung by imaging to ensure that the ring has not caused vascular damage/blocking. Focusing on imaging an area that is fairly stabilized by the vacuum ring to prevent motion artifact and distortion of the images by pulsing of the lung. Representative lung imaging is shown in Video 1 and Video 2.


      Figure 3. Image of microscope lens on lung suction ring connected to lung of live mouse

    13. After imaging turn off vacuum, remove microscope lens and imaging ring, and euthanize mouse.

      Video 1. Representative live imaging of Nur77-GFP high patrolling monocytes (Green), all CD45-APC+ immune cells (Blue), and LLC-RFP tumor (Red) in mouse lung. Nur77-GFP mice were IV injected with LLC-RFP tumor 24 h prior to imaging. CD45-APC was IV injected.

      Video 2. Representative three dimensional reconstruction of live imaging data using Imaris. Nur77-GFPhigh patrolling monocytes (Green), GR1(Ly6G/C)-APC+ neutrophils and classical monocytes (Blue), and LLC-RFP tumor (Red) in mouse lung. Nur77-GFP mice were IV injected with LLC-RFP tumor 7 days prior to imaging. GR1(Ly6G/C)-APC was IV injected.

Data analysis

Three dimensional reconstructions and drift correction of lung images were performed using Imaris software (version 7.1.1 x 64). Detailed instructions on the use of Imaris software can be found at: http://www.bitplane.com/download/manuals/ReferenceManual6_1_0.pdf. Images were smoothed by median filtering at kernel size 3 x 3 pixels. When needed, motion artifacts caused by breathing were corrected with rigid body registration using an ImageJ plug-in (McArdle et al., 2014; McArdle et al., 2015). Imaris software was also used to automatically process 3D video data by detecting cells in each fluorescence channel, then creating tracks by linking the detected cells over time (using the Imaris 'Track spots over time' function). Tracks were manually edited to improve accuracy. Data for all experiments were analyzed with Prism software. Unpaired t-tests and two-way analysis of variance were used for comparison of experimental groups. P values of less than 0.05 were considered significant. The data appeared to be normally distributed with similar standard deviation and error observed between and within experimental groups.

Notes

  1. Optional: Intravenously inject (retro-orbital) directly conjugated fluorescent antibodies (20 μg diluted to 100 μl in DPBS of either Ly6G: Neutrophils, CD3e: T Cells, CD31: endothelial cells, etc.) to label other immune cells or vasculature. Optimal color for antibody labeling will vary with microscope set-up but we find good imaging with APC/A647, FITC/A488, BV421 or PE labeling of antibodies. Antibodies should be injected just prior or during imaging. Good antibody labeling of cells can be detected in vivo for approximately 30 min to more than 3 h, and varies considerably depending on the antibody and fluorophore conjugate.
  2. This same protocol and vacuum ring can be applied to image other organs (spleen, kidney, pancreas, skin, etc.). Ventilator is not necessary for imaging organs other than the lung.

Acknowledgments

This protocol is adapted from an earlier protocol describing live imaging of the lung (Thornton et al., 2012). This work was supported by NIH grants R01 HL118765 and R01 CA202987 (both to C.C.H.), American Heart Association Scientist Development Grant 125SDG12070005 (to R.N.H.), the La Jolla Institute for Allergy and Immunology Board of Directors Fellowship (to R.N.H.).

References

  1. Hanna, R. N., Cekic, C., Sag, D., Tacke, R., Thomas, G. D., Nowyhed, H., Herrley, E., Rasquinha, N., McArdle, S., Wu, R., Peluso, E., Metzger, D., Ichinose, H., Shaked, I., Chodaczek, G., Biswas, S. K. and Hedrick, C. C. (2015). Patrolling monocytes control tumor metastasis to the lung. Science 350(6263): 985-990.
  2. McArdle, S., Chodaczek, G., Ray, N. and Ley, K. (2015). Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries. J Biomed Opt 20(2): 26005.
  3. McArdle, S., Acton, S. T., Ley, K. and Ray, N. (2014). Registering sequences of in vivo microscopy images for cell tracking using dynamic programming and minimum spanning trees. IEEE International Conference on Image Processing: 3547-3551.
  4. Thornton, E. E., Krummel, M. F. and Looney, M. R. (2012). Live imaging of the lung. Curr Protoc Cytom Chapter 12: Unit12 28.

简介

免疫治疗已经通过激活免疫系统抵抗癌症而显示出巨大的治疗潜力。然而,关于在肿瘤和免疫细胞之间发生的相互作用的具体动力学知之甚少。在这个协议中,我们描述了一种新的方法来可视化肿瘤和免疫细胞在活小鼠的肺中的相互作用,可以应用到其他器官。在该协议中,荧光标记的肿瘤细胞转移到表达荧光标记的免疫细胞的受体小鼠。然后通过共聚焦或双光子显微镜对肺中的肿瘤 - 免疫细胞相互作用成像。使用该方案分析肿瘤与免疫细胞的相互作用应当有助于更好地理解这些相互作用的重要性及其在开发免疫疗法中的作用。

[背景] 一些免疫疗法在治疗癌症方面表现出巨大的希望。了解这些肿瘤免疫相互作用如何发生的空间时间分辨率对于增强和开发新的免疫疗法是重要的。在这个协议中,我们描述了一种新的方法直接可视化肿瘤免疫细胞相互作用体内在小鼠肺。该方案最初在我们的工作中描述,其检查小鼠肺中巡逻单核细胞和肿瘤细胞的相互作用(Hanna等人,2015)。该荧光显微镜方案使用真空成像环来稳定和成像肺,这最初由Looney及其同事描述(Thornton等人,2012)。在该协议中,荧光标记的肿瘤细胞转移到表达荧光标记的免疫细胞的受体小鼠。然后使用真空成像环通过共聚焦或双光子荧光显微术将肺中的肿瘤免疫细胞相互作用成像。该协议允许通过静脉内(IV)注射荧光标记的抗体添加其他免疫细胞标记物,并且适于在其他器官中使肿瘤免疫细胞相互作用成像。可以使用该方案收集定量信息,例如定位,肿瘤材料吞噬,这些免疫细胞相互作用的时间,速度和频率。这个协议应该有助于更好地了解特定的免疫肿瘤细胞相互作用,这对于在未来开发更好的免疫治疗很重要。

关键字:活体成像, 癌症, 免疫学, 肺, 吞噬

材料和试剂

  1. 细胞培养瓶
  2. 30号胰岛素注射器(BD,目录号:328431)Nr4a1-GFP(The Jackson Laboratory,目录号:018974),CX3CR1-GFP(The Jackson Laboratory,目录号:005582)或用于可视化免疫细胞的其它荧光报告小鼠。
  3. 1/2微盖玻??璃(12mm直径)(电子显微镜科学,目录号:72230-01)
  4. PE-90管(BD,Intramedic TM ,目录号:427420)
  5. 小鼠
  6. 表达红色荧光蛋白(LLC-RFP)或其他荧光标记的肿瘤细胞系(AntiCancer.com)的Lewis肺癌细胞
  7. TrypLE TM超表达酶(Thermo Fisher Scientific,Gibco TM ,目录号:12604013)
  8. Dulbecco's磷酸盐缓冲盐水(DPBS)(GE Healthcare,Hyclone ,目录号:SH30038.02)
  9. 盐酸氯胺酮
  10. 甲苯噻嗪盐酸盐
  11. Vetbond胶(3M,目录号:1469SB)
  12. 氧气
  13. 乙醇
  14. Dow Corning高真空润滑脂(Sigma-Aldrich,目录号:Z273554)

设备

  1. 离心机
  2. 机械鼠标呼吸机(Harvard Apparatus,型号:845)
  3. 精细解剖镊子和剪刀(精细科学工具)
  4. 成像用吸引环(Mekilect,目录号:吸引环)
  5. 带压力调节器和压力表的真空管道
  6. 立式共焦或2光子显微镜与共振扫描仪(我们使用徕卡SP5),加热舞台和长距离20-25x水浸物镜适合实时成像。

软件

  1. Imaris软件(版本7.1.1 x 64)(Bitplane, http ://www.bitplane.com/download/manuals/ReferenceManual6_1_0.pdf
  2. Prism软件(GraphPad软件)

程序

  1. 肿瘤注射
    1. 收集在细胞培养瓶中生长的约70-80%汇合的LLC-RFP肿瘤细胞。用DPBS洗涤两次,然后轻轻地胰蛋白酶消化细胞约3-5分钟。我们发现,当收集用于注射时,具有70-80%汇合和高度存活(> 95%)的肿瘤的最大量的再现性。
      注意:如果有任何肿瘤细胞活力的问题,在肿瘤细胞收集后通过台盼蓝染色或其他方法检测活力。
    2. 重悬细胞在DPBS中,并在300×g离心5分钟。重复DPBS清洗。
    3. 计数细胞并确认高活力(> 95%),然后以3×10 6个细胞/ml将细胞重悬在DPBS中。
    4. 将悬浮在100μlDPBS中的表达红色荧光蛋白(LLC-RFP)或其它荧光标记的肿瘤细胞系的3×10 5个Lewis肺癌细胞静脉内(在尾静脉中)静脉内注射到尾静脉中30号注射器。

  2. 鼠标成像
    1. 用90mg/kg氯胺酮盐酸盐和15mg/kg盐酸赛拉嗪的初始剂量在0.5ml PBS中腹膜内注射小鼠以麻醉小鼠。在图像采集期间保持麻醉,一半剂量皮下增强每45分钟长达3小时。
    2. 放置在仰卧位置在加热的显微镜载物台上的小鼠,实现直肠温度37°C。
    3. 一旦诱导了足够的麻醉(通过爪收缩测试),暴露气管并与周围结缔组织分离。
    4. 在平行于软骨环的前气管中切开一个小开口,并将以45度角修剪的PE-90管插入气管朝向肺。用Vetbond胶固定管道并闭合气管周围的皮肤,并连接到机械呼吸机。应用8至10μl/g体重(室内空气或更高的氧气分数)的潮气量,呼吸频率约为每分钟120次呼吸。这是平均大小的成年小鼠(25-30g)的近似正常呼吸率。
    5. 重新评估麻醉深度与爪压缩。
    6. Dab小鼠轻轻用70%乙醇围绕左肋骨和去除皮肤暴露肋骨。
    7. 轻轻地插入剪刀之间的两个最低的肋骨和切割0.5厘米的洞进入胸腔。
    8. 用镊子拔出孔上方的第二个肋,然后移取大约300微升PBS进入孔,以帮助从胸腔左侧肺叶。
    9. 继续用镊子向胸腔施加向上的压力,以保持肺远离肋骨,防止肺损伤,同时去除肋骨。小心地清除覆盖左肺叶的三个前肋的约1.5cm,如图1所示。


      图1.步骤B9中描述的肺左叶曝光图像

    10. 在真空环的内边缘涂一薄层高真空润滑脂,然后将一个用高真空油脂密封的12 mm玻璃盖玻片贴在真空吸环上。使用连接到压力表和调节器的专用真空管线对环施加20至25mmHg的真空吸力以调节压力。
    11. 小心地将胸部吸气窗降低到左肺叶的平坦表面,然后进入胸部窗口并稳定成像,如图2所示。


      图2.肺左叶吸气窗的图像

    12. 添加?200微升DPBS到幻灯片,将显微镜透镜放在成像环上,如图3所示,并根据需要获取成像。通过成像确认肺中的正常血流量,以确保环没有造成血管损伤/阻塞。聚焦于通过真空环相当稳定的区域的成像,以防止由于肺的脉冲引起的图像的运动伪影和失真。代表性肺成像显示在视频1和视频2中

      图3.连接到活小鼠肺的肺吸气环上的显微镜透镜的图像

    13. 成像后关闭真空,取下显微镜镜头和成像环,并安乐死鼠标。

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      视频1. Nur77-GFP高巡逻单核细胞(绿色),所有CD45-APC + 免疫细胞(蓝色)和LLC-RFP肿瘤(红色)在小鼠肺中的代表性实时成像。 在成像前24小时,用LLC-RFP肿瘤IV注射Nur77-GFP小鼠。 IV注射CD45-APC。
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      视频2.使用Imaris的实时成像数据的代表性三维重建 Nur77-GFP 巡逻单核细胞(Green),GR1(Ly6G/C)-APC 中性粒细胞和经典单核细胞(蓝色)和LLC-RFP肿瘤(红色)。在成像之前7天,用LLC-RFP肿瘤IV注射Nur77-GFP小鼠。 IV注射GR1(Ly6G/C)-APC。
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数据分析

使用Imaris软件(版本7.1.1 x 64)进行肺图像的三维重建和漂移校正。有关使用Imaris软件的详细说明,请访问: http://www.bitplane.com/download/manuals/ReferenceManual6_1_0.pdf。在内核大小为3 x 3像素的情况下,通过中值滤波平滑图像。当需要时,由使用ImageJ插件的刚体配准来校正由呼吸引起的运动伪像(McArdle等人,2014; McArdle等人,2015)。 Imaris软件还用于通过检测每个荧光通道中的细胞来自动处理3D视频数据,然后通过随时间链接所检测的细胞来创建轨迹(使用Imaris的"随时间变化的斑点"功能)。手动编辑轨道以提高精度。用Prism软件分析所有实验的数据。未配对的测试和双向方差分析用于实验组的比较。 P 值小于0.05被认为是显着的。数据似乎是正常分布的,在实验组之间和之内观察到类似的标准偏差和误差。

笔记

  1. 任选:静脉内注射(后眼)直接缀合的荧光抗体(20μg稀释至100μl,在DPBS中的Ly6G:嗜中性粒细胞,CD3e:T细胞,CD31:内皮细胞等)其他免疫细胞或脉管系统。抗体标记的最佳颜色将随着显微镜设置而变化,但是我们发现APC/A647,FITC/A488,BV421或PE标记抗体的良好成像。应在成像前或成像期间注射抗体。可以在体内检测细胞的良好抗体标记大约30分钟至大于3小时,并且根据抗体和荧光团缀合物显着变化。
  2. 该相同的方案和真空环可以应用于对其他器官(脾,肾,胰腺,皮肤,等)成像。对于除肺以外的器官,呼吸器不是必需的。

致谢

该协议改编自描述肺活体成像的早期方案(Thornton等人,2012)。这项工作由NIH授予R01 HL118765和R01 CA202987(均为C.C.H.),美国心脏协会科学家发展授权125SDG12070005(授予R.N.H),La Jolla过敏和免疫学研究所董事会研究员(授予R.N.H)支持。

参考文献

  1. Hanna,D。,Rasquinha,N.,McArdle,S.,Wu,R。,Peluso, E.,Metzger,D.,Ichinose,H.,Shaked,I.,Chodaczek,G.,Biswas,SK和Hedrick,CC(2015)。  Patrolling monocytes control tumor metastasis to the lung。 Science 350(6263):985- 990.
  2. McArdle,S.,Chodaczek,G.,Ray,N.和Ley,K.(2015)。  活体细胞触发的成像系统揭示了动脉粥样硬化动脉中的单核细胞巡逻和巨噬细胞迁移。生物选择 20(2):26005。 />
  3. McArdle,S.,Acton,ST,Ley,K.和Ray,N。(2014)。 
  4. Thornton,EE,Krummel,MF和Looney,MR(2012)。  肺的实时成像。 12:
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hanna, R. N., Chodaczek, G. and Hedrick, C. C. (2016). In vivo Imaging of Tumor and Immune Cell Interactions in the Lung. Bio-protocol 6(20): e1973. DOI: 10.21769/BioProtoc.1973.
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