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Quantification of Tumor Material Uptake
肿瘤物质摄取的量化   

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参见作者原研究论文

本实验方案简略版
Science
Nov 2015

Abstract

Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.

Keywords: Cancer (癌症), Phagocytosis (吞噬), Imaging (成像), Immunology (免疫学), Lung (肺)

Background

Recent studies have demonstrated that extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris shed from tumors are important mediators of tumor metastasis, growth and evasion of the immune response (Vader et al., 2014; Pucci and Pittet, 2013; Pucci et al., 2016). Immune cells have the ability to remove, respond and transport this circulating tumor material (Hanna et al., 2015; Pucci et al., 2016; Headley et al., 2016). This protocol offers a novel approach to quantify tumor material uptake by specific immune cell populations, and may be adapted to test immune targets that regulate tumor material uptake. This protocol may assist in better understanding the immune response to extracellular tumor material, with the hope of eventually developing novel therapies targeting extracellular tumor material in cancer development.

Materials and Reagents

  1. 30 gauge insulin syringe (BD, Ultra-FineTM, catalog number: 328431 )
  2. 15 ml tubes
  3. 70 μm cell strainers (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22363548 )
  4. 15 mm Petri dish
  5. 5 ml syringe (BD, Luer-LokTM, catalog number: 309646 )
  6. 96-well v-bottom plate
  7. Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP), B16F10 green fluorescent protein (B16F10-GFP) or other fluorescent-tagged tumor cell line (AntiCancer.com, http://www.anticancer.com/Fluorescent_protein_cell_lines_April_2010.pdf)
  8. Syngeneic recipient mice. C57BL/6J mice (The Jackson Laboratory, catalog number: 000664 ) for LLC and B16F10 tumors.
  9. TrypLE (Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 )
  10. Dulbecco's phosphate-buffered saline (DPBS) (GE Healthcare, HycloneTM, catalog number: SH30013.02 )
  11. UltraPureTM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  12. Red blood cell (RBC) lysis buffer (10x) (BioLegend, catalog number: 420301 )
  13. Fc Block (CD16/32) (BD, PharmingenTM, catalog number: 553141 )
  14. Fluorochrome conjugated antibodies:
    CD11b-FITC (BioLegend, catalog number: 101206 )
    CD115-APC (BioLegend, catalog number: 135510 )
    Ly6C-APC-Cy7 (BioLegend, catalog number: 128026 )
    CD45-BV421 (BioLegend, catalog number: 103134 )
  15. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  16. Live/Dead Fixable Blue (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L34962 )
  17. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  18. Flow buffer (FB) (see Recipes)
  19. RBC lysis buffer (see Recipes)

Equipment

  1. Cell culture flask
  2. Centrifuge
  3. Multiparameter flow cytometer with optimal excitation and detectors for tumor fluorophore (BD, LSRII or similar) or Amnis imaging cytometer (ImageStream X Mark II)

Software

  1. ImageJ software (ImageJ)
  2. FlowJo software (version 9.2)
  3. 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 methods after tumor collection.
    2. Resuspend cells in DPBS and centrifuge for 5 min at 300 x g. Repeat DPBS wash.
    3. Wash tumor cell with DPBS two times and then gently trypsinize cells. Bring up cells in DPBS and centrifuge for 5 min at 300 x g. Repeat DPBS wash.
    4. Count cells and bring up at 3 x 106 cells/ml.
    5. 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. Recipient mice should be syngeneic for the tumor cell line (C57BL/6J mice for LLC and B16F10 tumors). Optimal internalization of tumor material in circulating myeloid cells is observed between 4 and 72 h after intravenous injection of tumor, but tumor material observer can be observed in lung tissue for at least 3 weeks after injection.

  2. Isolation of immune cells
    1. Collect samples in 15 ml tubes filled with DPBS (with 2 mM EDTA).
    2. Collect 200 μl-1 ml of blood, whole spleen, or whole lung.
    3. Centrifuge 300 x g for 10 min for blood, and then lyse RBC with 10 ml of RBC buffer for 5 min.
    4. Place a 70 μm filter in a 15 mm Petri dish and mechanical dissociate spleen or lung through filter using a 5 ml syringe plunger. Pour cell solution into 15 ml tube and then lyse with 2 ml of RBC buffer for 2-5 min.
    5. Wash 1x with 10 ml DPBS and centrifuge 300 x g for 10 min (repeat lysis for blood if necessary).
    6. Resuspend cells around 1 x 107 cells/ml in flow buffer (FB).

  3. Staining of immune cells
    1. Place ~2 x 106 cells/well in a 96-well v-bottom plate.
    2. Wash cells in 200 μl in FB, centrifuge for 5 min at 300 x g, flick out wash (swing plate over sink to remove liquid).
    3. Resuspend cells in 100 μl Fc Block (CD16/32) at a dilution of 1:200 (Make a master mix and add to cells using a multichannel pipettor).
    4. Let sit on ice for 5 min.
    5. Make a master mix of fluorochrome conjugated antibodies and viability dye to stain immune cell population of interest in FB. Avoid other red channels (PE, PE-Cy7 and Percp5.5) if assaying for LLC-RFP uptake and avoid green channels (FITC/A488/GFP) if assaying for B16F10-GFP. For example, to label myeloid cell populations after LLC-RFP injection we use CD11b-FITC, CD115-APC, Ly6C-APC-Cy7, CD45-BV421, and Live/Dead Blue, but this panel can vary depending on your cell of interest and tumor injected.
    6. Wash cells in 200 μl FB, centrifuge for 5 min at 300 x g, flick out wash.
    7. Resuspend cells in 100 μl of fluorochrome conjugated antibody master mix.
    8. Stain compensation controls/beads with single antibodies.
    9. Incubate on ice for 30 min.
    10. Wash cells 2 x with 200 μl FB, centrifuge for 5 min at 300 x g, flick out wash.
    11. Resuspend in 200 μl of FB
    12. Transfer 200 μl to analysis tubes and run samples on the flow cytometer or Amnis imaging cytometer.
    13. Gate on live cells, then CD45+ immune cell population of interest, and then RFP+ or GFP+ tumor material found in the immune cell population by flow cytometry (Figure 1) or image tumor material localization using imaging cytometer (Figure 2).


      Figure 1. Gating of LLC-RFP+ tumor material in CD11b+ myeloid cells isolated from blood and analyzed by flow cytometry. Cells were gated on Live, CD45+ immune cells before this analysis.


      Figure 2. Imaging of B16F10-GFP tumor material uptake (Green) by CD11b+ myeloid cells (Orange) isolated from blood and analyzed by imaging cytometry. Cells were gated on Live, CD45+ immune cells before this analysis. Scale bar in lower right corner is 10 μm.

Data analysis

For our flow and imaging cytometry analysis we usually quantify the percent of a cell population with tumor material or the relative amount or size of tumor material in each cell. Calculations of percentages of CD45+ immune cells were based on live cells as determined by forward and side scatter and viability analysis. Average size of tumor material in cells can be measured from imaging cytometry data using ImageJ software. Cell fluorescence was assessed with an LSRII and was analyzed with FlowJo software (version 9.2). 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. Always keep the immune cells on ice during collection and staining.
  2. Work fast and don’t let the samples sit too long before analysis as the internalized tumor material can degrade or lose fluorescence quickly. Analyze sample within 3 h of collection for optimal results.
  3. Set detector fairly high on flow cytometer in channel detecting tumor material in order to detect small fragments of tumor engulfed by cells. Though there is no universal range for setting these detectors since cytometers setup and fluorophore brightness varies, detector settings should put tumor material in the upper log scale of the cytometers flow plots without going off scale.
  4. For reproducibility, remove any mouse from analysis that was questionably IV injected with tumor as variability in the amount of tumor material injected can greatly affect the amount of tumor uptake measured in immune cells.

Recipes

  1. Flow buffer (FB)
    10 g 1.0% BSA
    1 g 0.1% sodium azide
    1 L 1x DPBS
    Store at 4 °C.
  2. RBC lysis buffer
    Dilute 10x from stock.

Acknowledgments

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. Headley, M. B., Bins, A., Nip, A., Roberts, E. W., Looney, M. R., Gerard, A. and Krummel, M. F. (2016). Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature 531(7595): 513-517.
  3. Pucci, F. and Pittet, M. J. (2013). Molecular pathways: tumor-derived microvesicles and their interactions with immune cells in vivo. Clin Cancer Res 19(10): 2598-2604.
  4. Pucci, F., Garris, C., Lai, C. P., Newton, A., Pfirschke, C., Engblom, C., Alvarez, D., Sprachman, M., Evavold, C., Magnuson, A., von Andrian, U. H., Glatz, K., Breakefield, X. O., Mempel, T. R., Weissleder, R. and Pittet, M. J. (2016). SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions. Science 352 (6282): 242-246.
  5. Vader, P., Breakefield, X. O. and Wood, M. J. (2014). Extracellular vesicles: emerging targets for cancer therapy. Trends Mol Med 20(7): 385-393.

简介

包括外来体,微泡和凋亡性肿瘤碎片的细胞外肿瘤材料可以帮助癌症侵入新器官。增强免疫细胞对细胞外肿瘤材料的去除代表了用于预防癌症转移的新型免疫治疗方法。该方案定量免疫细胞从循环和组织吸收和去除细胞外肿瘤物质。在该测定中,将荧光肿瘤细胞转移到小鼠中,然后通过流式细胞术或成像细胞计量术来定量免疫细胞对肿瘤材料的摄取。

[背景] 研究已经证明,包括从肿瘤脱落的外来体,微泡和凋亡性肿瘤碎片的细胞外肿瘤材料是肿瘤转移,生长和逃避免疫应答的重要介质(Vader等人,2014; Pucci和Pittet ,2013; Pucci等人,2016)。免疫细胞具有去除,应答和转运这种循环肿瘤材料的能力(Hanna等人,2015; Pucci等人,2016; Headley等人。,2016)。该协议提供了一种新的方法来量化特定免疫细胞群体的肿瘤材料摄取,并且可以适于测试调节肿瘤材料摄取的免疫靶标。该协议可以帮助更好地理解对细胞外肿瘤材料的免疫应答,希望最终开发针对癌症发展中的细胞外肿瘤材料的新疗法。

关键字:癌症, 吞噬, 成像, 免疫学, 肺

材料和试剂

  1. 30号胰岛素注射器(BD,Ultra-Fine TM ,目录号:328431)
  2. 15 ml管
  3. 70微米细胞过滤器(Thermo Fisher Scientific,Fisher Scientific,目录号:22363548)
  4. 15 mm培养皿
  5. 5ml注射器(BD,Luer-Lok TM ,目录号:309646)
  6. 96孔v型底板
  7. 表达红色荧光蛋白(LLC-RFP),B16F10绿色荧光蛋白(B16F10-GFP)或其他荧光标记的肿瘤细胞系的Lewis肺癌细胞(AntiCancer.com, http://www.anticancer.com/Fluorescent_protein_cell_lines_April_2010.pdf
  8. 同基因受体小鼠。 LLC和B16F10肿瘤的C57BL/6J小鼠(The Jackson Laboratory,目录号:000664)。
  9. TrypLE(Thermo Fisher Scientific,Gibco TM ,目录号:12604013)
  10. Dulbecco's磷酸盐缓冲盐水(DPBS)(GE Healthcare,Hyclone ,目录号:SH30013.02)
  11. UltraPure TM 0.5M EDTA,pH 8.0(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  12. 红细胞(RBC)裂解缓冲液(10x)(BioLegend,目录号:420301)
  13. Fc区(CD16/32)(BD,Pharmingen TM ,目录号:553141)
  14. 荧光染料共轭抗体:
    CD11b-FITC(BioLegend,目录号:101206)
    CD115-APC(BioLegend,目录号:135510) Ly6C-APC-Cy7(BioLegend,目录号:128026) CD45-BV421(BioLegend,目录号:103134)
  15. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  16. 活/死可固定蓝(Thermo Fisher Scientific,Molecular Probes TM ,目录号:L34962)
  17. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  18. 流缓冲器(FB)(参见配方)
  19. RBC裂解缓冲液(参见配方)

设备

  1. 细胞培养瓶
  2. 离心机
  3. 具有最佳激发和肿瘤荧光团(BD,LSRII或类似物)或Amnis成像细胞仪(ImageStream X Mark II)的多参数流式细胞仪

软件

  1. ImageJ软件(ImageJ)
  2. FlowJo软件(版本9.2)
  3. 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%汇合且高度可行(> 95%)的肿瘤的最大量的再现性。
      注意:如果肿瘤细胞活力存在任何问题,在肿瘤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. 用DPBS洗涤肿瘤细胞两次,然后轻轻地胰蛋白酶消化细胞。将细胞在DPBS中并在300×g离心5分钟。重复DPBS清洗。
    4. 计数细胞并以3×10 6细胞/ml升高
    5. 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。受体小鼠应该对于肿瘤细胞系是同基因的(C57BL/6J小鼠对于LLC和B16F10肿瘤)。在静脉内注射肿瘤后4和72小时之间观察到循环骨髓细胞中肿瘤材料的最佳内化,但是在注射后可以在肺组织中观察到肿瘤材料观察者至少3周。

  2. 免疫细胞的分离
    1. 将样品收集在装有DPBS(含2mM EDTA)的15ml管中
    2. 收集200μl-1ml血液,整个脾脏或整个肺
    3. 离心300×10 5分钟用于血液,然后用10ml RBC缓冲液裂解RBC 5分钟。
    4. 放置一个70微米过滤器在15毫米培养皿和机械解离脾或肺通过过滤器使用5毫升注射器柱塞。将细胞溶液倒入15ml试管,然后用2ml RBC缓冲液裂解2-5分钟
    5. 用10 ml DPBS洗涤1次,并离心300×g 10分钟(如果必要,重复裂解血液)。
    6. 在流动缓冲液(FB)中重悬细胞约1×10 7个细胞/ml
  3. 免疫细胞染色
    1. 在96孔v-底板中放置?2×10 6个细胞/孔
    2. 洗涤细胞在200微升在FB,离心5分钟,在300×g ,弹出洗涤(摇摆板在水槽上以移除液体)。
    3. 重悬细胞在100微升Fc块(CD16/32)稀释1:200(制作主混合,并使用多通道移液器加入细胞)。
    4. 让我们坐在冰上5分钟。
    5. 使荧光染料结合的抗体和活力染料的主混合物染色FB中感兴趣的免疫细胞群体。如果测定LLC-RFP摄取,避免其他红色通道(PE,PE-Cy7和Percp5.5),如果测定B16F10-GFP,避免绿色通道(FITC/A488/GFP)。例如,为了在LLC-RFP注射后标记骨髓细胞群体,我们使用CD11b-FITC,CD115-APC,Ly6C-APC-Cy7,CD45-BV421和活/死蓝,但是该面板可以根据感兴趣的细胞和肿瘤注射。
    6. 在200μlFB中洗涤细胞,在300×g离心5分钟,轻轻吹洗。
    7. 重悬细胞在100μl的荧光染料结合的抗体主混合物。
    8. 用单一抗体染色补偿对照/珠子。
    9. 在冰上孵育30分钟。
    10. 用200μlFB洗涤细胞2次,以300×g离心5分钟,轻拂洗出。
    11. 重悬于200μl的FB
    12. 转移200微升到分析管,并在流式细胞仪或Amnis成像细胞仪上运行样品
    13. 在活细胞上,然后在感兴趣的CD45 +免疫细胞群中,然后在免疫细胞群中发现的RFP + 或GFP + 肿瘤材料通过流式细胞术(图1)或使用成像细胞计数器的图像肿瘤材料定位(图2)。


      图1. LLC-RFP + 肿瘤材料在从血液分离的CD11b + sup/+骨髓细胞中的门控,并通过流式细胞术分析。活,CD45 + 免疫细胞

      图2.通过从血液分离的CD11b +髓样细胞(Orange)成像B16F10-GFP肿瘤材料摄取(绿色),并通过成像细胞计数分析。将细胞在活体上门控,CD45 + 免疫细胞。右下角的比例尺为10μm。

数据分析

对于我们的流式细胞术和成像细胞计数分析,我们通常用肿瘤材料或每个细胞中肿瘤材料的相对量或大小来量化细胞群的百分比。 CD45 +免疫细胞的百分比的计算基于通过正向和侧向散射和存活力分析确定的活细胞。可以使用ImageJ软件从成像细胞计数数据测量细胞中肿瘤材料的平均尺寸。用LSRII评估细胞荧光,并用FlowJo软件(版本9.2)分析。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. 在收集和染色过程中始终保持免疫细胞在冰上
  2. 工作速度快,不要让样品在分析前停留太久,因为内化肿瘤材料可能迅速降解或失去荧光。在收集的3小时内分析样品以获得最佳结果。
  3. 在流式细胞仪上在通道检测肿瘤材料中设置检测器相当高,以便检测被细胞吞噬的肿瘤的小片段。虽然没有通用的范围设置这些检测器,因为细胞仪设置和荧光灯亮度变化,检测器设置应该将肿瘤材料在细胞仪流量图的上对数标度,而不会超出规模。
  4. 为了再现性,从可疑IV注射肿瘤的分析中除去任何小鼠,因为注射的肿瘤材料的量的变化性可极大地影响在免疫细胞中测量的肿瘤摄取量。

食谱

  1. 流缓冲器(FB)
    10g 1.0%BSA 1g 0.1%叠氮化钠 1 L 1x DPBS
    储存于4°C。
  2. RBC裂解缓冲液
    从库存稀释10倍

致谢

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. Headley,MB,Bins,A.,Nip,A.,Roberts,EW,Looney,MR,Gerard,A.and Krummel,MF(2016)。  对肺中先驱转移细胞的即时免疫应答的可视化。 531 ):513-517。
  3. Pucci,F。和Pittet,MJ(2013)。  分子途径:肿瘤来源的微泡及其与免疫细胞在体内的相互作用。 Clin Cancer Res 19(10):2598-2604。
  4. Pucci,F.,Garris,C.,Lai,CP,Newton,A.,Pfirschke,C.,Engblom,C.,Alvarez,D.,Sprachman,M.,Evavold,C.,Magnuson, Andrian,UH,Glatz,K.,Breakefield,XO,Mempel,TR,Weissleder,R。和Pittet,MJ(2016)。  SCS巨噬细胞通过限制肿瘤衍生的囊泡-B细胞相互作用来抑制黑素瘤。 352(6282):242 -246。
  5. Vader,P.,Breakefield,XO and Wood,MJ(2014)。  Extracellular vesicles:emerging targets for cancer therapy。 Trends Mol Med 20(7):385-393。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hanna, R. N. and Hedrick, C. C. (2016). Quantification of Tumor Material Uptake. Bio-protocol 6(20): e1974. DOI: 10.21769/BioProtoc.1974.
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