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Tumor metastases develop when disseminated intravascular cancer cells acquire the ability to arrest by adhering to the capillary walls of distant organs, actively extravasate into their parenchyma, proliferate and establish secondary colonies. The extravasation assay described here is an in vivo technique aimed to analyze the ability of tumor cells to achieve early colonization of the lungs following tail vein injection in mice. Importantly, tumor cells need to be easily visible, therefore either they are fluorescent (e.g. expressing RFP or GFP) or they have to be pre-labelled with a fluorescent tracker prior to injection. Lungs are analyzed at different time points, experimentally determined by the researcher, depending on cell features and malignancy. Generally, an early time point is required to check equal lodging in the pulmonary vasculature for the various cells injected. At one or more later time points (from 6 to 48 h) extravasated cells dispersed in the lung parenchyma are quantitated. With our protocol extravasation is directly evaluated in the whole lungs ex vivo considering cell fluorescence. However, immunofluorescence stainings for endothelial markers and microscopic analyses of lung sections are recommended to evaluate positioning and status of tumor cells (i.e. inside, outside the vessels or associated to them; single cells or clusters). Since extravasation is not only influenced by tumor cell motility but also by their survival ability, the results obtained with this technique should be complemented with proliferation and apoptosis analyses.

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In vivo Extravasation Assay
癌细胞外渗的体内试验

癌症生物学 > 侵袭和转移 > 动物模型 > 细胞侵袭
作者: Elisa Penna
Elisa PennaAffiliation: Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy
Bio-protocol author page: a1173
 and Daniela Taverna
Daniela TavernaAffiliation: Deptartment of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy
For correspondence: daniela.taverna@unito.it
Bio-protocol author page: a1174
Vol 4, Iss 4, 2/20/2014, 4048 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1051

[Abstract] Tumor metastases develop when disseminated intravascular cancer cells acquire the ability to arrest by adhering to the capillary walls of distant organs, actively extravasate into their parenchyma, proliferate and establish secondary colonies. The extravasation assay described here is an in vivo technique aimed to analyze the ability of tumor cells to achieve early colonization of the lungs following tail vein injection in mice. Importantly, tumor cells need to be easily visible, therefore either they are fluorescent (e.g. expressing RFP or GFP) or they have to be pre-labelled with a fluorescent tracker prior to injection. Lungs are analyzed at different time points, experimentally determined by the researcher, depending on cell features and malignancy. Generally, an early time point is required to check equal lodging in the pulmonary vasculature for the various cells injected. At one or more later time points (from 6 to 48 h) extravasated cells dispersed in the lung parenchyma are quantitated. With our protocol extravasation is directly evaluated in the whole lungs ex vivo considering cell fluorescence. However, immunofluorescence stainings for endothelial markers and microscopic analyses of lung sections are recommended to evaluate positioning and status of tumor cells (i.e. inside, outside the vessels or associated to them; single cells or clusters). Since extravasation is not only influenced by tumor cell motility but also by their survival ability, the results obtained with this technique should be complemented with proliferation and apoptosis analyses.
Keywords: Metastasis(转移), Endothelial cells(内皮细胞), Tumor cells(肿瘤细胞), Melanoma(黑色素瘤), Breast cancer(乳腺癌)

[Abstract]

Materials and Reagents

  1. Tumor cells (in particular, this protocol was set up for A375P, MC-1, MA-2, WK-Mel and B16 melanoma cells and for MDA-MB-231 breast cancer cells)
  2. CD1 athymic nude female mice (6 to 9 week-old)
  3. Phosphate buffered saline (PBS)
  4. Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 15400-054 )
  5. DMSO (Sigma-Aldrich, catalog number: 41648 )
  6. CellTrackerTM Orange CMRA (Life Technologies, Molecular Probes®, catalog number: C34551 ); alternatively tumor cells themselves are required to express red fluorescent protein (RFP)
  7. 4% Paraformaldehyde (PFA) in PBS
  8. 30% Sucrose in PBS
  9. Freezing resin (Killik, Bio-Optica Milano SpA, catalog number: 05-9801B )
  10. Isopentane
  11. Dry ice
  12. Anti-CD31 antibody (BD Biosciences, catalog number: 550274 ) or alternatively anti-Von Willebrandt Factor antibody (Dako, catalog number: A0082 ) to stain blood vessels
  13. Anti-rat or anti-rabbit Alexa-Fluor-488 secondary antibody (Life Technologies, Molecular Probes®, catalog numbers: A11006 and A11008 )
  14. FBS (Beromed, GmbH)
  15. Serum-free medium (see Recipes)
  16. Complete medium (Life Technologies, Gibco®) (see Recipes)

Equipment

  1. Cell culture set up, laminar flow hood, centrifuge, cell counting chamber
  2. 50 ml tubes
  3. P1000 and P200 tips
  4. Mouse housing and handling set up
  5. U-100 syringe with 29 G x 1/2 inch needle
  6. Scissor-handle straight Kelly hemostatic forceps
  7. Fluorescence stereomicroscope (e.g. Leica Microsystems, model: MZ16F )
  8. Cryostat
  9. Fluorescence microscope (e.g. ZEISS AxioObserver with ApoTome Module)
  10. Burker counting chamber (Marienfeld-superior)

Software

  1. Image analysis software (e.g. ImageJ, http://rsbweb.nih.gov/ij/)

Procedure

  1. Remove the medium from dishes or wells containing 80-90% confluent monolayers of tumor cells (either transfected/treated according to your purposes or not), wash the cells with PBS and detach them by incubating with 1 ml 0.05% trypsin-EDTA for 3-8 min; stop trypsinization with complete medium.
  2. (Only for non fluorescent cells) Spin down the cells at 800 rpm/100 rcf for 3 min and resuspend the pellet in 3-4 ml of serum-free medium in 50 ml tubes.
  3. (Only for non fluorescent cells) Resuspend the entire lyophilized CellTrackerTM Orange CMRA in 9.1 μl DMSO and add 3-4 μl CellTrackerTM Orange CMRA (1:1,000 dilution) to the cells in each tube. Incubate 35-45 min at 37 °C. During the incubation gently rock the tube every 10-15 min.
  4. (Only for non fluorescent cells) Spin down the cells at 800 rpm/100 rcf for 5 min and resuspend the pellet in 15-25 ml of PBS, depending on the initial confluency. The cells will appear pink/red due to the incorporation of the tracker.
  5. Count the cells with a Burker counting chamber.
  6. Prepare cell mixtures for injection. You will inject 1 to 1.7 x 106 cells in a volume of 200 μl PBS per mouse. The number of cells to be injected per mouse should be previously determined by the researcher in a pilot experiment and depends mainly on cell type, size, aggressiveness and on the propensity to form cell clusters. For example we inject 1 x 106 B16 cells, which are extremely metastatic and are prone to form big clusters and 1.6 x 106 A375P cells which are smaller and less metastatic.
  7. Spin down (at 800 rpm/100 rcf for 3 min) the proper number of cells needed for your experiment according to the number of mice you will inject. Resuspend the cells in PBS by pipetting the cell suspension up and down several times with P1000 and P200 tips and maintain the tube on ice until injection. To avoid cell cluster formation, which is potentially dangerous for the mice and could alter the cell behavior during extravasation and disturb your analyses, it is essential that the cells are in homogeneously single cell status.
  8. Warm up the CD1 nude mice under a red light lamp for some minutes to obtain vasodilation for easier processing. Inject 200 μl of cell mixture (1.4 to 1.7 x 106 cells) per mouse in the lateral tail vein with a U-100 syringe with 29 G x 1/2 inch needle.
  9. Analyze the mouse lungs (as indicated in steps 10-14) at an early time point after injection as a "loading control" (10 min to 2 h) and at later time points (12 h to 72 h) to detect extravasated cells. For melanoma (e.g. A375P, WK-Mel) and breast cancer (e.g. MDA-MB-231) cells we performed analyses at 2 and 48 h respectively (see Figure 1).


    Figure 1. Lung extravasation macroscopic and microscopic analyses. A, D. Representative pictures of whole lungs 2 h (A) or 48 h (D) following tail vein injections of CMRA-labelled (red) melanoma cells in nude mice. Bar: 500 μm. B, C, E, F. Representative fields of murine lung sections 2 h (B, C) or 48 h (E, F) post-injections of CMRA-labelled (red) melanoma cells, stained for Von Willebrandt factor to highlight blood vessels (green) and counter-stained with DAPI (blue). Bar: 30 μm

  10. Sacrifice the mice by cervical dislocation and dissect the animal chest to expose the trachea with sharp-edged microdissection tweezers and scissors, by eliminating all the connectival tissues surrounding the trachea, until the white cartilage rings are well visible. It is worth to proceed very gently to avoid trachea perforation. Make a longitudinal cut in the diaphragm (but without completely opening the chest) to allow the lungs to inflate. Clamp the trachea the most cranially you can with hemostatic forceps and gently inject 0.8-1 ml ice cold 4% PFA inside the trachea with a U-100 syringe with 29 G x 1/2 inch needle, with the needle parallel to the trachea. You will clearly see the lungs inflating. Complete the dissection and extract the lungs.
  11. Wash briefly the whole lungs in clean PBS to eliminate blood and fix them in 8-10 ml of 4% PFA inside 15 ml tubes at 4 °C for 2-3 h. Do not exceed 5 h otherwise a fluorescent background will disturb your analyses.
  12. Take photos (at least 3 fields/lung) of the whole lungs under a fluorescence stereomicroscope. In particular, rinse well the lungs in clean PBS and eliminate the excess liquid on a paper towel, then put the entire organ in a common cell culture petri dish (without the lid) under the microscope. Cells will appear as several red fluorescent clusters disposed along lung vasculature at early time points and as dispersed small dots at late time points, as you can observe in Figure 1A and 1D.
  13. Perform a quantitative and statistical analysis of the extravasated cells by counting the number of cells and/or calculating the area of red fluorescence in the photos, by means of an image analysis software (e.g. ImageJ, http://rsbweb.nih.gov/ij/).
  14. To verify the precise localisation of the tumor cells within the lungs, microscopic analyses should be performed.
    1. Wash extensively the fixed lungs in PBS and dehydrate them in a 30% sucrose solution for at least 24 h at 4 °C.
    2. Include the lungs in a freezing resin inside home-made tinfoil molds on dry-ice-cold (about -50 °C) isopentane and store them at -80 °C.
    3. Cryostat-cut the specimens in 6 μm thick sections on charged slides.
    4. For immunofluorescent stainings, thaw the slides, rinse them in PBS and draw a hydrophobic barrier around the specimen by using a PAP Pen.
      Note: Optionally, you may want to post-fix the specimens in acetone for 5-8 min to reduce the risk of tissue loss, but avoid the use of formaldehyde-derived fixatives).
      1. Samples are then blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature (RT).
      2. Incubated with anti-CD31 or anti-Von Willebrandt Factor antibody (1:100 or 1:50 dilution, respectively) for 1 h at RT or overnight at 4 °C, rinsed 3 times in PBS.
      3. Incubated with anti-rat or anti-rabbit Alexa-Fluor-488 (green) secondary antibody (1:500 dilution) for 45 min at RT.
      4. Counterstained with a nuclear marker such as DAPI and finally rinsed well in PBS and protected by mounting a coverslip.
      5. Examine specimens using a standard fluorescence microscope. Representative images are shown in Figure 1B-F.

Recipes

  1. Serum-free medium
    DMEM GlutamaxTM supplemented with 1 mM sodium pyruvate, 25 mM HEPES (pH 7.4) and 100 μg/ml gentamicin
  2. Complete medium
    DMEM GlutamaxTM supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 25 mM HEPES (pH 7.4), 100 μg/ml gentamicin (for MDA-MB-231) and with 1x MEM Non-Essential Amino Acids (for WK-Mel and B16) and with 1x MEM Non-Essential Amino Acids and 1x MEM Vitamin Solution (for A375P, MC-1 and MA-2)

Acknowledgments

This method was adapted from Casazza et al. (2011). We acknowledge Andrea Casazza for suggestions during the development of the protocol. We also thank Francesca Orso for useful discussion. Our work is supported by grants from the Compagnia San Paolo (2008.1054/DT), PRIN 2008/DT, AIRC 2010 (IG10104/DT), and FIRB giovani 2008 (RBFR08F2FS-002/FO). Elisa Penna is a FIRC fellow (2012–2014).

References

  1. Casazza, A., Finisguerra, V., Capparuccia, L., Camperi, A., Swiercz, J. M., Rizzolio, S., Rolny, C., Christensen, C., Bertotti, A., Sarotto, I., Risio, M., Trusolino, L., Weitz, J., Schneider, M., Mazzone, M., Comoglio, P. M. and Tamagnone, L. (2011). Sema3E-Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice. J Clin Invest 121(7):2945.
  2. Penna, E., Orso, F., Cimino, D., Vercellino, I., Grassi, E., Quaglino, E., Turco, E. and Taverna, D. (2013). miR-214 coordinates melanoma progression by upregulating ALCAM through TFAP2 and miR-148b downmodulation. Cancer Res 73(13): 4098-4111.
  3. Penna, E., Orso, F., Cimino, D., Tenaglia, E., Lembo, A., Quaglino, E., Poliseno, L., Haimovic, A., Osella-Abate, S., De Pitta, C., Pinatel, E., Stadler, M. B., Provero, P., Bernengo, M. G., Osman, I. and Taverna, D. (2011). microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C. EMBO J 30(10): 1990-2007.

材料和试剂

  1. 肿瘤细胞(特别地,该方案针对A375P,MC-1,MA-2,WK-Mel和B16黑素瘤细胞和MDA-MB-231乳腺癌细胞建立)
  2. CD1无胸腺裸鼠(6至9周龄)
  3. 磷酸盐缓冲盐水(PBS)
  4. 胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:15400-054)
  5. DMSO(Sigma-Aldrich,目录号:41648)
  6. CellTracker TM Orange CMRA(Life Technologies,Molecular Probes ,目录号:C34551); 或者肿瘤细胞本身需要表达红色荧光蛋白(RFP)
  7. 4%多聚甲醛(PFA)在PBS中的溶液
  8. 30%蔗糖的PBS溶液
  9. 冷冻树脂(Killik,Bio-Optica Milano SpA,目录号:05-9801B)
  10. 异戊烷
  11. 干冰
  12. 染色血管的抗CD31抗体(BD Biosciences,目录号:550274)或抗Von Willebrandt因子抗体(Dako,目录号:A0082)
  13. 抗大鼠或抗兔Alexa-Fluor-488二抗(Life Technologies,Molecular Probes ,目录号:A11006和A11008)
  14. FBS(Beromed,GmbH)
  15. 无血清培养基(见配方)
  16. 完全培养基(Life Technologies,Gibco ®)(参见配方)

设备

  1. 细胞培养设置,层流罩,离心机,细胞计数室
  2. 50ml管子
  3. P1000和P200提示
  4. 鼠标外壳和处理设置
  5. 带29 G x 1/2英寸针的U-100注射器
  6. 剪刀柄直凯利止血钳
  7. 荧光立体显微镜(例如,徕卡显微系统公司,型号:MZ16F)
  8. 冷冻切片机
  9. 荧光显微镜(例如具有ApoTome模块的ZEISS AxioObserver)
  10. Burker计数室(Marienfeld-superior)

软件

  1. 图像分析软件(例如 ImageJ, http://rsbweb.nih.gov/ij/

程序

  1. 从含有80-90%的肿瘤细胞(根据您的目的转染/处理)的80-90%铺满单层的皿或孔中移除培养基,用PBS洗涤细胞,并通过与1ml 0.05%胰蛋白酶-EDTA孵育3 -8分钟;用完全培养基终止胰蛋白酶消化。
  2. (仅用于非荧光细胞)以800rpm/100rcf旋转细胞3分钟,并将沉淀物重悬于50ml管中的3-4ml无血清培养基中。
  3. (仅用于非荧光细胞)重悬在9.1μlDMSO中的整个冻干的CellTracker TM Suppress Orange CMRA,并加入3-4μlCellTracker TM sup Orange TM CMRA (1:1,000稀释)加入每个管中的细胞。在37℃孵育35-45分钟。在孵育期间,每10-15分钟轻轻摇动试管。
  4. (仅用于非荧光细胞)以800rpm/100rcf旋转细胞5分钟,并将沉淀物重悬在15-25ml PBS中,取决于初始融合。由于跟踪器的合并,单元格将显示为粉红色/红色。
  5. 用布尔计数室计数细胞。
  6. 准备注射用的细胞混合物。您将在每只小鼠200μlPBS的体积中注射1至1.7×10 6个细胞。每只小鼠注射的细胞数目应事先由研究者在试验性实验中确定,并且主要取决于细胞类型,大小,侵袭性和形成细胞簇的倾向。例如,我们注射1×10 6个B16细胞,其是极度转移的并且易于形成大的簇和1.6×10 6个 A375P细胞,其较小且转移性较小。
  7. 根据您将注射的小鼠数量,旋转(在800 rpm/100 rcf 3分钟)您的实验所需的适当数量的细胞。通过吸取细胞悬液上下数次用P1000和P200尖端重悬细胞在PBS中,并保持管在冰上直到注射。为了避免细胞簇形成,其对于小鼠是潜在危险的,并且可能在外渗期间改变细胞行为并干扰分析,所以细胞处于均一的单细胞状态是必要的。
  8. 加热CD1裸鼠在红灯下几分钟,以获得血管舒张,更容易处理。使用具有29G×1/2英寸针的U-100注射器在外侧尾静脉中注射200μl的每只小鼠的细胞混合物(1.4至1.7×10 6个细胞)。
  9. 在注射后的早期时间点,作为"加载对照"(10分钟至2小时)和在稍后的时间点(12小时至72小时)分析小鼠肺(如步骤10-14所示)以检测外渗细胞。对于黑色素瘤(例如,A375P,WK-Mel)和乳腺癌(例如,MDA-MB-231)细胞,我们分别在2小时和48小时进行分析(参见图1) 。


    图1.肺外渗显微镜和显微镜分析。 A,D。在尾静脉注射CMRA标记的(红色)黑素瘤细胞后,全肺的代表性图片2小时(A)或48小时在裸鼠中。条:500μm。 B,C,E,F。鼠肺部切片的2小时(B,C)或48小时(E,F)后注射的代表性区域,CMRA-标记的(红色)黑色素瘤细胞,对Von Willebrandt因子染色以突出血液血管(绿色)并用DAPI(蓝色)进行反染色。酒吧:30μm

  10. 通过颈部脱位牺牲小鼠,通过消除气管周围的所有结缔组织,解剖动物胸部以用尖锐的显微切割镊子和剪刀暴露气管,直到白色软骨环是明显可见的。值得非常轻轻地进行,以避免气管穿孔。在隔膜上做纵向切口(但没有完全打开胸部),以允许肺部充气。使用止血钳夹住气管最多的头部,轻轻地注射0.8-1毫升冰冷的4%PFA在气管内用U-100注射器与29 G x 1/2英寸针,平行于气管的针头。你会清楚地看到肺膨胀。完成解剖并拔出肺。
  11. 在清洁的PBS中短暂清洗整个肺,以消除血液,并将其固定在8-10毫升的4%PFA内15毫升管在4℃下2-3小时。不要超过5小时否则荧光背景将扰乱您的分析。
  12. 在荧光立体显微镜下拍摄全肺的照片(至少3个视野/肺)。特别地,在干净的PBS中冲洗肺,并在纸巾上消除多余的液体,然后将整个器官在显微镜下放置在共同的细胞培养皿(没有盖子)中。在早期时间点,细胞将显示为沿着肺脉管系统设置的几个红色荧光团,以及在晚期时间点的分散小点,如图1A和1D所示。
  13. 通过借助于图像分析软件(例如,ImageJ, http://rsbweb.nih.gov/ij/)。
  14. 为了验证肺内肿瘤细胞的精确定位,应进行显微镜分析。
    1. 在PBS中大量洗涤固定的肺,并在30%蔗糖溶液中在4℃下将其脱水至少24小时。
    2. 包括肺在冷冻树脂在自制锡箔模具在干冰冷(约-50℃)异戊烷和存储在-80℃。
    3. 在带电载玻片上以6μm厚的切片冷冻切片。
    4. 对于免疫荧光染色,解冻载玻片,在PBS中冲洗它们,并通过使用PAP笔在样品周围形成疏水屏障。
      注意:或者,您可能需要将样本在丙酮中后固定5-8分钟,以减少组织损失的风险,但避免使用甲醛衍生的固定剂)。
      1. 然后将样品在室温(RT)下在5%牛血清白蛋白(BSA)中封闭1小时
      2. 与抗CD31或抗Von Willebrandt因子抗体(分别为1:100或1:50稀释)一起在室温下孵育1小时或在4℃下过夜,在PBS中漂洗3次。
      3. 在室温下用抗大鼠或抗兔Alexa-Fluor-488(绿色)第二抗体(1:500稀释)温育45分钟。
      4. 用核标记物例如DAPI复染色,最后在PBS中漂洗并通过安装盖玻片来保护。
      5. 使用标准荧光显微镜检查标本。代表图像如图1B-F所示。

食谱

  1. 无血清培养基
    补充有1mM丙酮酸钠,25mM HEPES(pH 7.4)和100μg/ml庆大霉素的DMEM Glutamax TM sup。
  2. 完成媒介
    补充有10%热灭活的FBS,1mM丙酮酸钠,25mM HEPES(pH 7.4),100μg/ml庆大霉素(用于MDA-MB-231)和用1×MEM Non(Invitrogen)的DMEM Glutamax TM - 必需氨基酸(用于WK-Mel和B16)和1x MEM非必需氨基酸和1x MEM维生素溶液(用于A375P,MC-1和MA-2)

致谢

该方法改编自Casazza等人(2011)。我们感谢Andrea Casazza在协议开发过程中的建议。我们也感谢Francesca Orso有益的讨论。我们的工作得到Compagnia San Paolo(2008.1054/DT),PRIN 2008/DT,AIRC 2010(IG10104/DT)和FIRB giovani 2008(RBFR08F2FS-002/FO)的资助。 Elisa Penna是FIRC研究员(2012-2014)。

参考文献

  1. Casazza,A.,Finisguerra,V.,Capparuccia,L.,Camperi,A.,Swiercz,JM,Rizzolio,S.,Rolny,C.,Christensen,C.,Bertotti,A.,Sarotto, ,M.,Trusolino,L.,Weitz,J.,Schneider,M.,Mazzone,M.,Comoglio,PM和Tamagnone,L。(2011)。 Sema3E-Plexin D1信号驱动小鼠的人类癌细胞侵袭和转移扩散。 em> J Clin Invest 121(7):2945。
  2. Penna,E.,Orso,F.,Cimino,D.,Vercellino,I.,Grassi,E.,Quaglino,E.,Turco,E.and Taverna,D。(2013)。 miR-214协调黑素瘤进展 通过上调ALCAM通过TFAP2和miR-148b下调。 Cancer Res 73(13):4098-4111。
  3. Penna,E.,Orso,F.,Cimino,D.,Tenaglia,E.,Lembo,A.,Quaglino,E.,Poliseno,L.,Haimovic,A.,Osella-Abate, C.,Pinatel,E.,Stadler,MB,Provero,P.,Bernengo,MG,Osman,I.and Taverna,D。(2011)。 microRNA-214通过抑制TFAP2C促进黑色素瘤肿瘤进展 EMEMO J(10):1990-2007。
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How to cite this protocol: Penna, E. and Taverna, D. (2014). In vivo Extravasation Assay. Bio-protocol 4(4): e1051. DOI: 10.21769/BioProtoc.1051; Full Text



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