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The Chick Embryo Chorioallantoic Membrane as an in vivo Model to Study Metastasis
基于鸡胚绒毛尿囊膜的体内癌症转移模型   

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Abstract

Metastasis is a complex process that includes several steps: neoplastic progression, angiogenesis, cell migration and invasion, intravasation into nearby blood vessels, survival in the circulatory system, extravasation followed by homing into distant tissues, the formation of micrometastases, and finally the growth into macroscopic secondary tumors. This complexity makes metastases difficult to investigate and quantify in animal models. The chick embryo is a unique in vivo model that overcomes many limitations for studying the metastatic process, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located under the eggshell, that is receptive to the xenografting of mammalian tumor cells, including human. Since the chick embryo is naturally immunodeficient at this stage, the CAM can support the engraftment of tumor cells, and their growth therein can faithfully recapitulate most of the characteristics of the carcinogenic process including: growth, invasion, angiogenesis and colonization of distant tissues (Deryugina and Quigley, 2008; Zijlstra et al., 2002). The CAM sustains rapid tumor formation within 5-7 days after cancer cell grafting. This feature provides a unique experimental model for a rapid study of the intravasation and colonization steps of the metastatic cascade. Furthermore, using quantitative PCR to detect species-specific sequences, such as Alu, the chick embryo CAM model can be used to monitor and quantify the presence of the xenografted, ectopic tumor cells in distant tissues. Thus, the chick embryo model has proved a valuable tool for cancer research, in particular for the investigation of molecules and pathways involved in cancer metastasis and to analyze the response of metastatic cancer to potential therapies (Herrero et al., 2015; Casar et al., 2014). In this respect, the use of the rapid and quantitative spontaneous metastasis chick embryo model can provide an alternative approach to conventional mouse model systems for screening anti-cancer agents.

Keywords: Metastasis model(转移模型), CAM(凸轮), Tumor growth(肿瘤的生长), Cancer(癌症)

Materials and Reagents

  1. 20 G needles (BD, PrecisionGlideTM, catalog number: 305175 )
  2. 30 G needles (BD, PrecisionGlideTM, catalog number: 305128 )
  3. MicroAmp® optical 96 well PCR plate (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8010560 )
  4. MicroAmp® optical adhesive film (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311971 )
  5. Cotton tipped applicators, cotton swab, Iodine liquid (Thermo Fisher Scientific)
  6. Laboratory tape 1/2" x 500" (VWR, catalog number: 470144-262 )
  7. Fertilized chicken eggs (Gilbert farm, Tarragona, Spain)
  8. A375 melanoma cell line (ATCC, catalog number: CRL-1619 )
  9. SKMEL2 (ATCC, catalog number: HTB68 )
  10. RKO colorectal cancer cell line (ATCC, catalog number: CRL-2577 )
  11. HCT116 (ATCC, catalog number: CCL-247 )
  12. DEL 22379 (Vichem Chemie, Budapest)
  13. Trypsin 0.05% with EDTA (1 mM), liquid (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054 )
  14. Phosphate-buffered saline (PBS) (1x, pH 7.4), liquid (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  15. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, catalog number: 41965062 )
  17. Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  18. QIA amp genomic DNA purification kit (QIAGEN, catalog number: 158906 ; 158910 ; 158914 )
  19. Primers (HPLC purification, IDT DNA technologies)
    Alu (human) sense: 5’ ACGCCTGTAATCCCAGGACTT 3’
    Alu (human) antisense: 5’ TCGCCCAGGCTGGCTGGGTGCA 3’
    Chicken GAPDH sense: 5’ GAGGAAAGGTCGCCTGGTGGATCG 3’
    Chicken GAPDH antisense: 5’ GGTGAGGACAAGCAGTGAGGA ACG 3’
  20. SYBR® green mix real time PCR (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4472908 )

Equipment

  1. Incubator 37 °C, 60% humidity (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 51028117 )
  2. Rotating eggs trays (automatic eggs turner) (GQF, catalog number: 1611 )
  3. Tugon tube (Drifton, model: Tygon LMT )
  4. Egg candler (Lyon, model: 950-170 )
  5. Microsurgical kits, sterile forceps, push pin, dissection scissors, needle nose forceps (VWR, IntegraTM Miltex®, catalog number: 95042-542 )
  6. Dremel 100 rotary tool (Dremel, model: 100N/7 )
  7. Dremel cut off wheels number 36 (Dremel)
  8. Hemocytometer, Neubauer chamber (EMD Millipore)
  9. 2-20 µl pipette (Eppendorf, Eppendorf Research®, catalog number: 3120000038 )
  10. 20-200 µl pipette (Eppendorf, Eppendorf Research®, catalog number: 3120000054 )
  11. Automatic pipette (Eppendorf, Eppendorf Easypet® 3, catalog number: 4430000018 )
  12. Real time PCR instrument (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOne plus RT PCR )

Software

  1. Graph Pad Prism software

Procedure

A schematized summary of the spontaneous metastasis assay is illustrated in Figure 1.


Figure 1. The chick embryo for spontaneous metastasis model. After 10 days of incubation the CAM is dropping and tumor cells (green) are applied to it. After allowing xenografted tumor cells to grow, the tumor and chicken tissues are harvested on day 5-7 for Alu PCR.

  1. Preparing the eggs for xenografting tumor cells
    1. Freshly fertilized chicken eggs are incubated on their side in a rotating incubator at 37 °C and 60% humidity for 10 days. The eggs are rotated three times per hour (Figures 2.1 and 2.2)
    2. On day 10 the eggs are placed on their side on an egg rack (Figure 2.3). Use a tube lamp or other suitable light source to candle the eggs by shining the light at the blunt end of the egg where the air sack is located. The embryo must be located near the bottom of the egg and the air sack on its right. Localize and mark using a pencil the allantoic vein that is located at the top of the eggshell, right where several blood vessels cross (Figure 2.4).
    3. Clean the area, including and around the mark, using a cotton swab soaked with iodine (Figure 2.5).
    4. Drill a small hole through the eggshell into the air sack using a 30-gauge syringe needle (Figures 2.6 and 3A).
    5. Make another hole near the allantoic vein, which penetrates the shell membrane but not the CAM, using a Dremel rotary tool kit (Figures 2.7 and 3B). The CAM is attached to the inner surface of the shell, so care should be exercised at this point. Use a 20-gauge syringe needle with a small hook on the end to make a third very small hole in the eggshell membrane (Figure 3C, Note 1).


      Figure 2. A serial depiction of the model. 1 and 2. Incubate the eggs; 3. Prepare a clean area and set all the materials; 4. Mark the location of the allantoic vein; 5. Apply iodine using a cotton swab; 6 and 7. Drill holes in the eggshell; 8. Drop the CAM; 9. Cut an opening for tumor cell grafting; 10. Apply the tumor cells and wait for 5 min; 11. Seal the hole using laboratory tape; 12. Incubate the eggs.

    6. To suction, create with an automatic pipette aid fitted with a piece of Tygon tubing placed against the hole in the airsac. To separate the CAM from the shell and let it drop, apply a mild vacuum to the hole over the air sack so the blood vessel drops down, away from the eggshell and attaches to the embryo (Figures 2.8 and 3D).
    7. Use a cut off wheel (Dremel) to cut a square window (1 cm2) approximately 0.5 cm away from the branch point in the vein to expose the underlying CAM. A pair of needle-nose forceps can be used to remove a small section of eggshell in the square window and expose the CAM (Figures 2.9 and 3E).
    8. Once the CAM is dropped, seal the hole located near the chorioallantoic vein using laboratory tape.
    9. Then place the eggs in an egg rack into a stationary incubator at 37 °C and 60% humidity in anticipation for grafting the tumor cells.


      Figure 3. A six panel diagram illustrating the appearance of the chick embryo metastasis model in cross section at key steps. A. After 10 days of incubation, the allantoic vein is positioned against the top of the egg. Drill one hole in the eggshell into the air sack using a 30 G syringe needle. B. Make another hole using a Dremel rotary tool kit adjacent to the attachment point for the allantoic vein. C. Make a third small hole in the eggshell membrane using a 20 G syringe needle. D. Apply a mild vacuum into the hole over the air sack to evacuate the air and drop the CAM. E. Cut a square window (1 cm2) using a Dremel cut off wheel and remove a small section of eggshell to expose the CAM. F. Graft the tumor cells onto the CAM.

  2. Preparing tumor cells for grafting
    1. Detach tumor cells from their culture dishes using trypsin/EDTA and wash with phosphate buffered saline (PBS) twice in order to remove any residual media.
    2. Cells are counted using a Neubauer chamber and resuspended in serum-free DMEM at 40 million cells/ml depending on the cell line used (Note 2).
  3. Grafting the tumor cells onto the CAM
    1. Using a 20-200 μl pipette place 25 μl of the cell suspension onto a small area of the CAM (Figures 2.10 and 3F). The optimal number of cells should be determined empirically, but can range from 0.4 x 105 to 2 x 106 depending on the growth and ‘invasiveness’ characteristics of the tumor cell line used. Different tumors and cell suspensions derived from tumors have been implanted on the CAM (Tables 1 and 2). Tumor cell lines grow and intravasate to the vasculature with different efficiencies. For instance, suspensions of A375 melanoma (1 x 106), SKMEL2 melanoma (1 x 106), RKO colorectal carcinoma (2 x 106), HCT116 (0.5 x 106), U87 glioblastoma (0.2 x 106), PC3 prostate carcinoma (1 x 106), Hep3 epidermoid carcinoma (0.4 x 106), HT-1080 fibrosarcom (0.5 x 106) cell lines; have been grafted on the CAM of embryonated chicken eggs. All cell lines tested formed 4-8 mm size tumors, which recapitulated hallmarks of corresponding human specimens. Depending on the tissue origin, number of tumor cells and their proliferation capacity, primary CAM tumors can reach up to 500-600 mg in 6-7 days after cell inoculation.
    2. Seal the window in the egg tightly with laboratory tape and leave the eggs in a position with the embryos standing upright for 5-10 min, in order to allow the cells to settle (Figure 2.11).
    3. After 10 min, return the eggs to the stationary incubator (Figure 2.12).
    4. Allow cells to grow for 5-7 days depending on the nature of the tumor cell line used. This time should be enough for a macroscopic tumor to be visible.
    5. The CAM model allow experimental studies of potential anti-tumorigenic and anti-metastatic compounds. Tumors can be treated topically on the upper CAM with potential anti-cancer drugs. For instance, to evaluate the effects of DEL22379 in A375 and RKO tumor growth we inoculated tumor cells on the CAM and incubate the embryos for 4 days at 37 °C and 60% humidity. Then, we added 10 µM DEL22379 (prepare freshly, soluble in DMSO) topically on the CAM and treatment was refreshed every 12 h. Two days later tumors were harvested.

      Table 1. Different tumor samples implanted onto the CAM


      Table 2 .Tumor cells tested onto the CAM


      We describe the different steps for graft the tumor cells onto the CAM using the spontaneous metastasis model in Video 1.

      Video 1. The chick chorioallantoic membrane as an in vivo xenograft model to study tumor growth and metastasis

  4. Harvesting tumors and chick embryo tissues
    1. Perform dissections in an area free of contaminating DNA (Figure 4.1). To detect metastatic invasion this assay quantifies the presence of ectopic DNA in chicken organs using a highly sensitive PCR approach. Therefore, it is very important to prevent contamination with exogenous DNA.
    2. In order not to have cross-contamination of your samples, use three separated sets of surgery tools. One set for cutting the egg, one set for harvesting the primary tumor and one set for removing the internal tissues.
    3. Prepare three wash containers for sequential rinsing of the tools between each animal and change their liquid between different experimental groups. Sequentially these washes are: distilled water, 70% ethanol and 1x PBS.
    4. Get the eggs from the stationary incubator.
    5. Open a new window removing some of the eggshell such that the tumors become visible. At this point, you can take some pictures of the appearance of the macroscopic tumor (Figures 4-6).
    6. Resect the primary tumor from the CAM and weigh the tumor.
    7. Remove the chick embryo from the eggshell by cutting the shell radially into equal halves.


      Figure 4. Pictures showing tumor and chicken tissues harvest. 1. Perform dissection in a clean area. 2. Open a new window removing some of the eggshell and resect the primary tumor from the CAM. 3. Cut the eggshell radially into equal halves and harvest lower CAM. 4. Open the chick embryo cutting through the sternum and collect a piece of liver.

    8. Transfer the embryo to a clean weight boat. The animal must be dissected using a clean set of tools.
    9. Open the chick embryo cutting through the sternum. Once the embryo is open, collect a piece of the liver. To harvest the lung, cut the rib cage and separate from the breast.
    10. Harvested samples can be processed immediately for DNA isolation or stored at -80 °C for later extraction of DNA, if necessary.


      Figure 5. Different tumors have been implanted on the CAM. Tumors were formed using 0.5 x 106 HCT116 colorectal cancer cells or 1 x 106 SKMEL2 melanoma cells during 5 and 7 days respectively.

  5. Genomic DNA isolation
    1. To extract genomic DNA from tissues, we use the DNA kit from Qiagen. Purified DNA following manufactuer's instructions. DNA can be used immediately or stored at -20 °C. Dilute the DNA 1:50 or 1:25 to get 30 ng/µl in nuclease free water before using it for the PCR reaction.
  6. PCR analysis
    1. To detect human tumor cell DNA in the chick tissues we use quantitative PCR for human Alu sequences using Alu-specific primers and SYBR green mix amplification kit. Prepare the reaction mix in a final volume of 10 μl, with 0.4 μM of each primer. Optimize the amount of template DNA empirically, we usually use 30 ng. Use chicken GAPDH primers as an internal control. The PCR is run under the following conditions: 95 °C for 2 min, 40 cycles at 95 °C for 30 sec, 63 °C for 30 sec, 72 °C for 30 sec.
    2. Generate a standard curve using a dilution series of human DNA (102, 103, 104) from the original tumor cells. Use this standard curve to quantify human tumor cells in a chick embryonic metastasis assay.
    3. Measure Ct values in triplicate and against the standard curve calculate the number of tumor cells in each CAM and liver sample.
    4. Invaded cells are detected using either 30 ng of 50 ng of total CAM DNA. Use water as a negative control.
    5. To evaluate results and statistical significance of control and experimental groups, use Graph Pad Prism software and Student t-test or ANOVA analysis (Figure 6).


      Figure 6. DEL 22379 inhibits tumor growth and metastasis of cancer cells. Tumors were formed using 1 x 106 A375p melanoma cells (A) or 2 x 106 RKO colorectal cancer cells (B). Cells were applied to the CAM of day-10 embryos. Four days after applying the tumor cells half of the embryos were treated with DMSO and the other half with DEL22379 (10 µM). Two days later tumors were harvested to weigh. CAM and chick organs (lung) were harvested from embryos bearing tumors and subjected to quantitative Alu PCR. Conversely, quantitative PCR of chGAPDH was used as an internal control to confirm the presence of equivalent quantities of host genomic DNA. Data shows average ± SEM from 3 independent experiments. **P < 0.05; ***P < 0.005, by unpaired Student t-test (Herrero et al., 2015).

Notes

  1. A small hook on the end of the 20 G syringe needle is necessary to make the third hole in the eggshell membrane. Try to do it very softly and carefully not to tear the underlying CAM.
  2. Place the tube on ice during the experiment to avoid the formation of clusters of tumor cells.

Acknowledgments

This protocol was adapted from the previously published studies, Zijlstra et al. (2002), Deryugina et al. (2008), Casar et al. (2014) and it was used in Herrero et al. (2015). We are grateful to Dr. Elena Deryugina and Dr. James Quigley for teaching us the techniques and advise. We thanks to Dr. Marian Ros and Dr. María Felix for providing equipment, technical assistance, and advise. PC lab was supported by grant SAF-2015-63638-R from the Spanish Ministry of Economy-Fondos FEDER and by the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD/12/0036/0033), Spanish Ministry of Health. BC was supported by Fundación Francisco Cobos - CSIC.

References

  1. Casar, B., Rimann, I., Kato, H., Shattil, S. J., Quigley, J. P. and Deryugina, E. I. (2014). In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated β1 integrin and induction of FAK/PI3K/Akt motility signaling. Oncogene 33(2): 255-268.
  2. Deryugina, E. I. and Quigley, J. P. (2008). Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis. Histochem Cell Biol 130(6): 1119-1130.
  3. Herrero, A., Pinto, A., Colon-Bolea, P., Casar, B., Jones, M., Agudo-Ibanez, L., Vidal, R., Tenbaum, S. P., Nuciforo, P., Valdizan, E. M., Horvath, Z., Orfi, L., Pineda-Lucena, A., Bony, E., Keri, G., Rivas, G., Pazos, A., Gozalbes, R., Palmer, H. G., Hurlstone, A. and Crespo, P. (2015). Small molecule inhibition of ERK dimerization prevents tumorigenesis by RAS-ERK pathway oncogenes. Cancer Cell 28(2): 170-182.
  4. Zijlstra, A., Mellor, R., Panzarella, G., Aimes, R. T., Hooper, J. D., Marchenko, N. D. and Quigley, J. P. (2002). A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction. Cancer Res 62(23): 7083-7092.

简介

转移是一个复杂的过程,包括几个步骤:肿瘤进展,血管生成,细胞迁移和入侵,附近血管内渗,循环系统中的生存,外渗,然后归巢到远端组织,微转移的形成,最后生长宏观继发性肿瘤。这种复杂性使得转移瘤难以在动物模型中研究和量化。鸡胚是独特的体内模型,其克服了用于研究转移过程的许多限制,这是由于绒毛尿囊膜(CAM)的可接近性,所述绒毛尿囊膜是位于蛋壳下的良好血管化的胚胎外组织,其接受哺乳动物肿瘤细胞(包括人)的异种移植。由于鸡胚在此阶段是天然免疫缺陷的,所以CAM可以支持肿瘤细胞的植入,并且其中的生长可忠实地重现致癌过程的大多数特征,包括:生长,侵入,血管生成和远端组织的定居(Deryugina和Quigley,2008; Zijlstra等人,2002)。 CAM在癌细胞移植后5-7天内维持快速肿瘤形成。这个特征提供了用于快速研究转移性级联的灌注和定植步骤的独特实验模型。此外,使用定量PCR来检测物种特异性序列,例如Alu ,鸡胚CAM模型可用于监测和定量在远端组织中异种移植的异位肿瘤细胞的存在。因此,鸡胚模型已经证明是用于癌症研究的有价值的工具,特别是用于研究癌症转移中涉及的分子和途径,并分析转移性癌症对潜在疗法的反应(Herrero等人 。,2015; Casar 。,2014)。在这方面,使用快速和定量的自发转移鸡胚胎模型可以提供用于筛选抗癌剂的常规小鼠模型系统的替代方法。

关键字:转移模型, 凸轮, 肿瘤的生长, 癌症

材料和试剂

  1. 20G针(BD,PrecisionGlide TM ,目录号:305175)
  2. 30G针(BD,PrecisionGlide TM ,目录号:305128)
  3. MicroAmp光学96孔PCR板(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:N8010560)
  4. MicroAmp光学粘合剂膜(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4311971)
  5. 棉针头,棉签,碘液(Thermo Fisher Scientific)
  6. 实验室胶带1/2"×500"(VWR,目录号:470144-262)
  7. 受精的鸡蛋(Gilbert农场,西班牙塔拉戈纳)
  8. A375黑素瘤细胞系(ATCC,目录号:CRL-1619)
  9. SKMEL2(ATCC,目录号:HTB68)
  10. RKO结肠直肠癌细胞系(ATCC,目录号:CRL-2577)
  11. HCT116(ATCC,目录号:CCL-247)
  12. DEL 22379(Vichem Chemie,Budapest)
  13. 用EDTA(1mM),液体(Thermo Fisher Scientific,Gibco< sup>,目录号:25300-054)的胰蛋白酶0.05%
  14. 磷酸盐缓冲盐水(PBS)(1x,pH 7.4),液体(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  15. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,目录号:41965062)
  17. 胎牛血清(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  18. QIA amp基因组DNA纯化试剂盒(QIAGEN,目录号:158906; 158910; 158914)
  19. 引物(HPLC纯化,IDT DNA技术)
    Alu(人)意义:5'ACGCCTGTAATCCCAGGACTT 3'
    Alu(人)反义:5'TCGCCCAGGCTGGCTGGGTGCA 3'
    鸡GAPDH感觉:5'GAGGAAAGGTCGCCTGGTGGATCG 3'
    鸡GAPDH反义:5'GGTGAGGACAAGCAGTGAGGA ACG 3'
  20. SYBR 绿色混合实时PCR(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4472908)

设备

  1. 孵育器37℃,60%湿度(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:51028117)
  2. 旋转蛋托盘(自动蛋打蛋器)(GQF,目录号:1611)
  3. Tugon管(Drifton,型号:Tygon LMT)
  4. 蛋烛(里昂,型号:950-170)
  5. 显微外科套件,无菌镊子,推针,解剖剪,针尖钳(VWR,Integra TM,Miltex ,目录号:95042-542)
  6. Dremel 100旋转工具(Dremel,型号:100N/7)
  7. Dremel切断车轮36号(Dremel)
  8. 血细胞计数器,Neubauer室(EMD Millipore)
  9. 2-20μl移液管(Eppendorf,Eppendorf Research ,目录号:3120000038)
  10. 20-200μl移液管(Eppendorf,Eppendorf Research ,目录号:3120000054)
  11. 自动移液管(Eppendorf,Eppendorf Easypet 3,目录号:4430000018)
  12. 实时PCR仪(Thermo Fisher Scientific,Applied Biosystems ,型号:StepOne plus RT PCR)

软件

  1. Graph Pad Prism软件

程序

图1中示出了自发转移测定的示意性概述

图1.用于自发转移模型的鸡胚。孵育10天后,CAM下降并且将肿瘤细胞(绿色)施用于其上。在允许异种移植的肿瘤细胞生长后,在5-7天收获肿瘤和鸡组织用于Alu PCR。

  1. 准备蛋用于异种移植肿瘤细胞
    1. 新鲜受精的鸡卵在其侧面在37℃和60%湿度的旋转培养箱中孵育10天。鸡蛋每小时旋转三次(图2.1和2.2)
    2. 在第10天,将鸡蛋放在蛋架上(图2.3)。使用管灯或其他合适的光源,通过照亮鸡蛋的空气袋所在的蛋的钝端照亮鸡蛋。胚胎必须位于蛋的底部附近,右侧的空气袋。使用铅笔定位和标记位于蛋壳顶部的尿囊静脉,几个血管交叉(图2.4)。
    3. 使用浸有碘的棉签(图2.5)清洁包括标记周围的区域。
    4. 使用30号注射器针头在蛋壳上钻一个小孔进入空气袋(图2.6和3A)。
    5. 在尿囊静脉附近另一个孔,使用Dremel旋转工具套件(图2.7和3B)穿透壳膜而不是CAM。 CAM连接到外壳的内表面,因此在这一点上应该小心。使用20号注射器针头,在末端有一个小钩子,在蛋壳膜上形成第三个非常小的孔(图3C,注1)。


      图2.模型的连续描述 1和  2。孵化鸡蛋; 3.准备一个干净的区域,并设置所有的材料; 4.标记尿囊静脉的位置; 5.使用棉签涂抹碘; 6和  7。在蛋壳上钻孔; 8.放下CAM;切开肿瘤细胞移植的开口; 10.应用肿瘤细胞并等待5分钟; 11.使用实验室胶带密封孔; 12.孵化鸡蛋。

    6. 要吸入,创建与自动移液器辅助配备一块聚乙烯管放置在airsac的孔。为了将CAM与外壳分开并使其下降,对气囊上的孔施加温和的真空,使得血管下降,远离蛋壳并附着到胚胎(图2.8和3D)。
    7. 使用切割轮(Dremel)切割距离静脉中分支点大约0.5cm的正方形窗口(1cm <2> )以暴露下面的CAM。可以使用一对针头钳去除方形窗口中的一小部分蛋壳,并露出CAM(图2.9和3E)。
    8. 一旦CAM掉落,使用实验室胶带密封位于绒毛尿囊静脉附近的孔
    9. 然后将鸡蛋放在蛋架上,置于37°C和60%湿度的固定培养箱中,以预期移植肿瘤细胞。


      图3.显示在关键步骤的横截面中鸡胚胚转移模型的外观的六个面板图。A.孵育10天后,将尿囊静脉靠着蛋的顶部放置。使用30 G注射器针在蛋壳中钻一个孔进入空气袋。 B.使用Dremel旋转工具套件,在尿壶静脉附着点附近再做一个洞。 C.使用20G注射器针在蛋壳膜上制造第三个小孔。 D.在空气袋上的孔中施加温和的真空,以排空空气并使CAM掉落。 E.使用Dremel切割轮切割正方形窗口(1cm 2),并移除小部分蛋壳以暴露CAM。 F.将肿瘤细胞移植到CAM上
  2. 准备肿瘤细胞移植
    1. 使用胰蛋白酶/EDTA从其培养皿中分离肿瘤细胞,并用磷酸盐缓冲盐水(PBS)洗涤两次,以除去任何残余的培养基。
    2. 使用Neubauer室计数细胞,并根据所使用的细胞系(注2)重悬于40百万细胞/ml的无血清DMEM中。
  3. 将肿瘤细胞移植到CAM上
    1. 使用20-200微升吸管将25微升细胞悬液放置在CAM的一小块区域上(图2.10和3F)。细胞的最佳数量应当根据经验确定,但是根据肿瘤细胞的生长和"侵入性"特征,其范围可以为0.4×10 5至2×10 6个线使用。来自肿瘤的不同肿瘤和细胞悬浮液已植入CAM上(表1和2)。肿瘤细胞系以不同的效率生长并灌注到脉管系统中。例如,将A375黑素瘤(1×10 6个),SKMEL2黑素瘤(1×10 6个),RKO结肠直肠癌(2×10 6个/),HCT116(0.5×10 6个),U87胶质母细胞瘤(0.2×10 6个),PC3前列腺癌(1×10 6个/),Hep3表皮样癌(0.4×10 6个),HT-1080纤维肉瘤(0.5×10 6个)细胞系;已被嫁接在胚胎鸡蛋的CAM上。所有测试的细胞系形成4-8mm大小的肿瘤,其概括了相应的人类标本的标志。根据组织起源,肿瘤细胞数量及其增殖能力,在细胞接种后6-7天内,原发性CAM肿瘤可达到500-600mg。
    2. 用实验室胶带密封鸡蛋的窗口,将鸡蛋放置在胚胎直立5-10分钟的位置,以使细胞定居(图2.11)。
    3. 10分钟后,将蛋放回固定的培养箱(图2.12)
    4. 允许细胞生长5-7天,取决于使用的肿瘤细胞系的性质。这个时间应足以使肉眼可见的肿瘤可见
    5. CAM模型允许潜在的抗肿瘤发生和抗转移化合物的实验研究。肿瘤可以在上CAM上用潜在的抗癌药物局部治疗。例如,为了评估DEL22379在A375和RKO肿瘤生长中的作用,我们在CAM上接种肿瘤细胞,并在37℃和60%湿度下孵育胚胎4天。然后,我们在CAM上局部加入10μMDEL22379(新鲜制备,可溶于DMSO),并且每12小时更新处理。两天后收获肿瘤。

      表1.植入CAM上的不同肿瘤样本


      表2。在CAM上测试的肿瘤细胞


      我们描述了使用视频1中的自发转移模型将肿瘤细胞移植到CAM上的不同步骤
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  4. 收获肿瘤和鸡胚组织
    1. 在无污染DNA的区域进行解剖(图4.1)。为了检测转移性侵袭,该测定使用高度敏感的PCR方法定量鸡器官中异位DNA的存在。因此,防止外源DNA污染是非常重要的
    2. 为了不对样品造成交叉污染,使用三组分离的手术工具。一组用于切割卵,一组用于收获原发性肿瘤,一组用于切除内部组织
    3. 准备三个洗涤容器,用于在每个动物之间的工具的顺序冲洗和在不同的实验组之间改变他们的液体。接下来,这些洗涤是:蒸馏水,70%乙醇和1x PBS
    4. 从固定的孵化器获得鸡蛋。
    5. 打开一个新窗口,删除一些蛋壳,使肿瘤变得可见。在这一点上,你可以拍摄一些宏观肿瘤的图片(图4-6)
    6. 切除CAM的原发肿瘤,称重肿瘤。
    7. 通过将壳径向切成相等的两半,从蛋壳中取出鸡胚。



      图4.显示肿瘤和鸡组织收获的图片。1.在干净的区域进行切除。 2.打开一个新窗口,去除一些蛋壳,并从CAM中切除原发肿瘤。 3.将蛋壳径向切成相等的两半并收获下CAM。 4.打开雏鸡胚胎切开胸骨,收集一块肝脏。

    8. 转移胚胎到一个干净的重量船。动物必须使用一套干净的工具进行解剖。
    9. 打开小鸡胚胎切割通过胸骨。一旦胚胎开放,收集一块肝脏。要收获肺,切开胸廓,并从乳房分离
    10. 收获的样品可立即处理用于DNA分离或储存在-80℃下,以备后续提取DNA。


      图5.在CAM上植入不同的肿瘤。使用0.5×10 6个HCT116结肠直肠癌细胞或1×10 6个肿瘤细胞形成肿瘤, SKMEL2黑素瘤细胞分别在5和7天
  5. 基因组DNA分离
    1. 为了从组织中提取基因组DNA,我们使用来自Qiagen的DNA试剂盒。纯化的DNA按照制造商的说明书。 DNA可立即使用或储存于-20℃。稀释DNA 1:50或1:25以获得30 ng /μl的无核酸酶游离水,然后用于PCR反应。
  6. PCR分析
    1. 为了检测小鸡组织中的人类肿瘤细胞DNA,我们使用Alu特异性引物和SYBR绿色混合扩增试剂盒对人类Alu序列进行定量PCR。准备反应混合物在终体积为10μl,每个引物0.4μM。根据经验优化模板DNA的量,我们通常使用30 ng。使用鸡GAPDH引物作为内部对照。 PCR在以下条件下进行:95℃2分钟,40个循环,95℃30秒,63℃30秒,72℃30秒。
    2. 使用来自原始肿瘤细胞的人类DNA(10μL,10μL,10μL,10μL)的稀释系列产生标准曲线。使用此标准曲线在鸡胚胎转移测定中定量人肿瘤细胞
    3. 一式三份测量Ct值,并根据标准曲线计算每个CAM和肝样品中的肿瘤细胞数
    4. 使用30ng 50ng的总CAM DNA检测侵袭的细胞。用水作为阴性对照。
    5. 为了评价对照组和实验组的结果和统计学显着性,使用Graph Pad Prism软件和Student 测试或Anova分析(图6)。




      图6.ELD 22379抑制癌细胞的肿瘤生长和转移。使用1×10 6个A375p黑素瘤细胞(A)或2×10 6个肿瘤细胞 RKO结肠直肠癌细胞(B)。细胞应用于第10天胚胎的CAM。施用肿瘤细胞后4天,用DMSO处理一半胚胎,另一半用DEL22379(10μM)处理。两天后收获肿瘤称重。从具有肿瘤的胚胎收获CAM和鸡器官(肺),并进行定量的Alu PCR。相反,chGAPDH的定量PCR用作内部对照以证实等量的宿主基因组DNA的存在。数据显示来自3个独立实验的平均值±SEM。 ** P 0.05; *** P 0.005,通过不配对的Student - 测试(Herrero等人,2015)。

笔记

  1. 在20 G注射器针头的末端的一个小钩是必要的,以使蛋壳膜上的第三个洞。尝试非常轻柔,小心地不要撕裂下面的CAM
  2. 在实验期间将管置于冰上以避免形成肿瘤细胞簇。

致谢

此协议改编自以前发表的研究,Zijlstra等人。 (2002),Deryugina等人。 (2008),Casar等人。 (2014),并且其用于Herrero等人。 (2015)。我们感谢Elena Deryugina博士和James Quigley博士教我们的技术和建议。我们感谢Marian Ros博士和MaríaFelix博士提供设备,技术援助和建议。 PC实验室由西班牙经济部Fondos FEDER和西班牙卫生部红十字会(RTICC)(RD/12/0036/0033)授予SAF-2015-63638-R资助。 BC由弗朗西斯科科博斯基金会 - CSIC支持。

参考文献

  1. Casar,B.,Rimann,I.,Kato,H.,Shattil,SJ,Quigley,JP和Deryugina,EI(2014)。  在体内切割的CDCP1通过与活化的β1整联蛋白的复合和诱导FAK/PI3K/Akt运动性信号传导促进早期肿瘤传播。 Oncogene 33(2):255-268。
  2. Deryugina,EI和Quigley,JP(2008)。  Chick胚胎绒毛尿囊膜模型系统以研究和显现人肿瘤细胞转移。组织化学细胞生物学130(6):1119-1130。
  3. Herrero,A.,Pinto,A.,Colon-Bolea,P.,Casar,B.,Jones,M.,Agudo-Ibanez,L.,Vidal,R.,Tenbaum,SP,Nuciforo,P.,Valdizan, EM,Horvath,Z.,Orfi,L.,Pineda-Lucena,A.,Bony,E.,Keri,G.,Rivas,G.,Pazos,A.,Gozalbes,R.,Palmer,HG,Hurlstone, A.和Crespo,P.(2015)。  小分子抑制ERK二聚化阻止RAS-ERK通路癌基因的肿瘤发生。癌症细胞 28(2):170-182。
  4. Zijlstra,A.,Mellor,R.,Panzarella,G.,Aimes,RT,Hooper,JD,Marchenko,ND和Quigley,JP(2002)。< a class ="ke-insertfile"href ="http: //www.ncbi.nlm.nih.gov/pubmed/12460930"target ="_ blank">使用人特异性实时聚合酶链反应的转移级联中的速率限制步骤的定量分析。 em> Cancer Res 62(23):7083-7092。
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引用:Crespo, P. and Casar, B. (2016). The Chick Embryo Chorioallantoic Membrane as an in vivo Model to Study Metastasis. Bio-protocol 6(20): e1962. DOI: 10.21769/BioProtoc.1962.
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