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Apr 2016
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Qualitative in vivo Bioluminescence Imaging
体内定性生物发光成像技术   

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Abstract

Bioluminescence imaging (BLI) technology is an advanced method of carrying out molecular imaging on live laboratory animals in vivo. This powerful technique is widely-used in studying a variety of biological processes, and it has been an ideal tool in exploring tumor growth and metastatic spread in real-time. This technique ensures the optimal use of laboratory animal resources, particularly the ethical principle of reduction in animal use, given its non-invasive nature, ensuring that ongoing biological processes can be studied over time in the same animal, without the need to euthanize groups of mice at specific time points. In this protocol, the luciferase imaging technique was developed to study the effect of co-inoculating pericytes (contractile, αSMA+ mesenchymal stem cell-like cells, located abluminally in microvessels) on the growth and metastatic spread of ovarian cancers using an aggressive ovarian cancer cell line–OVCAR-5–as an example.

Keywords: Bioluminescence (生物发光), Tumor imaging (肿瘤显像), Luciferase Imaging (荧光素酶成像), Metastasis (转移), Ovarian cancer (卵巢癌)

Background

The principle of bioluminescence imaging (BLI) is based on the light-emitting properties of a relatively simple biochemical process, i.e., luciferase-mediated oxidation of the molecular substrate luciferin to produce light. In cancer research, BLI is a popular tool (Contag et al., 2000) used to study the metastatic spread of luciferase-transduced cancer cells in live animals in vivo. Most animal tissues have little to no baseline bioluminescent properties, which ensures that there is a very high signal to noise ratio in BLI experiments. Nevertheless, it is always pragmatic to ensure that BLI experiments are conducted with non-substrate injected rodents as a negative control group. A few crucial factors are key to ensuring best practice in performing BLI experiments on rodents for the detection of luciferase-tagged cancer cells. Firstly, BLI is a powerful tool that allows easy detection of luciferase expressing-cells by imaging emitted bioluminescence under anesthesia (Figure 1). However, this technique is essentially qualitative and attempts to quantitate BLI signal can be misleading given that the strength of signal is dependent on several factors including duration of exposure, anesthetic technique, time elapsed after injecting luciferin, etc. Moreover, published evidence indicates that light emission (i.e., quantity of photons) is not directly related to luciferase activity (Rice et al., 2001). Secondly, the location or depth of the tissue of interest, particularly its distance from the skin’s surface, and the size of the metastatic cell mass are important considerations in planning a BLI experiment (Weissleder, 2001). This is because photon loss occurs as the signal travels through the tissue mass–consequently, luciferase-tagged cells/tissues that are closer to the skin’s surface tend to appear brighter as do larger metastases. Micrometastases can be detected but may require sacrificing the animal and imaging the organs directly after skin removal in place of intact animals. Notwithstanding these and other challenges, BLI is an efficient and powerful, non-invasive technique for studying biological processes in vivo.


Figure 1. Imaging primary subcutaneous OVCAR-5 tumors and metastases. A. GFP-luciferase labeled OVCAR-5 cells, xenografted alone (OVCAR-5) or co-injected with pericytes (OVCAR-5+P) generated tumors that were imaged at regular intervals. The increased BLI signal observed in images of pericyte co-injected xenografts show that pericytes promote OVCAR-5 tumor growth rate and induce metastases compared to the control group. B. At day 28 the BLI signal from the primary tumor is saturated, and the mice were sacrificed and the primary tumors (along with the abdominal skin excised to permit clearer imaging of the metastatic nodules–metastatic nodules marked with red rings).

Materials and Reagents

  1. Materials
    1. Pipette tips (Interpath Services, catalog numbers: 39770 , 39730 )
    2. Hamilton® syringe ( Hamilton, catalog number: 80366
    3. Tissue culture plasticware (6-well plates BD; 2, 5 and 10 ml pipettes SARSTEDT)
    4. Steritop-GP polyethersulfone with low binding PES membrane (0.22 μm pore size) (Merck, Millipore, catalog number: SCGPS05RE )
    5. Pasteur pipette (Biologix, catalog number: 30-0138A1 )

  2. Biological materials
    1. Lentiviral vector pFUGW-Pol2-ffLuc2-eGFP (Addgene, catalog number: 71394 )
    2. OVCAR-5 cell line was obtained from NCI, and authenticated using short tandem repeat markers to confirm cell identity against the Genome Project Database (Wellcome Trust Sanger Institute)
    3. HIV-1 packaging vector pCMV-deltaR8.2 (Addgene, catalog number: 8455 ), a kind gift from Dr. Cameron Johnstone, Anderson Lab, Peter MacCallum Cancer Centre, Melbourne
    4. Packaging cell line HEK293T (ATCC, catalog number: CRL-3216 ), a kind gift from Dr. Cameron Johnstone, Anderson Lab, Peter MacCallum Cancer Centre, Melbourne
    5. Mice: 6-8 weeks old female athymic nude Balb/c mice were obtained from Walter Eliza Hall Institute, housed in a pathogen-free 12 h light–dark environment, fed ad libitum were used for tumorigenicity assays. This age range of mice is optimal for good tumor take rates which decline if older mice (e.g., 10 weeks old) are used.

  3. Reagents
    1. Bovine serum albumin (Sigma-Aldrich, catalog number: A9418-500G
    2. DAPI:4’, 6’-Diamidino-2-Phenylindole Dihydrochloride (Sigma-Aldrich, catalog number: D9542-5MG )
    3. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
    4. Diflucan (Fluconozole) (Sigma-Aldrich, catalog number: F8929 )
    5. D-Luciferin: Sodium salt 4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid sodium salt (Gold Biotechnology, catalog number: LUCNA-1G )
    6. FuGENE 6 (Roche Diagnostics, Mannheim, Germany)
    7. Endothelial basal media (EBMTM-2) (Lonza, catalog number: CC-3156 )
    8. Endothelial growth media (EGMTM-2) SinglequotsTM (Lonza, catalog number: CC-4147
    9. Fetal Calf Serum (FCS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
    10. Forthane/Isoflurane (Sigma-Aldrich, catalog number: 792632 )
    11. HEPES pH 7.4 (Sigma-Aldrich, catalog number: H0887-100ML )
    12. MatrigelTM (standard) (BD, catalog number: 356234 )
    13. PBS without Ca2+ and Mg2+ (GE Healthcare, Hyclone, catalog number: SH30256.FS )
    14. Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    15. Polybrene (Sigma-Aldrich, catalog number: 107689 )
    16. Potassium chloride (Sigma-Aldrich, catalog number: P5405 )
    17. Potassium dihydrogen phosphate (Sigma-Aldrich, catalog number: P5655 )
    18. RMPI-1640 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11875 )
    19. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
    20. Sodium chloride (Astral Scientific, catalog number: AMX190 )
    21. Sodium hydrogen phosphate (Sigma-Aldrich, catalog number: S5136 )
    22. Trypsin 0.05% (Thermo Fisher Scientific, GibcoTM, catalog number: 25300120 )
    23. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    24. Phosphate buffered Saline (PBS) (see Recipes)
    25. RPMI-1640 media (see Recipes)

Equipment

  1. Pipettes (Corning, catalog numbers: 4487 , 4488 , 4489 )
  2. Hemocytometer (ProSciTech, catalog number: SVZ2NI0U )
  3. Cell culture incubator (NuAire, model: NU-5510/E )
  4. Centrifuge (Beckman Coulter, model: Allegra X-12 )
  5. Electronic calipers (Fisher Scientific, catalog number: 14-648-17 )
  6. SW28 Rotor (Beckman Coulter, catalog number: 342207 )
  7. Ultracentrifuge (Beckman Coulter, model: OptimaTM XE-100 )
  8. Fluorescent Microscope (Nikon Instruments, model: Nikon A1+ Confocal Microscope )
  9. Xenogen Realtime Imaging System (IVIS Lumina II)
  10. Becton Dickinson Biosciences FACS DivaTM cell sorter

Software

  1. Prism 6 (GraphPad Inc.)
  2. Photoshop CS 6.0 (Adobe Inc.)
  3. Metamorph (Molecular devices)
  4. ImageJ (NIH software)

Procedure

In the protocol below we describe luciferase imaging to measure the metastatic spread of OVCAR-5 cells after the establishment of primary tumors following subcutaneous injection in immunocompromised mice. However, other cell types are equally amenable to this approach, and we have used this protocol to track OVCAR-8 cells for metastatic spread and monitor the persistence and survival of pericytes in vivo, when co-injected with OVCAR cells (Sinha et al., 2016).

  1. Lentiviral vector
    All work with lentiviruses was performed with due regard to biosafety concerns in line with guidelines set down by the Office of the Gene Technology Regulator of the Government of Australia and with Institutional approval (#09/2006). The replication defective 3rd generation lentiviral vector pFUGW-Pol2-ffLuc2-eGFP (Addgene plasmid #71394) expressing green fluorescent protein (GFP) from jellyfish Aequorea victoria and the firefly luciferase gene (Day et al., 2009) is used to transduce OVCAR-5 cells in this protocol, by co-transfection of the HIV-1 packaging vector and VSV-G envelope glycoprotein (Sinha et al., 2016).

  2. Lentiviral production
    The packaging cell line HEK293T is used to produce lentivirus for transduction of OVCAR-5 cells (Sinha et al., 2016). Briefly, HEK293T cells are plated at 2 x 105 cells per 60 mm plate in 3 ml DMEM containing 10% FCS, and allowed to adhere and grow overnight at 37 °C in a humidified incubator containing 5% (v/v) CO2. HEK293T cells are then transfected using FuGENE 6 (Roche Diagnostics, Mannheim, Germany) at a FuGENE (μl): pFUGW-Pol2-ffLuc2-eGFP plasmid DNA (μg) ratio of 3:1 as follows:
    1. Add an appropriate volume of FuGENE 6 to serum free media and incubate for 5 min at room temperature. 
    2. Determine the concentration of plasmid DNA using 260 nm absorption; then add it to the FuGENE/media mix at the appropriate concentration, and incubate for 15 min at room temperature. 
    3. Add the FuGENE/DNA/media mixture dropwise to the plated cells at 100 μl per 60 mm plate. Incubate the plate for 24-48 h at 37 °C. 
    4. Collect viral supernatant from HEK293 cells, place it in a sterile tube and concentrate by spinning at 1,000 x g at 25 °C for 90 min in an ultracentrifuge using an SW28 rotor; aspirating the media and resuspending the viral pellet in 1 ml of RPMI-1640 media.

  3. Lentiviral transduction
    1. One day prior to infection, plate OVCAR-5 cells into 6-well plates at 1 x 105 cells per well in RPMI-1640 (Sinha et al., 2016).
    2. For each well of OVCAR-5 cells to be transduced, add 2.5 μl of 10 mg/ml polybrene to 2 ml of media, and add to 500 μl of viral supernatant. This is the infection cocktail. 
    3. Overlay OVCAR-5 cells with infection cocktail for 12 h at 37 °C with 5% CO2, aspirate virus-containing medium and replace with fresh medium.
    4. Assess transduction efficiency of OVCAR-5 cells using fluorescence microscopy, as judged by the number of GFP positive cells. 
    5. Propagate transduced cells and expand in culture for up to two passages monitoring GFP transduction efficiency by fluorescence microscopy, prior to sorting for enrichment for GFP-positive cells. To sort, trypsinize cells and resuspend in blocking buffer (2% [v/v] FCS and 2% BSA in PBS without Ca2+ and Mg2+) containing DAPI at a final concentration of 2 μg/ml. Viable (DAPI negative) GFP+ cells are sorted and collected on a BD FACS DivaTM sorter and expanded prior to use in luciferase imaging experiments. Lentiviral transduction efficiencies are extremely high and transduction efficiencies of 85-90% were routinely achieved in our laboratory even with primary cells such as pericytes.

  4. GFP-luciferase+ OVCAR-5 cell culture and preparation for injection into mice
    1. Culture GFP-luciferase+ OVCAR-5 cells to 80-90% confluency.
    2. Aspirate culture media using sterile Pasteur pipette.
    3. Wash cell monolayer with a small volume of pre-warmed PBS without Ca2+ and Mg2+.
    4. Add an appropriate volume of 0.05% trypsin at 37 °C and incubate for 1-2 min.
    5. Tap plate gently to dislodge adherent cells.
    6. Quench trypsin activity with an equal volume of media containing FCS.
    7. Transfer cell suspension to centrifuge tube and spin at 400 x g for 5 min at 4 °C.
    8. Discard supernatant.
    9. Perform cell count using trypan blue stain and hemocytometer.
    10. Resuspend 8 x 105-8 x 106 cells in 100 μl of a PBS/MatrigelTM (5 μg/ml) mixture per mouse on ice.

  5. Animal preparation
    1. Inject immunocompromised mice with 100 μl cell suspension subcutaneously under the front flank using a Hamilton® syringe with a 26 G needle. (See video clip of procedure at https://youtube.com/watch?v=nrOTzLiWC8U)
    2. Keep mice under daily observation after injection to ensure full recovery, looking for signs of stress.
    3. Monitor tumor volume by taking metric measurements using electronic calipers.
      Note: OVCAR-5 tumors were monitored every alternate day. Modify protocol depending on the tumor growth rate of cell lines in use.

  6. Luciferase Imaging
    1. Inject mice subcutaneously on the non-tumor bearing flank with 150 μg per gram of body weight in 100 μl sterile PBS of D-luciferin, the substrate for the luciferase enzyme.
      Note: Acquire imaging within 10-12 min of injecting with D-Luciferin.
    2. Allow mice free movement for 6-8 min.
    3. Anesthetize mice with 2.5% Forthane (commercial isoflurane anesthetic) in oxygen.
      Note: Take care not to anesthetize for longer than 3 min.
    4. Place anesthetized mice to inhalation cones in induction chamber of Xenogen Real-Time Imaging System on the heated platform.
    5. Close chamber door securely.
    6. Acquire images at the same exposure length and time interval for all experimental groups within 10-12 min of luciferin injection prior to signal decay.

  7. End point
    1. Repeat above luciferase imaging (Steps F1-F6) every 7 days to continue to track metastatic spread.
    2. At the experimental end point, the luminescent signal from large primary tumors is saturated and can block the signal from the smaller metastatic nodules. In order to overcome this, inject mice with D-luciferin and allow free movement for 6-8 min, euthanize the animals and surgically excise the primary tumors. Open the peritoneal cavity surgically, remove skin tissue over the anatomical sites to be imaged and proceed with BLI, adjusting exposure length to ensure signals from smaller metastatic nodules are captured.
      Note: All experiments were performed in compliance with Institutional ethics committee guidelines which stipulate that animals should not be subjected to undue distress–experimental animals were monitored daily for signs of distress and were sacrificed when tumors reached a volume of 1,500 mm3 (where tumor volume = [width]2 x length x 0.5) even if this occurs before the planned end point of the experiment. No experiments were taken beyond Day 35 after injection. 

Data analysis

  1. Metastatic spread detected by BLI is a qualitative assay permitting the investigator to determine whether particular experimental conditions (in this case co-injection with pericytes) leads to metastasis or not. It is also possible to compare the time of onset of metastases under specific experimental conditions–in this case establishing that co-injection of OVCAR-5 cells with pericytes, leads to earlier onset of metastases than OVCAR-5 cells alone, by imaging at different time points. However, to establish that OVCAR-5 cells consistently yield metastatic nodules when co-injected with pericytes at specific time points, 10 mice per experimental group were imaged at several time points and each experiment replicated twice. BLI can also be used to track the persistence of non-tumor cells–as shown in Figure 2, by transducing pericytes with the GFP-luciferase lentivirus and co-injecting with untagged OVCAR-5 cells.


    Figure 2. Tracking pericytes in OVCAR-5 xenografts in vivo (Sinha et al., 2016). BLI images of xenografts generated by unlabeled OVCAR-5 cells alone (A); or OVCAR-5 cells co-injected with GFP-luciferase labeled pericytes (B). Imaging was conducted in 3 mice per group per time point, in 2 replicate experiments. Unlabeled OVCAR-5 tumors acted as a negative control, giving no signal despite luciferin injection (A). Sequential BLI of OVCAR-5 tumors co-injected with GFP-luciferase labeled pericytes displaying a positive BLI signal from Day 6 to Day 35, suggesting the persistence of pericytes in xenografts (B).

  2. BLI can be used to quantitate the number of metastatic nodules obtained with OVCAR-5 cells alone versus OVCAR-5 cells co-injected with pericytes but is best determined at the endpoint of the experiment after sacrificing the animals and enumerating the number of nodules detected by BLI from 10 mice per group from two independent experiments. Data are then expressed as mean number ± SEM of metastatic nodules per mouse and statistical analysis for differences between experimental groups performed using one-way ANOVA (Figure 3). In addition, metastases must be verified independently to confirm their OVCAR-5 cell origin by an independent means–thus each metastatic nodule is harvested, processed for histological analysis via paraffin sections that are immunostained for GFP expression using an antibody to GFP.


    Figure 3. Quantification and immunohistochemical verification of metastatic nodules (Sinha et al., 2016). A. The number of metastatic nodules obtained under varying experimental conditions was counted at the experimental endpoint, revealing that OVCAR-5 cells do not yield metastases at Day 28 whereas co-injection with pericytes does. Increasing the ratio of pericytes: tumor cells leads to an increase in metastatic nodules (compare OVCAR-5: pericyte ratio of 10:1 versus 1:1). B. GFP staining of GFP-luciferase+OVCAR-5 tumors generated from OVCAR-5 cells alone or with pericyte co-injection (OVCAR-5+P) in the liver and spleen verifying that the metastatic nodules detected by BLI are derived from OVCAR-5 cells and not attributed to spurious signal.

Notes

  1. The absolute amount of BLI signal detected is dependent on the number of GFP-luciferase+ cells being imaged and the duration of exposure after luciferin injection. Thus, imaging smaller tumors at early stages of tumor development or the lower number of pericytes can be enhanced by increasing the duration of exposure after luciferin injection. However, care has to be taken to ensure that the same exposure is used across all experimental groups of animals compared within an experiment. 
  2. Detection of metastatic nodules by BLI is limited by their size and depth of location within the body–the deeper their location from the imaged surface, the lower their chance of detection. Low signal from small metastatic nodules can also be obliterated by the strong signal from the primary tumor. Thus, while BLI is a good indicator of the presence or absence of metastasis, it is advisable to use the endpoint of experiments to establish the full extent and number of metastases. At this stage, animals are sacrificed, the primary tumor excised and the skin removed over the region to be imaged for metastases. 
  3. Use of different surgical tools to excise primary tumors from each experimental animal is advisable due to the possibility of transferring luciferase positive cells from one animal to the next, giving rise to false positive BLI readings. This also makes it critical to verify that each “metastatic nodule” detected by BLI is indeed derived from GFP-luciferase labeled tumor cells by harvesting the BLI positive metastasis, processing for histological sectioning and immunohistochemical analyses after staining for GFP as shown in Figure 3B.

Recipes

  1. Phosphate buffered Saline (PBS)
    1. Dissolve 1 g KCl; 1 g KH2PO4; 5.75 g Na2HPO4 and 40 g NaCl in approximately 2 L of MillQ H2O
    2. Make volume up to 5 L with MilliQ H2O and stir well until all ingredients have dissolved
    3. Measure pH and adjust to pH 7.0-7.5. Ensure osmolarity is 270 ± 13 mM
    4. Filter sterilize using a 20 L pressure tank and Sartobran filter
    5. Store at 4 °C
  2. RPMI-1640 media
    1. Supplement RPMI-1640 with 25 mM HEPES, 1% penicillin-streptomycin, 1.5% Diflucan and 10% (v/v) heat inactivated FCS
    2. To heat-inactivate FCS, incubate FCS at 56 °C for 45 min
    3. Filter sterilize the RPMI-1640 medium using a 20 L pressure tank and Sartobran filter
    Note: In this study, RPMI-1640 is used to culture the OVCAR-5 cell line.

Acknowledgments

The techniques described in this article were optimized during research work supported by grants from the CASS Foundation, Cancer Council of Victoria #807184 and the NHMRC #1025874 to PK; and an International HDR Ph.D. scholarship from ANU, Canberra to DS. We thank Prof Robin Anderson and Dr. Clare Slaney for valuable discussions and technical advice on BLI. ZP and PK are supported by funds awarded by the Faculty of Health Sciences, Curtin University. This protocol was adapted from a previous study from our laboratory published in Clinical cancer Research (Sinha et al., 2016).

Competing interests

The authors have no conflicts of interest or competing interests to declare.

Ethics

All animal experimentation was conducted with approval from the Peter MacCallum Animal Research Ethics Committee–AEEC (#E394 and #E504) within reference to guidelines of ethical experimentation on animals laid down by the National Health & Medical Research Council of Australia.

References

  1. Contag, C. H., Jenkins, D., Contag, P. R. and Negrin, R. S. (2000). Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2(1-2): 41-52. 
  2. Day, C. P., Carter, J., Bonomi, C., Esposito, D., Crise, B., Ortiz-Conde, B., Hollingshead, M. and Merlino, G. (2009). Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study. Pigment Cell Melanoma Res 22(3): 283-295.
  3. Rice, B. W., Cable, M. D. and Nelson, M. B. (2001). In vivo imaging of light-emitting probes. J Biomed Opt 6(4): 432-440.
  4. Sinha, D., Chong, L., George, J., Schluter, H., Monchgesang, S., Mills, S., Li, J., Parish, C., Bowtell, D., Kaur, P. and Australian Ovarian Cancer Study, G. (2016). Pericytes promote malignant ovarian cancer progression in mice and predict poor prognosis in serous ovarian cancer patients. Clin Cancer Res 22(7): 1813-1824.
  5. Weissleder, R. (2001). A clearer vision for in vivo imaging. Nat Biotechnol 19(4): 316-317.

简介

生物发光成像(BLI)技术是一种在体内实验室动物上进行分子成像的先进方法。 这种强大的技术广泛应用于研究各种生物过程,是实时探索肿瘤生长和转移扩散的理想工具。 该技术确保实验室动物资源的最佳利用,特别是减少动物使用的伦理原则,考虑到其非侵入性,确保可以在同一动物中随时间研究正在进行的生物过程,而无需安乐死 小鼠在特定的时间点。 在该方案中,开发了荧光素酶成像技术以研究共同接种周细胞(收缩性,αSMA + 间充质干细胞样细胞,位于微血管内的细胞)对生长和转移性扩散的影响。 卵巢癌使用侵袭性卵巢癌细胞系-OVCAR-5-作为例子。

【背景】生物发光成像(BLI)的原理是基于相对简单的生化过程的发光特性,即,荧光素酶介导的分子底物荧光素氧化产生光。在癌症研究中,BLI是一种流行的工具(Contag et al。,2000),用于研究荧光素酶转导的癌细胞在活体动物体内的转移性扩散。大多数动物组织几乎没有基线生物发光特性,这确保了BLI实验中存在非常高的信噪比。然而,确保用非底物注射的啮齿动物作为阴性对照组进行BLI实验总是实用的。一些关键因素是确保在啮齿动物上进行BLI实验以检测荧光素酶标记的癌细胞的最佳实践的关键。首先,BLI是一种功能强大的工具,通过在麻醉下成像发射的生物发光,可以轻松检测荧光素酶表达细胞(图1)。然而,这种技术基本上是定性的,并且尝试定量BLI信号可能是误导的,因为信号的强度取决于若干因素,包括暴露持续时间,麻醉技术,注射荧光素后经过的时间,等。此外,已发表的证据表明,光发射(即,光子数量)与荧光素酶活性没有直接关系(Rice et al。,2001)。其次,感兴趣组织的位置或深度,特别是其与皮肤表面的距离,以及转移细胞块的大小是计划BLI实验的重要考虑因素(Weissleder,2001)。这是因为当信号穿过组织块时发生光子损失 - 因此,更接近皮肤表面的荧光素酶标记的细胞/组织倾向于看起来更大,因为更大的转移。可以检测微转移,但可能需要牺牲动物并在去除皮肤后直接对器官成像以代替完整的动物。尽管有这些和其他挑战,BLI是一种有效且强大的非侵入性技术,用于研究体内生物过程。


图1.成像原发性皮下OVCAR-5肿瘤和转移瘤。 A. GFP-荧光素酶标记的OVCAR-5细胞,单独异种移植(OVCAR-5)或与周细胞共同注射(OVCAR-5 + P )产生定期成像的肿瘤。在周细胞共注射异种移植物的图像中观察到的增加的BLI信号显示,与对照组相比,周细胞促进OVCAR-5肿瘤生长速率并诱导转移。 B.在第28天,来自原发性肿瘤的BLI信号饱和,并且处死小鼠并且切除原发性肿瘤(连同腹部皮肤切除以允许更清楚地成像转移性结节 - 用红环标记的转移性结节)。

关键字:生物发光, 肿瘤显像, 荧光素酶成像, 转移, 卵巢癌

材料和试剂

  1. 材料
    1. 移液器吸头(Interpath Services,目录号:39770,39730)
    2. Hamilton ®注射器(Hamilton,目录号:80366) 
    3. 组织培养塑料器皿(6孔板BD; 2,5和10毫升移液器SARSTEDT)
    4. 具有低结合PES膜的Steritop-GP聚醚砜(孔径0.22μm)(Merck,Millipore,目录号:SCGPS05RE)
    5. 巴斯德吸管(Biologix,目录号:30-0138A1)

  2. 生物材料
    1. 慢病毒载体pFUGW-Pol2-ffLuc2-eGFP(Addgene,目录编号:71394)
    2. OVCAR-5细胞系获自NCI,并使用短串联重复标记进行鉴定,以确认细胞与基因组计划数据库(Wellcome Trust Sanger Institute)的身份
    3. HIV-1包装载体pCMV-deltaR8.2(Addgene,目录号:8455),来自墨尔本Peter MacCallum癌症中心的安德森实验室Cameron Johnstone博士赠送的礼物
    4. 包装细胞系HEK293T(ATCC,目录号:CRL-3216),由Cameron Johnstone博士,安德森实验室,墨尔本Peter MacCallum癌症中心赠送
    5. 小鼠:从Walter Eliza Hall Institute获得6-8周龄雌性无胸腺Balb / c小鼠,将其置于无病原体的12小时光 - 暗环境中,随意喂食用于肿瘤发生性测定。这种年龄范围的小鼠对于良好的肿瘤摄取率是最佳的,如果使用年龄较大的老鼠(例如,10周龄),它们会下降。

  3. 试剂
    1. 牛血清白蛋白(Sigma-Aldrich,目录号:A9418-500G) 
    2. DAPI:4',6'-二脒基-2-苯基吲哚二盐酸盐(Sigma-Aldrich,目录号:D9542-5MG)
    3. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11965092)
    4. Diflucan(Fluconozole)(Sigma-Aldrich,目录号:F8929)
    5. D-荧光素:4,5-二氢-2-(6-羟基-2-苯并噻唑基)-4-噻唑甲酸钠盐(Gold Biotechnology,目录号:LUCNA-1G)
    6. FuGENE 6(Roche Diagnostics,德国曼海姆)
    7. 内皮基底培养基(EBM TM -2)(Lonza,目录号:CC-3156)
    8. 内皮生长培养基(EGM TM -2)Singlequots TM (Lonza,目录号:CC-4147) 
    9. 胎牛血清(FCS)(赛默飞世尔科技,Gibco TM ,目录号:10099141)
    10. Forthane / Isoflurane(Sigma-Aldrich,目录号:792632)
    11. HEPES pH 7.4(Sigma-Aldrich,目录号:H0887-100ML)
    12. Matrigel TM (标准)(BD,目录号:356234)
    13. 没有Ca 2 + 和Mg 2 + 的PBS(GE Healthcare,Hyclone,目录号:SH30256.FS)
    14. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
    15. Polybrene(Sigma-Aldrich,目录号:107689)
    16. 氯化钾(Sigma-Aldrich,目录号:P5405)
    17. 磷酸二氢钾(Sigma-Aldrich,目录号:P5655)
    18. RMPI-1640(Thermo Fisher Scientific,Invitrogen TM ,目录号:11875)
    19. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
    20. 氯化钠(Astral Scientific,目录号:AMX190)
    21. 磷酸氢钠(Sigma-Aldrich,目录号:S5136)
    22. 胰蛋白酶0.05%(Thermo Fisher Scientific,Gibco TM ,目录号:25300120)
    23. 台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
    24. 磷酸盐缓冲盐水(PBS)(见食谱)
    25. RPMI-1640媒体(见食谱)

设备

  1. 移液器(康宁,目录号:4487,4488,4489)
  2. 血细胞计数器(ProSciTech,目录号:SVZ2NI0U)
  3. 细胞培养培养箱(NuAire,型号:NU-5510 / E)
  4. 离心机(Beckman Coulter,型号:Allegra X-12)
  5. 电子卡尺(Fisher Scientific,目录号:14-648-17)
  6. SW28转子(Beckman Coulter,目录号:342207)
  7. 超速离心机(Beckman Coulter,型号:Optima TM XE-100)
  8. 荧光显微镜(Nikon Instruments,型号:Nikon A1 + 共聚焦显微镜)
  9. Xenogen实时成像系统(IVIS Lumina II)
  10. Becton Dickinson Biosciences FACS Diva TM 细胞分选仪

软件

  1. Prism 6(GraphPad Inc.)
  2. Photoshop CS 6.0(Adobe Inc.)
  3. Metamorph(分子器件)
  4. ImageJ(NIH软件)

程序

在下面的方案中,我们描述了荧光素酶成像,以测量在免疫受损小鼠中皮下注射后建立原发性肿瘤后OVCAR-5细胞的转移性扩散。然而,其他细胞类型同样适用于这种方法,并且我们已经使用该协议来跟踪OVCAR-8细胞的转移性扩散并监测体内体内的持久性和存活率与共同注射时OVCAR细胞(Sinha et al。,2016)。

  1. 慢病毒载体
    根据澳大利亚政府基因技术监管机构办公室制定的指导方针和机构批准(#09/2006),所有与慢病毒的合作都是在适当考虑生物安全问题的情况下进行的。复制缺陷3 rd 代慢病毒载体pFUGW-Pol2-ffLuc2-eGFP(Addgene质粒#71394)表达来自水母 Aequorea victoria 的绿色荧光蛋白(GFP)和萤火虫荧光素酶通过HIV-1包装载体和VSV-G包膜糖蛋白(Sinha )的共转染,使用基因(Day et al。,2009)转导该方案中的OVCAR-5细胞。等人。,2016)。

  2. 慢病毒生产
    包装细胞系HEK293T用于产生用于转导OVCAR-5细胞的慢病毒(Sinha 等人,,2016)。简言之,将HEK293T细胞以每60mm平板2×10 5个细胞接种于含有10%FCS的3ml DMEM中,并使其在37℃下在含有5%的加湿培养箱中粘附并生长过夜。 (v / v)CO 2 。然后使用FuGENE 6(Roche Diagnostics,Mannheim,Germany)以FuGENE(μl):pFUGW-Pol2-ffLuc2-eGFP质粒DNA(μg)比例3:1转染HEK293T细胞,如下:
    1. 在无血清培养基中加入适量的FuGENE 6,在室温下孵育5分钟。 
    2. 使用260nm吸收确定质粒DNA的浓度;然后将其以适当的浓度添加到FuGENE /培养基混合物中,并在室温下孵育15分钟。 
    3. 将FuGENE / DNA /培养基混合物逐滴添加到每60mm平板100μl的铺板细胞中。将板在37°C孵育24-48小时。 
    4. 收集来自HEK293细胞的病毒上清液,将其置于无菌管中,并使用SW28转子在超速离心机中在25℃下以1,000×g g / g旋转90分钟;吸出培养基并将病毒沉淀重悬于1ml RPMI-1640培养基中。

  3. 慢病毒转导
    1. 在感染前一天,将RPC-1640中的OVCAR-5细胞以每孔1×10 5个细胞铺板到6孔板中(Sinha et al。,2016) 。
    2. 对于待转导的OVCAR-5细胞的每个孔,将2.5μl10mg/ ml聚凝胺加入2ml培养基中,并加入500μl病毒上清液。这是感染鸡尾酒。 
    3. 将OVCAR-5细胞与感染混合物在37℃下用5%CO 2 吸收12小时,吸出含有病毒的培养基并用新鲜培养基替换。
    4. 使用荧光显微镜评估OVCAR-5细胞的转导效率,通过GFP阳性细胞的数量来判断。 
    5. 在分选富集GFP阳性细胞之前,通过荧光显微镜传播转导的细胞并在培养物中扩增最多两次传代,监测GFP转导效率。分选,胰蛋白酶消化细胞并重悬于含有DAPI的封闭缓冲液(2%[v / v] FCS和2%BSA的PBS中,不含Ca 2+ 和Mg 2 + )最终浓度为2μg/ ml。在BD FACS Diva TM 分选仪上分选和收集活细胞(DAPI阴性)GFP + 细胞,并在用于荧光素酶成像实验之前进行扩增。慢病毒转导效率极高,我们的实验室常规实现85-90%的转导效率,即使是原代细胞如周细胞也是如此。

  4. GFP-荧光素酶 + OVCAR-5细胞培养及注射入小鼠的准备
    1. 培养GFP-荧光素酶 + OVCAR-5细胞至80-90%汇合。
    2. 使用无菌巴斯德吸管吸出培养基。
    3. 用少量预热的PBS洗涤细胞单层,不含Ca 2+ 和Mg 2 + 。
    4. 在37°C下加入适量0.05%胰蛋白酶,孵育1-2分钟。
    5. 轻轻敲打板以移除粘附的细胞。
    6. 用含有FCS的等体积培养基淬灭胰蛋白酶活性。
    7. 将细胞悬浮液转移至离心管中,并在4℃下以400 x g 旋转5分钟。
    8. 丢弃上清液。
    9. 使用台盼蓝染色和血细胞计数器进行细胞计数。
    10. 每只小鼠在100μlPBS/ Matrigel TM (5μg/ ml)混合物中重悬8×10 5个 5 -8×10 6 细胞在冰上。

  5. 动物准备
    1. 使用带有26 G针头的Hamilton ®注射器,在前侧皮下注射100μl细胞悬液的免疫受损小鼠。 (请参阅 https://youtube.com/watch?v=nrOTzLiWC8U 上的过程视频剪辑)
    2. 注射后保持小鼠每日观察,以确保完全恢复,寻找压力的迹象。
    3. 通过使用电子卡尺进行公制测量来监测肿瘤体积。
      注意:每隔一天监测OVCAR-5肿瘤。根据使用的细胞系的肿瘤生长速率修改方案。

  6. 荧光素酶成像
    1. 在100μl无菌的D-荧光素PBS(荧光素酶的底物)中,每只体重150μg,在非肿瘤的侧腹皮下注射小鼠。
      注意:注射D-Luciferin后10-12分钟内获得成像。
    2. 让小鼠自由活动6-8分钟。
    3. 用氧气中的2.5%Forthane(商业异氟烷麻醉剂)麻醉小鼠。
      注意:注意不要麻醉超过3分钟。
    4. 将麻醉的小鼠置于加热平台上的Xenogen实时成像系统的诱导室中的吸入锥体中。
    5. 牢固地关闭室门。
    6. 在信号衰减之前,在荧光素注射的10-12分钟内,以相同的曝光长度和时间间隔获取所有实验组的图像。

  7. 终点
    1. 每7天重复上述荧光素酶成像(步骤F1-F6)以继续跟踪转移扩散。
    2. 在实验终点,来自大的原发性肿瘤的发光信号饱和并且可以阻断来自较小转移性结节的信号。为了克服这个问题,给小鼠注射D-荧光素并允许自由运动6-8分钟,使动物安乐死并通过外科手术切除原发性肿瘤。手术打开腹腔,去除解剖部位上的皮肤组织进行成像,然后进行BLI,调整暴露长度以确保捕获来自较小转移性结节的信号。
      注意:所有实验均符合制度伦理委员会指导原则,该指南规定动物不应受到不适当的痛苦 - 每天监测实验动物的痛苦迹象,当肿瘤达到1,500 mm 3 (其中肿瘤体积= [宽度] 2 x长度x 0.5)即使这发生在实验的计划终点之前。注射后第35天以后未进行任何实验。

数据分析

  1. 通过BLI检测的转移扩散是定性测定,允许研究者确定特定实验条件(在这种情况下与周细胞共注射)是否导致转移。也可以比较特定实验条件下转移的发作时间 - 在这种情况下,确定OVCAR-5细胞与周细胞共同注射,导致比单独的OVCAR-5细胞更早发生转移,通过在不同的情况下成像时间点。然而,为了确定OVCAR-5细胞在特定时间点与周细胞共注射时始终产生转移性结节,每个实验组10只小鼠在几个时间点成像,每个实验重复两次。 BLI还可用于追踪非肿瘤细胞的持久性 - 如图2所示,通过用GFP-荧光素酶慢病毒转导周细胞并与未标记的OVCAR-5细胞共注射。


    图2.体内OVCAR-5异种移植物中的周细胞追踪(Sinha et al。,2016)。单独由未标记的OVCAR-5细胞产生的异种移植物的BLI图像(A);或者用GFP-荧光素酶标记的周细胞共注射OVCAR-5细胞(B)。在2个重复实验中,每组时间点每组3只小鼠进行成像。未标记的OVCAR-5肿瘤充当阴性对照,尽管注射了荧光素,但没有发出信号(A)。 OVGAR-5肿瘤的顺序BLI与GFP-荧光素酶标记的周细胞共注射,从第6天至第35天显示阳性BLI信号,表明在异种移植物中存在周细胞的持续存在(B)。

  2. BLI可用于定量单独使用OVCAR-5细胞获得的转移性结节数量与同时注射周细胞的OVCAR-5细胞的数量,但最好在实验终点确定后处死动物并计算检测到的结节数量。来自两个独立实验的每组10只小鼠的BLI。然后将数据表示为每只小鼠的转移性结节的平均数±SEM,并使用单向ANOVA进行实验组之间差异的统计分析(图3)。此外,必须独立验证转移以通过独立方式确认其OVCAR-5细胞来源 - 因此收获每个转移性结节,通过使用针对GFP的抗体对GFP表达进行免疫染色的石蜡切片进行组织学分析。


    图3.转移性结节的定量和免疫组织化学验证(Sinha et al。,2016)。 A.变异结节的数量变化在实验终点计数实验条件,揭示OVCAR-5细胞在第28天不产生转移,而与周细胞共同注射。增加周细胞的比例:肿瘤细胞导致转移性结节增加(比较OVCAR-5:周细胞比率为10:1对比1:1)。 B.GFP-荧光素酶 + OVCAR-5肿瘤的GFP染色从单独的OVCAR-5细胞或在肝脏和脾脏中用周细胞共注射(OVCAR-5 + P)产生,证实转移性结节由BLI检测到的是来自OVCAR-5细胞并且不归因于假信号。

笔记

  1. 检测到的BLI信号的绝对量取决于成像的GFP-荧光素酶 + 细胞的数量和荧光素注射后的暴露持续时间。因此,通过增加荧光素注射后的暴露持续时间,可以在肿瘤发展的早期阶段成像较小的肿瘤或较低数量的周细胞。但是,必须注意确保在实验中比较所有实验动物组的相同暴露量。 
  2. 通过BLI检测转移性结节受到其体内大小和位置深度的限制 - 它们从成像表面的位置越深,其检测机会越低。来自原发性肿瘤的强信号也可以消除来自小转移性结节的低信号。因此,虽然BLI是转移存在与否的良好指标,但建议使用实验终点来确定转移的全部程度和数量。在此阶段,处死动物,切除原发肿瘤,并在该区域移除皮肤以进行转移成像。 
  3. 由于可能将荧光素酶阳性细胞从一只动物转移到另一只动物,从而产生假阳性BLI读数,因此建议使用不同的手术工具从每只实验动物切除原发性肿瘤。这也使得关键是通过收获BLI阳性转移,在GFP染色后进行组织切片和免疫组织化学分析,验证BLI检测到的每个“转移性结节”确实来自GFP-荧光素酶标记的肿瘤细胞,如图3B所示。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    1. 溶解1克KCl; 1克KH 2 PO 4 ; 5.75g Na 2 HPO 4 和40g NaCl在约2L MillQ H 2 O中
    2. 用MilliQ H 2 O使体积达到5L并充分搅拌直至所有成分溶解
    3. 测量pH并调节至pH 7.0-7.5。确保渗透压为270±13 mM
    4. 使用20升压力罐和Sartobran过滤器过滤灭菌
    5. 储存在4°C
  2. RPMI-1640媒体
    1. 补充RPMI-1640含25 mM HEPES,1%青霉素 - 链霉素,1.5%Diflucan和10%(v / v)热灭活FCS
    2. 为了使FCS热灭活,将FCS在56℃下孵育45分钟
    3. 使用20升压力罐和Sartobran过滤器对RPMI-1640培养基进行过滤灭菌
    注意:在本研究中,RPMI-1640用于培养OVCAR-5细胞系。

致谢

本文描述的技术在CASS基金会,维多利亚癌症委员会#807184和NHMRC#1025874授予PK的研究工作中得到优化;和国际HDR博士学位澳大利亚国立大学,堪培拉奖学金到DS。我们感谢Robin Anderson教授和Clare Slaney博士对BLI的宝贵讨论和技术建议。 ZP和PK由科廷大学健康科学学院授予的资金支持。该方案改编自我们在临床癌症研究中发表的实验室的先前研究(Sinha et al。,2016)。

利益争夺

作者没有利益冲突或竞争利益申报。

伦理

所有动物实验均在Peter MacCallum动物研究伦理委员会-AEEC(#E394和#E504)的批准下进行,参考国家卫生组织制定的动物伦理实验指南。澳大利亚医学研究委员会。

参考

  1. Contag,C.H.,Jenkins,D.,Contag,P。R.和Negrin,R。S.(2000)。 使用报告基因进行体内肿瘤疾病的光学测量。 / a> Neoplasia 2(1-2):41-52。 
  2. Day,C.P.,Carter,J.,Bonomi,C.,Esposito,D.,Crise,B.,Ortiz-Conde,B.,Hollingshead,M。和Merlino,G。(2009)。 慢病毒介导的双功能细胞标记体内黑色素瘤研究。 Pigment Cell Melanoma Res 22(3):283-295。
  3. Rice,B.W.,Cable,M。D.和Nelson,M。B.(2001)。 发光探针的体内成像。 J Biomed Opt 6(4):432-440。
  4. Sinha,D.,Chong,L.,George,J.,Schluter,H.,Monchgesang,S.,Mills,S.,Li,J.,Parish,C.,Bowtell,D.,Kaur,P。and 澳大利亚卵巢癌研究,G。(2016年)。 Pericytes促进小鼠恶性卵巢癌进展并预测浆液性卵巢癌患者预后不良。 Clin Cancer Res 22(7):1813-1824。
  5. Weissleder,R。(2001)。 更明确的体内成像愿景。 Nat Biotechnol 19(4):316-317。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sinha, D., Pieterse, Z. and Kaur, P. (2018). Qualitative in vivo Bioluminescence Imaging. Bio-protocol 8(18): e3020. DOI: 10.21769/BioProtoc.3020.
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Farrah Aram
None
Hello,

In this protocol, it states that for lentiviral production, the HEK293T cells are transfected using FuGENE 6 and the FUGLW plasmid. May you clarify at which step the packaging and envelope vectors are added?
2019/3/21 11:59:02 回复
Pritinder Kaur
School of Pharmacy & Biomedical Sciences, Faculty of Health Sciences, Curtin Health Innovation Research Institute, Curtin University, Australia, Australia,

Hi Farrah

Apologies for not making this clear in the protocol. The HEK293 cells are co-transfected with FUGLW AND the packaging vectors to generate replication-defective lentivirus. We cited Sinha et al., 2016 which explains this methodology a bit better.

Sincerely
Pritinder kaur

2019/3/24 19:20:18 回复


Farrah Aram
None

Hello,

Thank you for your reply to my previous question. I do, however, have an another one. Can you suggest any commercially available alternatives to the FULGW plasmid used in this protocol? Thank you in advance.

Sincerely,
Farrah

2019/3/31 7:09:26 回复