生物发光成像：肿瘤发展、疾病传播和治疗效果的体内监测

生物发光成像（BLI）是一种非侵入式光学成像方法，旨在实现组织中生物发光信号的可视化和量化。¹ BLI的根本是当酶作为分子报告基因在体内表达时，对底物的酶介导氧化过程中产生的可见光进行检测。

与使用终端终点、侵入性操作或放射性标记的传统成像和生物分布研究相比，BLI提供了可靠、灵敏和高通量的替代方案。 We were the first CRO to offer BLI in 2003 and in the 17 years since we have amassed considerable experience and know-how in this optical imaging field. 在此技术聚焦中，我们将介绍生物发光成像的原理，并重点介绍该临床前服务在癌症检测、监测疾病进展和体内抗肿瘤功效评估方面所提供的优势。

BLI要求细胞表达萤光素酶，该词源自拉丁语lucifer——发光物质，例如由萤火虫或海肾产生的发光物质。萤火虫萤光素酶基因于1985²年初次实现克隆，自那时起，绿色最常用于荧光色成像。萤火虫萤光素酶需要注入其底物D-萤光素，该荧光素会产生在562 nm时达到峰值的生物发光信号，该信号由放置在不透光柜中的高光子转换效率电荷耦合器件相机（CCD）捕获。 The sequence of events leading from engineering tumor cells to express luciferase to imaging animals in vivo is illustrated in Figure 1. We use IVIS® In Vivo Imaging Systems (PerkinElmer, Waltham, MA) which allow high sensitivity and high resolution in vivo BLI and fluorescence imaging (FLI) across a wide range of wavelengths. 在2%异氟烷气体麻醉下，一次最多可对五个动物进行成像。为每只小鼠注射D-萤光素，并在注射后10-15分钟内对小鼠进行成像。BLI信号在肿瘤、特定区域（例如颅骨、胸腔或腹部）、整个身体或离体组织周围绘制的感兴趣区域（ROI）中进行量化，并且该信号按照光子/秒表达，代表来自用户定义区域的全向辐射通量。图像使用Living Image 4.3.1（PerkinElmer，马萨诸塞州沃尔瑟姆）软件分析。

图1：使用发光活细胞的体内生物发光成像。将携带有启动子驱动荧光素酶基因的载体转染到肿瘤细胞中。稳定转染的细胞扩增并植入小鼠体内。肿瘤生长后，给动物注射荧光素并在麻醉下在IVIS成像系统中成像。荧光素酶细胞会发光，并可进行时空跟踪。

监测原位实体瘤生长

过去20年里，凭借出色的有效性和敏感性，BLI方法和工具的应用越来越广泛，包括：蛋白质间相互作用、基因筛选、细胞周期调节剂、寄生虫感染、干细胞植入跟踪、NK细胞迁移以及临床前肿瘤学。大量研究表明，BLI成像可以暂时评估肿瘤的发展。³当肿瘤生长无法通过卡尺测量确定，并且依赖于体重减轻等临床体征或动物安乐死后的肿瘤负荷评估时，这一点尤为重要。在下述示例中，所有动物护理和使用均根据动物福利法规在AAALAC认可的研究中心进行，并经过IACUC协议审查和批准。

图2中的实例突出了非侵入性BLI在监测深层组织肿瘤发展方面的价值。将NCI-H460-Luc2人类肺癌异种移植物原位植入（OT）（1x10⁵细胞/小鼠）雌性裸鼠的左肺。BLI（图2，左上）显示了最早在肿瘤细胞植入后8天就可以检测到的胸腔肿瘤（因在第28天可查看的最大信号阈值而在图像中不可见），而且肿瘤负荷在接下来的2周内增加。在整个研究过程中，小鼠的平均体重减轻百分比为18.3%，可能与疾病有关（未显示），并且小鼠在肿瘤细胞植入后28天安乐死。尸检揭示了原发性肿瘤的明显生长以及整个胸腔肿块的实质性发展。在图2（左下）中，雌性白化病C57BL/6小鼠颅内植入GL261-luc2同系鼠神经胶质瘤细胞（1x10⁶细胞/小鼠）。 植入后7天，大脑中可检测到肿瘤。与疾病进展相关的体重减轻在该模型中很常见，并且在植入肿瘤后11天就可以检测到，而死亡中位时间约为21天。在雌性裸鼠中进行心内注射后，人类乳腺腺癌细胞系MDA-MB-231-luc-D3H2LN形成骨病变（1x10⁵细胞/小鼠）。在注射肿瘤细胞21天后，骨病变清晰可见，并且在接下来的3周内，肿瘤数量和肿瘤总负荷都有所增加（图2，右侧，顶部和底部）。到第32天，小鼠体重最多减轻16.2%。体重减轻很大程度上与模型的侵袭性有关（未显示）。 The takeaway message from these studies is that BLI can be a powerful tool for investigating a variety of tumor types in real-time and non-invasively without relying solely on clinical signs of disease which may, or may not, accompany increasing tumor burden.

Figure 2: Representative BLI images across several in vivo models. Top left, NCI-H460-Luc2 human lung carcinoma implanted OT (left lung) in female nude mice. Lower left, syngeneic murine glioma GL261-luc2 cells implanted OT (brain) in female albino C57BL/6 mice and right side, human mammary tumor MDA-MB-231-luc-D3H2LN bone lesions after intracardiac injection in female nude mice showing whole body images in the prone (top) and supine (lower) positions.

Imaging Hematologic Malignancies

With over 80 unique hematological malignancy cell lines, we lead the industry with market-relevant hematologic malignancy models, particularly pertinent in the adoptive cell therapy (ACT), also known as cellular immunotherapy, field.⁴ BLI is critically valuable when evaluating progression and treatment response in hematological disseminated malignancies. Longitudinal studies performed in 4 different human hematological cancer cells injected IV into NSG mice showed increased tumor burden over time in all models (Figure 3) underscoring BLI’s value in repeated assessment of disease progression and severity, as well as understanding the biodistribution of tumor signal throughout the body.

Figure 3: Representative BLI data across several in vivo human hematologic malignancies following IV injection in NSG mice.

ACT are continuously evolving, providing better and more personalized options for patients. CAR-T cells are autologous or allogeneic T cells specifically targeting antigens or markers expressed by tumor cells. Preclinical in vivo assessment of the efficacy and safety of CAR-T cells is crucial before translating these new therapies into the clinic. The study shown in Figure 4 was designed to test different CAR-T preparations and doses in human Raji-luc B cell lymphoma cells implanted IV in NSG mice. The 3 different CAR-T constructs significantly inhibited tumor burden and prolonged survival, highlighting the value BLI brings to the development of these new cellular immunotherapies.

Figure 4: Effect of CAR-T therapy against human Raji-luc B cell lymphoma implanted IV into NSG mice. Top: Schematic diagram of tumor cell inoculation and CAR-T therapy. Bottom: Tumor burden assessed by BLI and overall survival.

Monitoring Treatment Efficacy

放射

Clearly, one of the key advantages of BLI is the ability to track the efficacy of anti-tumor therapies over time in the same animal, leading to a reduction in the number of animal used. In the example shown in Figure 5, female nude mice were implanted intracranially with NCI-H1975-luc human non-small cell lung carcinoma in order to mimic lung metastasis to the brain. Mice received two courses of fractionated radiation (2Gy; 5 days on, two days off for two cycles), delivered by a RadSource RS-2000 irradiator. The control group was sham irradiated. BLI images were acquired over time and demonstrated that radiation significantly reduced tumor burden (Figure 5, p<0.05 on day 19) and increased life span by 160% (p<0.05, not shown). In this experiment, we were able to quantify treatment response in vivo, critical when monitoring efficacy as well as designing and refining new therapeutic approaches. 

Figure 5: Effect of localized radiation treatment (2Gy; 5 days on, two days off for two cycles) against intracranially implanted NCI-H1975-luc human non-small cell lung carcinoma in female nude mice.

Checkpoint Inhibition and Combination Therapy

The effect of checkpoint inhibition, alone or in combination with radiation, was tested in murine syngeneic GL261-luc2 glioma cells implanted intracranially in female albino C57BL/6 mice. Mice were left untreated, treated with radiation, delivered by the Small Animal Radiation Research Platform (SARRP; Xstrahl Inc.), at a single 7.5Gy dose, anti-mouse PD-1 (clone RMP1-14, 10 mg/kg) or the combination of both therapies.  As shown in Figure 6 (left), BLI signal was already detectable 6 days post-tumor cell implantation and progressed over the next several weeks. Quantification of the resulting bioluminescent signal (right) showed the effect of treatments on tumor burden. Anti-mPD-1 treatment led to 2.6 days tumor growth delay and 1/8 tumor free survivor (TFS) while radiation resulted in 16.6 days tumor growth delay and 2/8 TFS. The combination of radiation and anti-mPD-1 treatment resulted in significant 38.6 days of tumor growth delay and complete remission with 7/8 mice. Traditional methods of gauging anti-tumor response in malignancies located deep within the body are based on terminal and often, time consuming read-outs. As shown in these examples, the integration of in vivo BLI imaging has allowed us to shift from a “black box” of tumor therapy to real-time assessment of individual responses, providing the flexibility to adjust and refine treatments more rapidly.

Figure 6: Effect of focal radiation, anti-mPD-1 and combination therapy in intracranial GL261-luc tumors in female C57BL/6 albino mice.

Multimodality

BLI can be coupled with other imaging modalities to interrogate different biological pathways. To illustrate this application, we used syngeneic 4T1-luc2-1A4 mouse breast adenocarcinoma cells injected OT into the mammary fat pad of immunologically competent BALB/c mice. When tumors reached ~300 mm³, a near-infrared imaging agent, ProSense^TM750 was injected IV to detect cathepsin activity associated with aggressive breast cancer growth. ProSense^TM750 is an optically silent agent which becomes fluorescent upon cleavage with cathepsins. As shown in Figure 7, BLI and FLI tumor signals were readily detectable in tumors. However, BLI and FLI signals were not perfectly superimposable which is expected given that luciferase is produced by all living tumor cells while the fluorescent signal is confined not only to tumor cells but also other tumor-associated cells, mainly macrophages, responsible for the cleavage and uptake of the cathepsin ProSense^TM750 probe. Multimodality can thus be used to interrogate different biologies in vivo, simultaneously.

Figure 7: Multimodality imaging of 4T1-luc2-1A4 tumors by BLI (left) and FLI (right) 24h after IV injection of the cathepsin-specific ProSenseTM750 fluorescence probe. Below are closeups of the corresponding tumors.

总结

Mouse models of deep-tissue cancer and cancer metastasis rely predominantly on terminal assessments like tissue weight, nodule counts, and/or histologic analysis for the assessment of tumor burden. Since its introduction well over 20 years ago, BLI has become an invaluable technique to track the growth dynamics of bioluminescently-tagged cancer cell lines over time and in response to various therapeutic approaches in vivo, minimizing the number of animals on study since the same subjects can be imaged over time without the need for terminal assessments. BLI has generated significant contributions to a variety of scientific fields, in addition to providing an alternative to the use of established invasive or radioisotope-dependent non-invasive imaging modalities.

Contact the scientists to request the full data set or to learn more about our BLI service and how it can be applied to your preclinical research.

参考资料

¹Contag CH, Spilman SD, Contag PR, Oshiro M, Eames B, Dennery P, Stevenson DK, Benaron DA. Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 1997; 66: 523–531.

²de Wet JR, Wood KV, Helinski DR, DeLuca M. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1985; 82, 7870–7873.

³ Jenkins DE, Oei Y, Hornig YS, Yu SF, Dusich J, Purchio T, Contag PR. 生物发光成像（BLI）可以改善和完善传统的鼠类肿瘤生长和转移模型。《Clin Exp Metastasis》，2003；20(8):733-744。

⁴ https://www.cancerresearch.org/immunotherapy/treatment-types/adoptive-cell-therapy