Introduction into the power of bioluminescence imaging 

Bioluminescence imaging (further referred to as BLI in this article) is an in vivo imaging technique that allows non-invasive longitudinal monitoring of in vivo processes, commonly applied in preclinical oncology research and general drug development. BLI allows monitoring and collection of qualitative and quantitative data of biological processes at the cellular and molecular levels in a living organism, such as an animal model of disease. Therefore, BLI is a highly suitable in vivo imaging method, frequently used in oncology research to monitor tumour growth and metastasis as well as monitoring expression of genes, protein-protein interactions and can even be used to evaluate biodistribution and pharmacodynamics of novel molecule carriers (such as lipid nanoparticles [LNPs]).  

In this blog post, we provide an overview of bioluminescence imaging, its application in different fields with emphasis on preclinical cancer research and drug development, with example case studies of orthotopic tumour models and novel drug delivery methods.  

The difference between bioluminescent and fluorescent imaging 

Although both bioluminescence and fluorescence imaging are often described in the same context of in vivo imaging systems, they both offer different and unique characteristics for optical imaging applications. Bioluminescence refers to the natural biological process of light emissions in living organisms, requiring enzyme (luciferase), substrate (luciferin) and oxygen which in turn result in light emission. BLI capitalises on this native light emission capability from bioluminescent organisms by using the luciferase enzymes. Three most commonly used luciferases in biomedical research are the firefly luciferase, renilla luciferase and gaussia luciferase. In contrast to light emission and capturing from bioluminescence, fluorescence imaging requires light excitation of fluorophores in order to result in light emission or light signal.  

To visualise this light emission and to convert it into data, an in vivo imaging system (IVIS, Figure 1) is used. For in vivo imaging, animals are anesthesized and placed into the warmed stage of the imager. Subsequently, an image of the animal is generated, capturing the bioluminescent or fluorescent signal which is then overlaid on the image of the animal. The signal is then expressed in photons per second and displayed as an intensity map (see examples in Figures 2-3 below) 

BLI imager, IVIS imaging, preclinical imager

FIGURE 1. In vivo imaging system (IVIS). The image above shows the BLI instrumentations, of which main instruments of the BLI system include luminometer, CCD camera, gas anaesthesia manifold, emission filter and imaging shelf/stage and imaging chamber.  

The Importance of Bioluminescent Imaging in Preclinical Research

Experiments performed during preclinical research help inform the next stages of research, ultimately informing whether a candidate drug can progress to first-in-human studies and later on to clinical trials. Therefore, well-designed experiments that can make use of longitudinal and non-invasive techniques are crucial. Thanks to its non-invasive nature, BLI helps gain insights into the in vivo processes taking place in an experimental animal model.  

Some examples of studies or endpoint assessments in preclinical research studies that may benefit from inclusion of BLI are listed below:  

  • Imaging of tumorigenesis and metastases (for e.g., in mouse models of cancer or in response to a novel compound in mouse model of cancer). 
  • Gene expression monitoring (for e.g., when assessing efficacy, pharmacodynamics and biodistribution of gene therapy).  
  • Transgene expression monitoring (for e.g., when evaluating the suitability of a new preclinical transgenic mouse models). 
  • Efficacy, pharmacodynamics and biodistribution of novel therapy types (for e.g., RNA therapy including RNAi, siRNA, mRNA etc.,). 
  • When evaluating delivery carrier platforms for novel therapy types, BLI provides real-time biodistribution and pharmacokinetic analysis of the delivery carrier formulation, effectiveness of target organ and off-target organ delivery (also in ex vivo isolated organs). Example of a delivery carrier (LNP) biodistribution evaluation using BLI is provided in Figure 2.  
  • Can be applied for visualization and distribution of compounds (antibodies, ADCs, nanoparticles, small molecules).  
  • Evaluation of (stem) cell therapies, offering valuable information on survival, functional behaviour and stability of implanted (stem) cells.    
  • Protein-protein interactions, protein folding, protein splicing, enzyme quantification

FIGURE 2. BLI allows real-time evaluation of biodistribution and pharmacokinetics of novel delivery methods. Following delivery of novel drug delivery vehicles such as LNPs, the BLI signal allows to discriminate the distribution between the liver and spleen, providing real-time biodistribution and delivery information of novel therapeutic agents over timeWe evaluate the intensity and duration of the luminescence signal in vivo at both the injection site and distally, in the liver and spleen (ex vivo). BLI can give insight into formulation (stability, biodistribution kinetics), different administration routes, different dosing regimens, deliverability into target tissue, evaluation of target expression in cells and tissues and immune response profiling. 

Advantages of Bioluminescent Imaging in Preclinical Research

BLI is a technique of choice among many researchers in the preclinical field due to several advantages:  

  • BLI offers acquisition of imaging data with high-sensitivity, high-resolution and high signal-to-noise ratio due to a low background signal noise in animals.  
  • Implementing BLI in research reduces the number of animals (in line with 3Rs of animal research) required for an experiment because multiple measurements can be made in the same animal over time in a within-subjects’ study design.  
  • BLI proves to be a cost-effective due to the reduction in animal numbers needed, this is especially important if you and your team are considering outsourcing your BLI studies to a preclinical contract research organization (CRO).  

Limitations of Bioluminescent Imaging in Preclinical Research

Although BLI proves to be a crucial imaging technique in preclinical research, there are few limitations that researchers aiming to use this technique should consider:  

  • Simple quantification of light emission may not provide a true representation of the biological effect studied.  
  • Similarly, if ATP, oxygen, or exogenously administered luciferin is not abundantly present, light emission may not be a true representation of luciferase activity. This may be the case when tumours have become necrotic.  
  • Substrate availability may also be influenced by the administration route (intraperitoneal vs intravenous).  
  • When assessing tumorigenesis, one may have to keep in mind that bigger tumors usually have higher uptake of substrate; this could lead to higher signal and false data interpretation.  
  • On the other side, high metabolic activity of cancer cells and the tumour microenvironment can also lead to higher bioluminescent signal.  
  • Luciferase reporters that are commonly used in research exhibit a 10-fold decrease in sensitivity per cm of depth, meaning that BLI is usually most suitable and accurate when imaging small laboratory animals such as mice.  

Application of Bioluminescent Imaging in Oncology Research 

As we have summarised above, as BLI offers quantitative, sensitive, and longitudinal real-time data acquisition, it is the in vivo modality of choice in preclinical oncology research. BLI has been broadly applied in oncology field can be applied to study fundamental cancer mechanisms (tumourigenesis, metastasis, angiogenesis, hypoxia, cancer cell metabolism) but to also evaluate the response of tumours to established and novel therapies. Here, we focus on the application of BLI in preclinical oncology field and drug development, with special focus on preclinical cancer models.  

Examples of studies in animal models of cancers that benefit from preclinical BLI are summarised below:  

  • Validation of cancer cell lines, helping to establish improved preclinical cancer mouse models.  
  • Transgenic animal models carrying the specific luciferase reporter genes.  
  • Animal models of cancers whereby cancer cells are transduced virally to the animal.  
  • Syngeneic (rodent cell lines) and/or xenograft (human cell lines) tumour models, offering improved sensitivity to traditional calliper measurements in helping detect (early) tumour growth regression following drug treatment.   
  • Orthotopic tumour models, enabling assessment of tumour growth in a model with relevant tumour microenvironment in mouse models of colorectal cancer, lung cancer, glioblastoma etc.,   
  • Metastatic tumour models, enabling the sensitive assessment and extent of metastasis in a various range of cancer types.  
  • Studies used to evaluate novel (immuno)therapies such as CAR-T cells, NK cells, checkpoint inhibition, combination and radiation therapies.   
  • Studies evaluating novel therapy’s mechanism of action complemented with immune cell profiling via flow cytometry.   

Bioluminescence Imaging to Monitor Tumour Growth and Metastasis

In preclinical oncology research, BLI is frequently used to monitor therapy’s efficacy in reducing tumour growth and metastasis in human cell-line derived tumour xenograft models and syngeneic cell-line derived metastatic or orthotopic models. To do this, tumour cells (both commonly used rodent and human tumour cell lines) are engineered to express luciferase and cultured to obtain necessary number of cells needed to inject into the animal. Following injection of luciferase expressing tumour cells (orthotopically or subcutaneously), tumorigenesis is followed-up at pre-determined times to track the efficacy of novel test compounds on either tumour growth and/or metastasis. To obtain the BLI signals at pre-determined times, the now-cancer cell injected animals are injected with luciferin substrate to allow implanted cells to bioluminescence and anesthetised to ensure that there are no movement artifacts during imaging. Following substrate injection, imaging can be started quickly due to the fast biodistribution kinetics of the substrate.  

Below are some examples of tumour models evaluated at InnoSer. To inquire about other cancer histotypes, or if you’re unsure which one is the most appropriate for your application, please reach out to our team.  

Example data: Bioluminescent Imaging in Colorectal Cancer Orthotopic Model (MC-38 colorectal cancer cell line)

MC38 colorectal carcinoma model represents a common anti-PD-1-responsive tumour model. Disease progression is assessed via observation of clinical signs, body weight and tumour volume measurements.

This model is available as both subcutaneous and orthotopic (MC-38 luc) model at InnoSer. We have previously shown and confirmed the findings that this model shows efficient growth kinetics and response to anti-PD1 therapy, resulting in a model with long dosing window to test novel immunotherapy candidates.

Injecting the MC-38 colorectal carcinoma cells orthotopically provides a valuable model of studying novel immunotherapies responses in a model with relevant tumour microenvironment (Figure 3).  

MC38 colorectal carcinoma orthotopic cancer model BLI measurement of tumour growth

FIGURE 3. BLI allows longitudinal measurement of tumour progression in the orthotopic colorectal carcinoma (MC38-luc) model. BLI images show tumour progression in a subset of two mice.  

Example data: Bioluminescence Imaging in Breast Cancer Orthotopic Model (4T1 breast cancer cell line)

4T1 breast carcinoma model represents a commonly used, highly tumorigenic model used to evaluate novel immunotherapies against breast cancer.

At InnoSer, we offer this model both as a subcutaneous model (cells are injected into the flank) and orthoptic model (cells are injected into the mammary fat pad; Figure 3).  

FIGURE 4. BLI allows longitudinal measurement of tumour progression and distant metastases in the breast cancer carcinoma (4T1) mouse model. BLI images show representative images of tumour progression.  

Application of bioluminescence imaging beyond oncology research – conclusion

To conclude our article, we have provided a brief summary of the value that BLI holds in preclinical research, with a special focus of animal models of cancer and drug development space. We have provided example studies which the experts at the InnoSer team have carried out. To inquire about performing a potential study at InnoSer we invite you to contact us via the contact form below. To view any other relevant material such as InnoSer’s capabilities in the general drug development space spanning from target identification and validation to pre-IND services, as well as InnoSer’s therapeutic expertise in immuno-oncology, we invite you to click on the respective hyperlinks.