Compared to the traditional method of euthanizing animals in batches to obtain experimental data, real-time bioluminescence imaging (BLI) technology allows for qualitative and quantitative observations at the cellular and molecular levels of a living organism. In the process of developing and analyzing in situ tumor CDX models, live imaging is more conducive to tracking disease progression and tumor metastasis, making dynamic monitoring of tumor models less restrictive, low-cost, and even noninvasive — all while providing a greater range of real-time research data for your experimental analysis.

In Vivo Bioluminescent Imaging Technology

In order to visualize and track potential anti-tumor therapies throughout the model organisms with in vivo imaging, researchers often use fluorescence-based optical tagging methods. Such optical labeling methods may be implemented to visualize and provide contrast for the tumor, health tissues, and even potential therapies. Fluorescent labeling works with large biological macromolecules, but has limitations when it comes to small molecule drugs. Small molecule drugs require a method of labeling that doesn’t impact the biodistribution of these drugs as fluorescent tags would, thereby relying on radioactive isotope labeling imaging (PET or SPECT).

The commonly used optical labeling methods for in vivo tumor imaging include:

1. Using Firefly Luciferase or fluorescent proteins as reporter genes: Tumor cells are labeled in vitro through transgenic techniques, allowing direct observation of changes in tumor progression or studying the role of specific genes in tumor development.

2. Via injection of exogenous functional fluorescent probes: Molecular events during tumor development can then be observed,  thereby reflecting the changes in tumor development.

Applications of In Vivo Imaging in Tumor Research & Oncology

In a macroscopic context, the application of small animal live optical imaging technology in tumor research primarily focuses on:

1. Long-term monitoring of tumor growth and metastasis;

2. Development of anti-tumor drugs;

3. Research into the molecular mechanisms and pathology of cancer.

In addition to clinical pharmacodynamics evaluations, this technology is also widely used in the preclinical study of drug targeting, distribution, and metabolism in the body. Such applications involve directly observing drugs, typically by labeling the drugs themselves with fluorescent probes and tracking the fluorescent signals to reflect the biodistribution of the drugs over time. For instance, in studying whether antibodies or peptide-based drugs can effectively target tumors, fluorescent dyes can be chemically attached to the target antibodies or peptides, and their tumor-targeting ability can be observed using real-time in vivo optical imaging.

Currently, the use of live fluorescence imaging technology for observing drug distribution is primarily limited to the study of large biological macromolecule drugs, whereas the distribution and metabolism of natural or small molecule drugs mainly rely on radioactive isotope labeling imaging (PET or SPECT). This is because labeling small molecule drugs with relatively large molecular weight fluorescent dyes can affect the distribution and metabolism of the drugs themselves, making it unsuitable for the application of live fluorescence imaging technology in such studies.

A Review of In Vivo and In Vitro Bioluminescence Experiments

Two common techniques used in small animal live imaging are bioluminescence and fluorescence. Bioluminescence imaging (BLI) involves labeling cells or DNA with luciferase genes and the luciferase enzymes produced by these genes naturally catalyze oxidation of the corresponding substrate (luciferin), producing photons of light which may then be visualized by the imaging equipment.

In Vitro Bioluminescence

Preparation of bioluminescence assay luciferin reagent: D-luciferin sodium salt is prepared as a 100 mM stock solution (200x), but it can also be prepared according to differential experimental requirements. Dissolve D-luciferin in prewarmed tissue culture medium to prepare a working solution with a concentration of 150 μg/ml. Dilute the stock solution 1:200 with tissue culture medium to prepare the working solution (final concentration 150 μg/mL).

Remove the culture medium from the cultured cells. Count the cells and adjust the cell concentration to 100,000 cells/ml (10,000 cells/100μl). Before image analysis, add 1× luciferin working solution to the cell culture plate, and then proceed with image analysis.

*Note: Short-term incubation of cells at 37°C before imaging can enhance the signal.

In Vivo Bioluminescence

Preparation of bioluminescence assay luciferin reagent: Prepare a working solution of D-luciferin potassium salt in sterile phosphate-buffered saline (PBS, 15mg/mL) and filter through a 0.2μm membrane filter to sterilize. Administer 150 mg/kg of luciferin working solution at a dose of 10 μL/g (e.g., inject 100μL of the working solution into a 10g mouse, giving 1.5mg of luciferin). Perform imaging 10-15 minutes after intraperitoneal injection.

Intraperitoneal injection procedure: Secure the animal with your left hand, abdomen facing up, and the animal's head downward. Gently shake the mouse 2-3 times to move undigested food in the intestines downward, creating an empty space in the lower quarter of the abdomen. Disinfect the injection site with 75% ethanol. Hold the syringe with your right hand, insert it into the mouse's abdomen, and inject at a point on the mouse's abdomen located 1.5cm to the side along a line drawn from the base of the hindlimb. Slowly insert the needle at an angle of about 45 degrees, with a needle insertion depth less than 1 cm. You will feel a slight resistance as the needle penetrates, and then quickly notice the sensation of the local skin depression as the needle disappears and injection may proceed.

Key Points for In Vivo Fluorescence Imaging Experiments with Mice

For mice with fur, it is recommended to shave them the day before imaging experiments to minimize background fluorescence from fur and other interference. The background fluorescence of mouse fur is particularly noticeable under short-wavelength excitation wavelength light, as seen in the two ICR mice in Figure 1 (white fur). The left mouse has been shaved, and the right one has not. Under 465 nm excitation light, the background fluorescence of the fur is clearly visible. It is generally advised to use a mouse shaver, as depilatory creams may contain aromatic ingredients that could introduce background fluorescence that interferes with data acquisition. Additionally, depilatory creams and chemicals like sodium sulfide can potentially cause burns on the mouse's skin, and these burns can sometimes be accompanied by strong background fluorescence.

Figure 1. The left mouse has been shaved, and the right one has not. Under 465 nm excitation light, the background fluorescence of the fur is clearly visible.

It is advisable to wipe the mouse's mouth, nose, paws, and urinary area with damp gauze soaked in warm water before imaging, as these areas often exhibit unnecessary background fluorescence. During fluorescence spectrum separation, weak signals that are difficult to identify can sometimes be a challenge. To address this, you can follow the experimental design as shown in Figure 2, which includes control mice, recipient mice, and a positive dye control.

Figure 2. Control mice, recipient mice, and a positive dye control.

Noise Issues with In Vivo Imaging

Background Light on the Sample

Oftentimes, the environmental background contains phosphorescent materials which create background phosphorescence and noise on the imaged sample. Problematic materials include plastics, pigments, organic substances, plastic ropes, and some plastic containers; certain contaminants, including animal urine, can also be a source of phosphorescence.

Background Light from the Sample

Background light from the sample refers to the natural emission of light from the sample, which is different from the signal that the sample is being detected for. Cell culture media may produce phosphorescence, and these need to be scanned before use to identify and eliminate problematic materials. This inherent emission of light, known as spontaneous bioluminescence, is always present in living animals and exists in the primary detection regions of in vivo bioluminescence imaging. Most animals exhibit low levels of spontaneous luminescence, and it typically only becomes problematic when detecting weak signals with high sensitivity.

Approximate background values, which may be obtained from similar control group animals or from non-detection areas of the mice being tested, can be subtracted from region-of-interest (ROI) measurements.

For in vivo applications, it is best to use a wavelength range above 600 nm. Light below 600 nm is easily absorbed by animal tissues, which limits the depth of light penetration. Fluorescent groups at depths of several millimeters or more may not be able to absorb the excitation light. Additionally, at wavelengths below 600 nm, tissue autofluorescence increases.

Preclinical Orthotopic Modeling and Analysis Solutions by Cyagen

In the field of orthotopic transplantation, there have been successful cases across multiple cancer types, including lung, colon, and liver cancers. Cyagen has successfully transplanted over 200 human cell lines into immunodeficient mice, including various implantation sites such as subcutaneous, intravenous, and orthotopic/in situ. With the support of high-sensitivity equipment like the PE Lumina III small animal in vivo imaging system, it is possible to conduct pharmacological experiments for therapies such as CAR-T, as well as to monitor the growth of orthotopic tumor models and the in vivo distribution of fluorescent nanomedicines.

We can perform custom cell and animal model development through preclinical analysis to jump-start your research — contact us today for a complimentary project consultation.