Application to CAR T-cell Cancer Therapy Research

Chimeric Antigen Receptor (CAR) T-cell therapies are transforming cancer treatment, yet preclinical evaluation still faces key challenges: monitoring tumor burden and tracking therapeutic T-cell dynamics often require separate assays or imaging sessions. This can increase variability, costs, and animal use.

Dual near-infrared (NIR) bioluminescence imaging with Infraluciferin overcomes these limitations by allowing both CAR T-cells and their cancer targets to be tracked and quantified simultaneously in the same animal.

This is made possible because Infraluciferin emits different wavelengths depending on the luciferase variant it reacts with. By pairing different luciferase enzymes with T-cells and tumor cells, two distinct “colors” of NIR bioluminescence can be captured in a single imaging session.

Advantages of Infraluciferin in CAR T-cell Research

1. Direct assessment of therapeutic activity
   Researchers can monitor the killing of cancer cells by CAR T-cells within living mice, enabling more accurate and dynamic evaluation of therapeutic performance. This dual readout accelerates preclinical testing of CAR T constructs across different tumor models and disease settings (Stowe et al., 2019).

2. Improved accuracy with far-red to NIR imaging
   Infraluciferin emits in the far-red to NIR range, which penetrates tissue more effectively than visible light. This results in higher sensitivity, better quantification of cell populations, and improved image resolution—crucial for evaluating therapy outcomes in deep tissue environments.

3. Reduced animal usage
   Because two biological parameters can be imaged within the same mouse, dual NIR bioluminescence has the potential to halve the number of animals required for CAR T-cell imaging studies. This represents not only a cost reduction but also an important step toward more ethical and efficient preclinical research.

4. Testing CAR T-cell safety switches
   Dual imaging is also ideal for evaluating engineered safety mechanisms such as “suicide switches.” Researchers can track the function of CAR T-cells alongside tumor response, providing a non-invasive method to confirm safety features in vivo.

Workflow: Dual NIR Imaging in CAR T-cell Research

1. Engineer and label cells

   • Tumor cells are engineered to express one variant of luciferase (e.g., red-shifted).

   • CAR T-cells are engineered to express a different luciferase variant (e.g., NIR-shifted).

2. Establish mouse model

   • Tumor cells are implanted into the mouse.

   • CAR T-cells are introduced (e.g., by infusion).

3. Administer Infraluciferin substrate

   • Bioflares’ Infraluciferin methyl ester is delivered systemically.

   • Once inside cells, esterases convert it into the active substrate.

4. Capture dual bioluminescence signals

   • Tumor cells and CAR T-cells emit distinct NIR signals.

   • Imaging systems detect and quantify both signals non-invasively.

5. Analyze therapeutic response

   • Monitor CAR T-cell expansion, persistence, and trafficking.

   • Track tumor regression (or relapse) in the same animal.

   • Assess safety features such as CAR T-cell “suicide switches.”

Advantages for CAR T-cell Studies

• Real-time efficacy assessment – Quantify tumor killing by CAR T-cells dynamically in living animals (Stowe et al., 2019).

• Superior accuracy – Far-red to NIR imaging penetrates deeper, improving quantification and resolution.

• Ethical and cost benefits – Dual imaging can halve animal usage by combining readouts into a single study.

• Safety validation – A powerful way to test built-in CAR safety mechanisms in vivo.

Summary

By combining Infraluciferin’s color-shifting properties with engineered luciferase variants (iLuc), dual NIR bioluminescence provides an unprecedented window into CAR T-cell therapy. Researchers can track tumor burden and immune activity together—non-invasively, longitudinally, and with high sensitivity—accelerating preclinical testing and improving translational outcomes.