Secreted luciferases as a minimally invasive 3R-compliant tool for accurate monitoring of tumor burden.

Preclinical mouse models are indispensable in cancer research, providing insights into tumor biology and therapeutic responses. This protocol describes a minimally invasive blood-based tumor monitoring approach using secreted luciferases for longitudinal tracking of tumor burden in transplantable xenografts and genetically engineered mouse models. Unlike intracellular luciferases used in bioluminescence imaging, secreted luciferases are actively released into circulation, enabling precise quantification from microliter-scale blood samples. We describe a transplantable model, where tumor cells are labeled in vitro using lentiviral transduction before engraftment. Orthogonal secreted luciferases enable multiplexed analysis of distinct tumor populations within a single host, reducing animal numbers and enhancing data density. We also describe an autochthonous lung cancer model, where intratracheal adenoviral delivery of Cre recombinase and CRISPR nucleases induces tumorigenesis through somatic genome editing while activating a conditional secreted luciferase reporter transgene. Tumor-bearing mice undergo routine blood sampling, with luciferase activity measured ex vivo to quantify viable tumor burden. Compared to imaging techniques, this method eliminates anesthesia and contrast agents, minimizing animal stress and enabling frequent monitoring with superior temporal resolution and reduced logistical complexity. The protocol requires only standard molecular biology skills and basic mouse handling expertise. While tumor labeling and growth duration is model dependent, blood sampling requires ~5 min per animal, with all samples from one cohort processed and measured together within 2 h. This approach provides an accessible, cost-effective and scalable alternative to imaging-based tumor monitoring, that is aligned with the 3Rs principles, offering a powerful and ethically sound platform for preclinical cancer research.