Challenges in tracer development for tumor microenvironment (TME) imaging.

Molecular imaging of the tumor microenvironment (TME) has emerged as a powerful approach to non-invasively interrogate immune cell composition, activation state and functional dynamics, thereby addressing key limitations of biopsy-based biomarkers in immuno-oncology. Early clinical successes, particularly in immune checkpoint and CD8⁺ T-cell imaging, underscore the translational potential of this strategy. However, the development of robust and clinically relevant TME-targeted radiopharmaceuticals is confronted with a distinct set of biological, technical and methodological challenges that fundamentally differentiate it from classical tumor imaging. These include low and dynamic target expression, tight coupling between tracer affinity, molar activity and receptor occupancy, limited suitability of standard in vitro assays, increasing complexity of in vivo models, and heightened vulnerability to pitfalls in image interpretation and quantification.This review provides a comprehensive analysis of these challenges across the tracer development pipeline, spanning target selection, molecular format choice, radiochemistry, preclinical evaluation and data analysis. Rather than representing prohibitive obstacles, these limitations delineate the biological and technical boundary conditions within which TME imaging strategies must be developed and interpreted. Emerging solutions include the growing role of peptides and engineered antibody fragments, stepwise and question-driven evaluation workflows, harmonized preclinical models and integrated targeting concepts that align biological relevance with technical feasibility. Together, these considerations provide a framework for sustainable and translational progress in TME imaging.