Speaker
Descrizione
Background: Cancer theranostics integrates molecular imaging and targeted radionuclide therapy using matched radiopharmaceutical pairs with identical molecular scaffolds labeled with diagnostic radionuclides such as fluorine-18 or gallium-68, and therapeutic emitters such as lutetium-177 [1]. Recent advances have led to the clinical translation of selective radiotracers exploiting differential protein overexpression between malignant and normal tissues [2]. However, monospecific agents often show limited efficacy due to intratumoral heterogeneity and dynamic tumor microenvironment interactions [3]. To overcome these constraints, heterodimeric radiotracers capable of simultaneously targeting two molecular determinants have been developed, improving specificity, uptake, sensitivity, and retention [1], [4], [5]. Among emerging targets, Fibroblast activation protein and Carbonic anhydrase IX are particularly promising tumor microenvironment associated targets for diagnostic and therapeutic applications [6], [7]. Despite their translational potential, preclinical evaluation of dual-targeted radiotracers remains challenging due to the limited availability of in vitro models that endogenously co-express both proteins at levels and patterns comparable to those observed in human tumors. Additionally, in vitro characterization of monomeric counterparts is critical to establishing a robust benchmark dataset while simultaneously highlighting critical biological and methodological challenges to be addressed in the design and evaluation of heterodimeric radiotracers.
Methods: We systematically assessed FAP and CAIX expression across a panel of cell lines to identify a biologically relevant dual-positive in vitro model. Monomeric radiotracers [68Ga]Ga-DOTA-FAPI-04 and [68Ga]Ga -DOTA-CAI were characterized for cellular uptake, specificity, and internalization. Binding assays, molecular dynamics simulations, and enzymatic inhibition studies were performed for CAIX-selective compound to elucidate the molecular basis of its interaction with carbonic anhydrase isoforms.
Results: U87MG cells were identified as the most suitable dual-positive model, exhibiting endogenous co-expression of FAP and CAIX. [68Ga]Ga-DOTA-FAPI-04 displayed specific uptake and predominant internalization in FAP-positive cells correlating with FAP expression. In contrast, in vitro studies of [⁶⁸Ga]Ga-DOTA-CAI, including uptake, blocking, and saturation assays, did not demonstrate CAIX-specific or saturable binding. Molecular dynamics simulations predicted stable interactions with CA II, IX, and XII and indicated that gallium complexation does not affect the sulfonamide-zinc anchoring mechanism. Enzyme inhibition assays confirmed nanomolar potency against tumor-associated CA IX and XII, showing preserved intrinsic affinity. In contrast, direct radiotracer binding assays showed limited association with tumor-associated isoforms, which was attributed to aggregation of recombinant CA IX and XII under the assay conditions.
Conclusions: This study identified a biologically relevant co-expressing cellular model and characterized monomeric FAPI- and CAI-based radiotracers, establishing a reference dataset for heterodimer evaluation. Future work will focus on further validation and optimization of CAIX-targeted tracer
