Serum‐Inert Tandem‐Locked Fluorescent Probe for Specific Imaging of Intratumoral Complement System

The complement system has recently been recognized to affect cancer initiation and progression. However, because its activity depends on a complex cascade of interacting proteins, conventional static analytical methods are inadequate for tracking its dynamic behavior within tumors, leading to ambiguous conclusions about its role in the tumor immune microenvironment. Although real-time imaging would be a superior approach, the abundant presence of complement proteins in serum and the lack of specific responsive substrates pose significant challenges for developing molecular optical probes. Herein, we synthesize a tandem-locked fluorescence probe for non-invasive monitoring of the intratumoral complement system. A substrate with high C1r specificity is identified via screening different peptide sequences and comparing their enzymatic kinetics toward C1r and tumor-overexpressed cathepsin B (CTSB). To prevent nonspecific activation by circulating serum complement proteins, the arginine residue within the C1r substrate is masked by a CTSB-cleavable peptide substrate, yielding the tandem-locked complement probe TECP_CTSB. TECP_CTSB remains silent in the blood circulation and emits a signal only where both CTSB and C1r are active. Accordingly, TECP_CTSB permits in vivo imaging of intratumoral complement activity and can be used as a flow cytometry reagent in conjunction with dye-labeled antibodies to profile C1r expression in the cells of tumor tissue, which was not possible before. The imaging results reveal a higher intratumoral complement activity in 4T1 tumors than in CT26 tumors, which could be part of the cause for the higher infiltration of myeloid-derived suppressor cells in 4T1 relative to CT26 tumors, and consequently a more severe immunosuppressive microenvironment. The flow cytometry profiling shows that complement activation-associated fluorescence predominates in cancer-associated fibroblasts, followed by tumor cells. This is consistent with the observation that the TECP_CTSB fluorescence is mainly localized at the tumor periphery, which, however, extends into the tumor interior after chemotherapy, probably due to enhanced complement activation induced by chemotherapy-mediated apoptosis. These findings provide new insights into intratumoral complement systems and underscore the potential of the optical probe for complement system research and clinical diagnostics.