Integrins, over-expressed in a broad range of cancer diseases, are widely utilized as a tumor biomarker. Metabolism investigation also plays important roles in tumor theranostics. Developing simple integrin-targetting probe and monitoring tumor metabolism will give opportunities to find ways for cancer treatment, however, the investigation of tumor metabolism with integrin receptor based probes has been rarely reported so far. Here, we developed an octavalent fluorescent probe Octa-RGD by convenient genetic method, based on one tetrameric far-red fluorescent protein (fRFP) linked with RGD peptides. We validated its intergin targeting by confocal imaging in vitro. Then we screened a variety of tumor cells, and differentiated their binding affinity based on the fluorescence of the probe via flow cytometry. Among these cells, CNE-2 cells had the highest uptake of the probe, while B16 cells had the lowest, corresponding with their intergin expression levels. Next, the fluorescent and metabolic imaging was performed in HT1080 (intergin postive) tumor, where nicotinamide adenine dinucleotide hydrogen (NADH), flavoprotein (Fp) and fRFP fluorescent signals were collected. The tumor from mice intravenously injected with Octa-RGD probe displayed obviously higher NADH redox ratio NADH/ (FptNADH) and fRFP signal, than those with fRFP protein. It suggested that integrin targeting may have influence on the target cell metabolism, and further demonstrated Octa-RGD probe facilitated its uptake in the targeted tumor in vivo. This paper developed a useful probe, which can bind integrins specifically and efficiently in tumor cells, and together with tumor metabolic information, it may provide new insight for RGD targeting-based cancer therapeutics.

Photodynamic therapy (PDT) gains wide attention as a useful therapeutic method for cancer. It is mediated by the oxygen and photosensitizer under the specific light irradiation to produce the reactive oxygen species (ROS), which induce cellular toxicity and regulate the redox potential in tumor cells. Nowadays, genetic photosensitizers of low toxicity and easy production are required to be developed. KillerRed, a unique red fluorescent protein exhibiting excellent phototoxic properties, has the potential to act as a photosensitizer in the application of tumor PDT. Meantime, the course of tumor redox metabolism during this treatment was rarely investigated so far. Thus here, we investigated the effects of KillerRed-based PDT on tumor growth in vivo and examined the subsequent tumor metabolic states including the changes of nicotinamide adenine dinucleotide hydrogen (NADH) and flavoprotein (Fp), two important metabolic coenzymes of tumor cells. Results showed the tumor growth had been significantly inhibited by KillerRedbased PDT treatment compared to control groups. A home-made cryo-imaging redox scanner was used to measure intrinsic fluorescence and exogenous KillerRed fluorescence signals in tumors. The Fp signal was elevated by nearly 4.5-fold, while the NADH signal decreased by 66% after light irradiation, indicating that Fp and NADH were oxidized in the course of KillerRedbased PDT. Furthermore, we also observed correlation between the fluorescence distribution of KillerRed and NADH. It suggests that the KillerRed protein based PDT might provide a new approach for tumor therapy accompanied by altering tumor metabolism.