It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. IDO1 regulation in the mesenteric lymph nodes (MLNs), an off-tumor IDO1 target, where the percentage uptake of 11C-l-1MTrp accurately annotated the therapeutic efficacy of multiple combination immunotherapies in preclinical models. Remarkably, 11C-l-1MTrp signal intensity in the MLNs was inversely related to the specific growth rates of treated tumors, suggesting that IDO1 expression in the MLNs can serve as a new biomarker of the cancer-immune set point. Conclusions PET imaging of IDO1 with 11C-l-1MTrp is a robust method to assess the therapeutic efficacy of multiple combinatorial immunotherapies, improving our understanding of the merit and challenges of IDO1 regimens. Further validation of this animal data in humans is ongoing. We envision that our results will provide a potential precision medicine paradigm for noninvasive visualizing each patients individual response in combinatorial cancer immunotherapy, and tailoring optimal personalized combination strategies. expression patterns. When the tumors grew to a maximum tumor diameter of 9C12?mm, the mice were selected randomly for further studies. PET/CT scans Whole-body dynamic PET scans were performed after intravenous injection of 11C-l-1MTrp (16.2C19.5 MBq/0.1?mL, corresponding to 0.73C1.45?nmol of l-1MTrp) using a small-animal Siemens Inveon PET scanner (Siemens, Knoxville, Tennessee, USA) to acquire 159 transaxial slices with 0.796?mm (center-to-center) spacing, a 10?cm transaxial field of view (FOV), and a 12.7?cm axial FOV. Emission scans of the tumor models were acquired in three-dimensional list mode with an energy window of 350C650 keV under isoflurane anesthesia from 0 to 75?min after the injection. Ex vivo biodistribution studies After 11C-l-1MTrp (7.1C7.5 MBq/0.1?mL, corresponding to 0.44C0.88?nmol of l-1MTrp) intravenous injection, human xenograft tumor-bearing mice were sacrificed by cervical dislocation at 5, 15, 30, 60, and Mouse monoclonal to Complement C3 beta chain 90?min, while combinatorial immunotherapy-treated syngeneic mice were sacrificed by cervical dislocation at 60?min. Tumors PD153035 (HCl salt) and major tissues (blood, tumor, MLN, heart, lung, thymus, liver, pancreas, spleen, kidney, adrenal, intestine, muscle, testis, epididymis, and brain) were promptly excised, harvested, and weighed. Radioactivity was counted using a -counter, and results are expressed as %ID/g tissue. All radioactivity measurements were corrected for decay. Results Radiotracer 11C-l-1MTrp specifically accumulates in IDO1-expressing human tumors l-1MTrp is a well-known IDO1 inhibitor showing specific binding PD153035 (HCl salt) to the catalytic pocket of IDO1, and has been indicated to inhibit tumor growth in diverse tumor types in vivo (figure 1A).20 Radiotracer 11C-l-1MTrp was synthesized PD153035 (HCl salt) with satisfactory radio-characteristics (figure 1B and C).16 To study the potential of 11C-l-1MTrp to specifically visualize IDO1 in human tumors, we performed 11C-l-1MTrp PET imaging in mice inoculated with human tumors with different IDO1 expression levels. Immunohistological staining for IDO1 in tumor sections indicated that the NCI-H69 small cell lung cancer and MDA-MB231 breast cancer markedly differed in their IDO1 expression levels, with higher IDO1 expression observed in the NCI-H69 than in the MDA-MB231 (figure 1D). This trend was also observed at the mRNA level by qRT-PCR (figure 1E). Open in a separate window Figure 1 11C-l-1MTrp imaging of IDO1 in human tumor models. (A) Protein structure of IDO1 (PDB ID: 2D0U). The blue and green colors indicate the subunits of the IDO1 dimer, and the red color indicates the tryptophan catalytic site. (B) Chemical structure of 11C-l-1MTrp. (C) Radio-characteristics of 11C-l-1MTrp. The half-life indicates the physical half-life of 11C-l-1MTrp. The molar activity and radioactivity were determined at the end of the synthesis (n=80) and showed an averaged synthesis time of 40?min from the end of the bombardment. (D) Immunostaining for IDO1 expression in tumor tissue samples from mice subcutaneously injected with 2.5106 NCI-H69 or MDA-MB231 cells. Green: Alexa Fluor 488-labeled anti-IDO1 antibody. Scale bar: 100?m. (E) Quantification of IDO1 expression in NCI-H69 and MDA-MB-231 cells based on quantitative real-time reverse transcriptase-PCR. (F) Representative PET/CT images of tumor-bearing mice. Tumor positions: NCI-H69 tumor in the left flank and MDA-MB231 tumor in the right flank. Signal lower than 2.2% ID/g weight was subtracted of all PET images. (i) Representative coregistered 11C-l-1MTrp PET/CT image; (ii) Competition 11C-l-1MTrp PET/CT images after a coinjection of the cold (opposite to the radiolabeled 11C-l-1MTrp) IDO1 inhibitor l-1MTrp (50?mg/kg) or (iii) INCB (INCB024360, 10?mg/kg). PET images were summed from 60 min to 75 min after 11C-l-1MTrp injection. (G, H) TimeCactivity curves of 11C-l-1MTrp in (G) NCI-H69 and (H) MDA-MB231 xenografts. (I) Ex vivo biodistribution data collected at 30, 60, and 90?min after 11C-l-1MTrp injection into xenograft mice. All comparisons were performed.