Introduction The goal of this study was to examine whether 99mTc-labeled Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (-MSH) cross peptide targeting both melanocortin-1 (MC1) and v3 integrin receptors was superior in melanoma targeting to 99mTc-labeled -MSH or RGD peptide targeting only the MC1 or v3 integrin receptor. uptake of 99mTc-RGD-Lys-(Arg11)CCMSH was 2.49 and 2.24 times (p 0.05) the melanoma uptakes of 99mTc-RAD-Lys-(Arg11)CCMSH and 99mTc-RGD-Lys-(Arg11)CCMSHscramble at 2 h post-injection, respectively. Either RGD or (Arg11)CCMSH peptide co-injection could block 42% and 57% of the tumor uptake of 99mTc-RGD-Lys-(Arg11)CCMSH, whereas the co-injection of RGD+ (Arg11)CCMSH peptide mixture could block 66% of the tumor uptake of 99mTc-RGD-Lys-(Arg11)CCMSH. Conclusions Targeting both MC1 and v3 Dinaciclib tyrosianse inhibitor integrin receptors enhanced the melanoma uptake of 99mTc-RGD-Lys-(Arg11)CCMSH in M21 human melanoma xenografts. Flank M21 human melanoma tumors were clearly visualized by SPECT/CT TIAM1 imaging using 99mTc-RGD-Lys-(Arg11)CCMSH as an imaging probe, highlighting its potential use as a dual-receptor-targeting imaging probe for human melanoma detection. Competitive Binding Assay The IC50 values of RGD-Lys-(Arg11)CCMSH, RAD-Lys-(Arg11)CCMSH and RGD-Lys-(Arg11)CCMSHscramble for the MC1 receptor were determined according to our previously published procedure  with modifications. Briefly, the M21 cells were harvested Dinaciclib tyrosianse inhibitor and seeded into a 24-well cell culture plate (1.5 105 cells/well) and incubated at 37 oC overnight. After being washed with binding medium Modified Eagles medium with 25 mM em N /em -(2-hydroxyethyl)-piperazine- em N /em -(2-ethanesulfonic acid), pH 7.4, 0.2% bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline, the cells were incubated at 37 oC for 2 h with approximately 30,000 counts per minute (cpm) of 125I-(Tyr2)-NDP-MSH in the presence of increasing concentrations (10?12 to 10?5 M) of each peptide in 0.3 mL of binding medium. The reaction medium was aspirated after the incubation. The cells were rinsed twice with 0.5 mL of ice-cold pH 7.4, 0.2% BSA / 0.01 M phosphate buffered saline (PBS) and lysed in 0.5 mL of 1 1 N NaOH for 5 minutes. The radioactivities associated with cells were measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ). The IC50 values of the peptides for the MC1 receptor were calculated using the Prism software (GraphPad Software, La Jolla, CA). The IC50 values of RGD-Lys-(Arg11)CCMSH, RAD-Lys-(Arg11)CCMSH and RGD-Lys-(Arg11)CCMSHscramble for the v3 integrin receptor were determined according to the published procedure  with modifications. The M21 cells were harvested, washed twice with PBS, and resuspended (2 106 cells/mL) in binding buffer (20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 1 mmol/L MnCl2, 0.1% bovine serum albumin). The M21 cells (1 105 cells/well) were seeded in Millipore 96-well filter multiscreen DV plates (0.65 m pore size) and incubated at 25 oC for 2 h with approximately 30,000 cpm of 125I-Echistatin in the presence of increasing concentrations (10?11 to 10?4 M) of each peptide in 0.2 mL of binding medium. Following the incubation, the plates had been filtered through a multiscreen vacuum manifold and rinsed double with 0.5 mL of ice-cold pH 7.4, 0.2% BSA / 0.01 M PBS. The hydrophilic polyvinylidenedifluoride (PVDF) filter systems had been collected as well as the radioactivities had been measured inside a Wallac 1480 computerized gamma counter (PerkinElmer, NJ). The IC50 ideals from the peptides for the v3 integrin receptor had been determined using the Prism software program Dinaciclib tyrosianse inhibitor (GraphPad Software program, La Jolla, CA). Peptide Radiolabeling RGD-Lys-(Arg11)CCMSH, RAD-Lys-(Arg11)CCMSH and RGD-Lys-(Arg11)CCMSHscramble had been radiolabeled with 99mTc with a glucoheptonate transchelation response using methods referred to previously . Quickly, 100 L of 2 mg/mL SnCl2 in 0.2 M glucoheptonate aqueous solution and 200 L of refreshing 99mTcO4- solution (37C74 MBq) had been added right into a response vial Dinaciclib tyrosianse inhibitor and incubated at space temperatures (25 C) for 20 min to create 99mTc-glucoheptonate. After that, 10 L of just one 1 mg/mL each peptide aqueous option was added in to the response vial as well as the pH from the response blend was modified to 8.5 with 0.1 M NaOH. The response blend was incubated at 75 C for 40.