For example, BLI cannot be employed in tumor models lacking expression of bioluminescent reporters, such as in spontaneous- or chemicalinduced cancer models. Moreover, the introduction of a foreign reporter protein, such as luciferase, may induce an immune response directed against the reporter protein itself in syngeneic mouse models of cancer. SKI II Finally, for clinical applications, such as image-guided surgery, BLI is also not applicable as it requires genetic modification of the targeted cells. Recent advances in the development of tumor-specific injectable near-infrared fluorescent probes make FLI a promising alternative to BLI. The use of NIRF probes has several advantages. For example, in the near-infrared region, the absorption coefficient of major light absorbers in tissues is minimal, which improves the photon penetration depth. In this 4T1-luc2 model, we first examined the tumor detecting abilities of four different NIRF probes, which are able to detect general tumor cell characteristics. These include: increased expression of growth factor receptors, elevated glucose metabolism and up-regulated glucose transporters and an increased tissue proteolysis by the tumor, through upregulation of proteolytic enzymes such as MMP-2 and cathepsin B. In vitro, we examined the activity and binding properties of the four NIRF probes towards 4T1-luc2 cells and observed a dosedependent uptake of each probe. This indicates that 4T1-luc2 cells express both GLUTs and EGFRs and also possess cathepsin activity in vitro. This is in line with previous studies showing that several mammary carcinoma cells, including 4T1 cells, express GLUTs and EGFRs as well as MMP and cathepsin activity. Using multispectral microscopy, we demonstrated that the activatable as well as the targeting probes were localized intracellularly. Consistently, Kovar et al. previously showed that IRDye CW800 2-DG enters the cytoplasm via internalization of the probe/receptor complex shortly after binding to its receptor. In vivo, tumor development and progression was studied using BLI and FLI, exploiting dual-wavelength imaging of a combination of an activatable and a targeting NIRF probe. Tumors could be detected at an early stage of the development and a strong linear correlation between FLI and BLI measurements was observed for all probes tested. Metastases, present in the lungs, could only be detected ex vivo both by BLI and FLI and Zebularine signals co-localized. Histological examination confirmed the presence of subpleural lung metastases. To further examine the specificity of the NIRF probes, immunohistochemistry was performed. Expression of GLUT-1, EGFR, MMP-9 and Cathepsin B was found throughout the tumor and the expression patterns closely corresponded to those obtained with FLI. As can be appreciated from these immunohistological and FLI data, the distribution of the examined probes is rather inhomogeneous in this breast tumor model. The strong immunostained areas of each marker coincided with the more intense fluorescent areas. Thus, by using dual-wavelengths, combined with spectral unmixing, it is possible to detect multiple tumor features simultaneously. This may provide a more comprehensive image of the tumor, as different types of tumors are often heterogeneous in structure and molecular characteristics. Previous studies have documented the importance of genetic and epigenetic alterations of oncogenes, tumor suppressor genes and mismatch repair genes in the development of gastric cancer.