32 Fendos and Engelman: pHLIP and acidity as universal biomarker for cancer Figure 2. Intravenously and intraperitoneally injected pHLIP-infrared dye conjugates localize preferentially to tumors. A) o verlay of NIr fluorescence and X-ray images obtained the day after injection of 500µg/kg pHLIP-c y5.5 conjugate (right) or an equivalent amount of free c y5.5 (left) into mice bearing tumors in the right flank. Peptide injection was performed on the sixth day after an initial cancer-cell implantation of 5x104 cells subcutaneously. Localization of the fluo- rophore is seen in the tumor, kidneys, and spleen. B) pHLIP-Alexa750 fluorescence (green) in a mouse bearing a tumor in the right flank (6 days after 106 cell implant) imaged the day after peptide injection. r eflectance is denoted in red. Localization of the fluorophore is seen in the tumor (arrow), kidneys, and spleen. C) pHLIP-c y5.5 given as a single injection (200µg/kg) into the left side of mice initially diffuses, accumulating in the right flank tumor by 20 hours. Blue represents background and red represents the highest fluorescence. D) 3D representations of pHLIP-c y5.5 fluorescence from a tumor-bearing mouse at different days after injection of 500µg/kg peptide. Peptide injection was done on the seventh day after subcutaneous tumor im- plantation. The height (z axis) and intensity of red indicates the strength of NIr signal, showing that pHLIP retention lasts for several days. e ) The NIr fluorescence of kidneys 2 days after in- jection of 500µg/kg pHLIP-Alexa750 into mice fed water (left) or 80 mm NaHc o 3 at pH 8.2 (right). The alkaline treatment resulted in an approximate 50 percent reduction of pHLIP kidney retention without affecting tumor localization. Figures reprinted from [15]: Andreev o A, et al. mecha- nism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo. Proc Natl Acad Sci USA. 2007;104(19):7893-8, reused according to permissions guidelines. The pHLIP conjugate did, however, ex- hibit some off-site localization [15]. The most prominent off-site localization was found in the kidneys (Figure 2A-B), where the proximal tubule region is particularly well labeled (Figure 2E). This labeling is consistent with the fact that our conjugates are small enough to enter through the glomerular fenestrae and be treated as fil- trate. Since the proximal tubules of the kid- neys are acidified to drive sodium ions across the membrane, our conjugates are likely re- sponding to this acidity, inserting into kidney cells and being retained in the tubule tissue. We do not know exactly in what part of the cells this retention occurs, but we did find that feeding the mice carbonated water helps to alleviate kidney labeling (Figure 2E) with-