Fendos and Engelman: pHLIP and acidity as universal biomarker for cancer 31 [10], have been used in the last few decades to confirm the observation. Despite a good deal of information on the phenomenon, lit- tle systematic effort has been devised until recently to try to exploit this fundamental characteristic for use as a cancer biomarker. pHLIP AS A CAn Ce r BIo Se n So r On average, tumor tissues have been found to be roughly a half pH unit more acidic than their normal tissue counterparts [4]. This observation has been made for both mouse and human tumors and seems to be a universal trait for all solid tumors examined thus far in the literature. The degree of the pH difference is, of course, variable de- pending on tumor type with adenocarcino- mas typically giving the largest pH differences [9,10], sometimes upwards of 0.7 to 0.8 pH units. Quite by accident, our lab has discovered a small pH-sensitive pep- tide called pHLIP† (pH-Low Insertion Pep- tide) that seems to be sensitive enough to detect these small pH differences in vivo, al- lowing us to label areas of localized acidity in mice. pHLIP was first reported in 1997 [11] when we were using bacteriorhodopsin to study the two-stage model of membrane protein assembly [12]. At this time, our lab was interested in testing the possibility that the cleaved helices of bacteriorhodopsin could spontaneously reassemble into a func- tional protein in the presence of lipids. Dur- ing this process, it was found that one of the helices, the C3 helix, despite being quite hy- drophobic, would not insert into membranes unless the pH was reduced. This C3 helix is the peptide we now call pHLIP (Figure 1). Using a variety of biophysical methods, including tryptophan fluorescence [13], cir- cular dichroism [14], and Fourier-trans- formed infrared spectroscopy [11], our lab has extensively characterized the behavior of pHLIP in synthetic liposomes to deter- mine that pHLIP exists in three different bio- physical states depending on environmental context (Figure 1A). These three states are as follows: 1) in the absence of lipids and at neutral or slightly basic pH, pHLIP exists as a relatively unstructured monomer in aque- ous solution; 2) in the presence of lipid membranes at neutral or slightly basic pH, unstructured pHLIP partitions very favor- ably to the membrane surface; and 3) at low pH, pHLIP inserts spontaneously (and re- versibly) into the membrane as a spanning α-helix. It is this latter insertion and the tran- sition between states 2 and 3 that allows us to use pHLIP as a pH-sensitive biosensor. This is because local acidic milieu in vivo is enough to trigger the pH-dependent inser- tion of pHLIP (Figure 1C), allowing the pep- tide to insert and be retained in the membranes of cells found in areas of local- ized acidity. pHLIP In vIvo LABe LIn g By covalently attaching fluorophore mol- ecules to the N-terminal, non-inserting end of pHLIP, in a collaboration with groups at the University of Rhode Island, we have been able to intravenously and peritoneally inject pHLIP-fluorophore conjugates into live mice harboring human tumors and observe prefer- ential retention of the construct in cancerous tissue (Figure 2A-B) [15]. This retention was very pronounced for a variety of tumor sizes [15], even 5 days after an initial implantation of only 105 cancer cells subcutaneously in nude mice. As mentioned above, we attribute this retention to the well-characterized pH-de- pendent insertion of the peptide: The peptide senses localized acidity and responds by in- serting into cell membranes as a spanning α- helix, anchoring the fluorophore in place. The kinetics of pHLIP localization are such that most of the conjugate clears from mice within 24 hours (Figure 2C), leaving an obvious retention in the tumor that per- sists for more than a week (Figure 2D). Al- though we have not yet performed in-depth toxicology on the mice used in these stud- ies, careful monitoring of the animals showed that the conjugates resulted in no obvious adverse physiological or behavioral side effects. As far as we could detect, the conjugates seemed to label the entirety of the tumors with no adjacent tissue being la- beled at all [21].