A major problem in cancer therapy is that anti-cancer agents do not adequately penetrate into tumor tissue. This proposal is based on recently identified tumor-homing peptides that specifically penetrate into tumors. These peptides contain both a tumor-specific homing sequence and a tissue-penetrating and cell-internalizing C-end Rule (CendR) motif. The CendR element in the tumor-penetrating peptides is cryptic, and proteolytically activated at the target site. Drug, fluorophore, and nanoparticle cargos attached to these peptides accumulate in tumors and penetrate deep into extravascular tumor tissue. Recent evidence produced with the prototype tumor-penetrating peptide, iRGD, indicates that it is not necessary to couple a cargo to the peptide for tumor-selective delivery; free iRGD activates a bulk transport pathway in the tumor which carries a co-injected drug or nanoparticle through the vascular wall and deep into the tumor tissue. This application proposes studies to provide detailed understanding of the tumor penetration process. Specifically, we propose (i) to define the molecular pathway of the transport pathway, (ii) to identify protease(s) that activate cryptic CendR elements in the tumor-penetrating peptides, (iii) to optimize the ability of the peptides to selectively increase tissue permeability in tumors, and (iv) to test selected peptide-anti-cancer drug combinations in treatment studies. The ability to specifically increase the accumulation of anti-cancer drugs in tumors is an advance of broad ramifications. It promises to make it possible to deliver drugs to tumors at higher concentrations than permitted by standard therapies. According to our results, the increase can be as high as 40 fold. Because the increase in drug concentration only occurs in tumors and not in normal tissues, the efficacy of the treatment is increased while the side effects remain the same. Alternatively, the dose could be reduced without compromising the efficacy of the treatment. As the adjuvant peptide and anti-cancer drug are not coupled to one another, a validated and an approved tumor-penetrating peptide could be used to augment any cancer drug - a major advance in cancer therapy could ensue.