Abstract. The microenvironment of solid cancers is acidic. This is a proposal to continue to investigate the causes and consequences of this acidity. Over the past period of support, significant progress has been made in all three aims to (1) improve methods for measuring pH in vivo, (2) determine the causes of the acidity, and (3) characterize the consequences of this acidity that are relevant to cancer progression. In aim 1, a method has been developed with which to measure pH following a single infusion of a pH-dependent contrast agent cocktail containing Gd-DOTA-4AmP and Dy-DOTA. In aim 2, the root causes of acid pHe have been identified as increased aerobic glucose metabolism (the Warburg Effect), which can be caused by dysregulation in one of six interrelated pathways. Experiments of aim 3 have shown that the acid pH promotes invasion and metastasis and, most importantly, inhibiting this acidity with oral ad lib bicarbonate led to significant reductions in spontaneous metastases and increased survival of mice with breast cancer xenografts. An important addition to this program during the previous period has been the incorporation of mathematical modeling based on somatic evolution. These efforts have provided a theoretical framework important to interpreting experimental results. These models led to the prediction that inhibiting acidosis would inhibit metastasis, as has been observed. A further model to arise from this work is the evolutionary equivalence principle that recognizes that Darwinian forces select for phenotype, not genotype, and is thus can explain the multiple molecular mechanisms and pathways associated with increased aerobic glycolysis in tumors. For clarity and consistency, the titles of the aims of the current proposal remain the same, although the foci have been significantly altered to accommodate new data and new insights. Aim 1 will develop a bolus injection approach of a pH-sensitive contrast agent, with an anticipated endpoint of IND filing for imaging tumor pHe with a single agent in humans within the next period of support. Aim 2 will investigate the molecular mechanisms underlying increased glycolysis by cancers by comparing the control strengths of the six interrelated pathways in a panel of derivatized and parental cell lines. Aim 3 will be focused entirely on characterizing and modeling the effect of pH buffering on inhibiting metastasis to better define the boundary conditions and mechanisms of this approach. These will add needed data to planned clinical trials.