We are exploring the use of extracellular fluorescent indicators to measure analytes in the microdomains between adjacent cells in tissues. Our primary initial goal is to measure changes in H+ activity in the intercellular space of xenograft tumor models before and after drug treatment. It is thought that acidification in the core of tumors protects these cancers from the primarily weak base chemotherapeutic drugs. We are comparing the use of functionalized nanoparticles and expressible indicators as putative extracellular sensors. We have constructed functionalized, fluorescein-labeled nanoparticles that enable the particles to adhere to the surface of schwannoma cells.

We have also been exploring the use of expressible membrane bound H+ indicators, glycosylphosphatidylinositol (GPI)-anchored GFP mutants. In vitro testing of the indicators was accomplished by aggregating single cells into clusters (artificial tumors) using dielectrophoresis (DEP) and agarose molds. Preliminary results indicated that nanoparticles could be trapped within artificial tumors using DEP as long as electric fields are applied. Long term (7 days) intercellular pH sensing was achieved in vitro using GPI-GFP expressing schwannoma cells in an artificial tumor crafted with an agarose mold (Messerli et al., 2009). Currently, we are investigating whether a ratioable pH indicator, GPI-pHluorin, can be used to measure changes in pH in the intercellular space of tumors, engineered by DEP, agarose molds, and grown in mouse xenografts. Development of these techniques will provide valuable information regarding cellular defenses against chemotherapeutic drugs and mechanisms of therapeutic drug action. In addition, we are investigating the development of other sensors targeted to analytes such as ATP, Cl-, Na+, and K+.