Optical Imaging of Treatment-Related Tumor Cell Death Using a Heat Shock Protein-90 Alkylator

Abstract

The ability to assess in near-real time the tumor cell killing efficacy of chemotherapy regimens would improve patient treatment and survival. An ineffective regimen could be abandoned early in favor of a more effective treatment. We sought to noninvasively image treatment-related tumor cell death in mice using an optically labeled synthetic heat shock protein-90 (Hsp90) alkylator, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO). The Hsp90 chaperone is an important element in oncogene addiction and tumor cell survival, and its expression is enhanced by chemotherapy. These factors were predicted to favor the detection of tumor cell death using GSAO. GSAO specifically labeled apoptotic and necrotic tumor cells in culture and cells of comparable morphology in subcutaneous human pancreatic carcinoma tumors in mice. A near-infrared fluorescent conjugate of GSAO was used to noninvasively image cyclophosphamide-induced tumor cell death in murine orthotopic human mammary tumors. The GSAO conjugate did not accumulate in healthy organs or tissues in the mouse, and unbound compound was excreted rapidly via the kidneys. There was a significant increase in the GSAO fluorescence signal in the treated tumors measured either in vivo or ex vivo, and the fluorescence signal colocalized with apoptotic cells in sectioned tumors. The favorable biodistribution of optically labeled GSAO, the nature of its tumor cell target, and its capacity to noninvasively detect tumor cell death should facilitate the application of this compound in studies of the efficacy of existing and new chemotherapeutics.

Necrotic platelets provide a procoagulant surface during thrombosis

“…Rationale for the platelet necrosis marker GSAO is a tripeptide trivalent arsenical that has recently been characterized as an imaging agent for necrotic and late apoptotic nucleated cells [15][16][17] ( Figure 1A). When tagged with a reporter compound at the g-glutamyl residue of the glutathione moiety, the compound is unable to cross the plasma membrane of viable cells.…”

“…[15][16][17] We employed conjugates of GSAO with AF647, AF546, or Oregon Green in this study. A GSAO control compound, GSCA [4-…”

“…GSAO and GSCA were produced and conjugated to Alexa Fluor 647, as described previously. 15,16,18 On some occasions, washed platelets were also labeled with annexin V-Pacific Blue (5% vol/vol; Life Technologies).…”

“…Traditional markers of loss of membrane integrity that bind to nuclear material such as propidium iodide or Sytox dyes cannot be used in the anucleate platelet. Thus, we investigated the use of a cell death imaging agent that complexes with cytoplasmic ligands, 4-[N-(S-glutathionylacetyl) amino] phenylarsonous acid (GSAO), [15][16][17] to determine whether we could identify the procoagulant platelet in vitro and in vivo. Using GSAO, we show that necrotic platelets are generated within a few seconds of agonist stimulation in vitro and are procoagulant.…”

Biodistribution and imaging of an hsp90 ligand labelled with 111In and 67Ga for imaging of cell death

“…Fluorophore-labelled conjugates of GSAO have been shown to be able to image cyclophosphamide-induced tumour cell death in murine orthotopic human mammary tumours. There was a significant increase in the GSAO fluorescence signal in the treated tumours measured in vivo and ex vivo and the signal co-localized with apoptotic cells in sectioned tumours [22]. In another study, fluorophore-labelled conjugates of GSAO localized to murine cerebral cryolesions and the areas of fluorescence corresponded with areas of TUNEL staining ex vivo [23].…”