Discovery of the Antitumor Effects of a Porphyrazine Diol (Pz 285) in MDA-MB-231 Breast Tumor Xenograft Models in Mice.

A series of porphyrazines (Pzs) with chiral bis-acetal moieties in the ß-pyrrole positions ((2R,3R)-2,3-dimethyl-2,3-dimethoxy-1,4-diox-2-ene) have been synthesized and screened as antitumor agents in MDA-MB-231 breast tumor cells in vitro. The lead Pz 285 was further tested in a mouse tumor xenograft model with Td-tomato-luc2 fluorescent breast tumor cells (MDA-MB-231 LM24 Her2+) that are highly metastatic to the lungs. Pz 285 shows marked antitumor effects in vivo, with treated mice exhibiting longer median survival that we attribute to smaller primary tumor regrowth after resection and less occurrence of metastasis when compared to vehicle control groups. Pz 285 is further compared to the clinically approved chemotherapeutic doxorubicin (Dox). This report lays the groundwork for development of an understudied class of compounds for classical chemotherapy.

Fluorescent cancer-selective alkylphosphocholine analogs for intraoperative glioma detection.

BACKGROUND: 5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence aids brain tumor resections but is not approved for routine use in the United States. We developed and describe testing of 2 novel fluorescent, cancer-selective alkylphosphocholine analogs, CLR1501 (green) and CLR1502 (near infrared), in a proof-of-principle study for fluorescence-guided glioma surgery. OBJECTIVE: To demonstrate that CLR1501 and CLR1502 are cancer cell-selective fluorescence agents in glioblastoma models and to compare tumor-to-normal brain (T:N) fluorescence ratios with 5-ALA. METHODS: CLR1501, CLR1502, and 5-ALA were administered to mice with magnetic resonance imaging-verified orthotopic U251 glioblastoma multiforme- and glioblastoma stem cell-derived xenografts. Harvested brains were imaged with confocal microscopy (CLR1501), the IVIS Spectrum imaging system (CLR1501, CLR1502, and 5-ALA), or the Fluobeam near-infrared fluorescence imaging system (CLR1502). Imaging and quantitative analysis of T:N fluorescence ratios were performed. RESULTS: Excitation/emission peaks are 500/517 nm for CLR1501 and 760/778 nm for CLR1502. The observed T:N ratio for CLR1502 (9.28±1.08) was significantly higher (P<.01) than for CLR1501 (3.51±0.44 on confocal imaging; 7.23±1.63 on IVIS imaging) and 5-ALA (4.81±0.92). Near-infrared Fluobeam CLR1502 imaging in a mouse xenograft model demonstrated high- contrast tumor visualization compatible with surgical applications. CONCLUSION: CLR1501 (green) and CLR1502 (near infrared) are novel tumor-selective fluorescent agents for discriminating tumor from normal brain. CLR1501 exhibits a tumor-to-brain fluorescence ratio similar to that of 5-ALA, whereas CLR1502 has a superior tumor-to-brain fluorescence ratio. This study demonstrates the potential use of CLR1501 and CLR1502 in fluorescence-guided tumor surgery.

Alkylphosphocholine analogs for broad-spectrum cancer imaging and therapy.

Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with (124)I-CLR1404 or (131)I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy.

Immunolabeling for correlative light and electron microscopy on ultrathin cryosections.

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.

Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles.

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.

Fluorescence quenching by colloidal heavy metals nanoparticles: implications for correlative fluorescence and electron microscopy studies.

Labels for correlative immunolabeling in light (LM) and electron microscopy (EM) employing colloidal metal nanoparticles (gold or palladium) and fluorescent dyes (Alexa Fluor, AF) were investigated. The fluorescence signals from direct conjugates (cAu-IgG-AF) and from an indirect label system (cAu-IgG-anti IgG-AF) were studied using scanning spectrofluorometry and fluorescence light microscopy. Direct conjugation of protein--AF, IgG-AF or FGN-AF to 18 and 5 nm colloidal gold (cAu18 and cAu5) or 12 nm colloidal palladium particles (cPd12) resulted in nearly completely quenched fluorescence signals (>99 %) at excitation wavelengths of 488, 546 and 594 nm. In contrast, indirect conjugation, when colloidal metal particles and AF were conjugated to primary or secondary antibody, respectively (cAu-IgG-antiIgG-AF), sufficient fluorescence signal was detected. Commercially available conjugates, consisting of IgG-AF-cAu5 and IgG-AF-cAu10, were also tested and proved to be a mixture of IgG-AF (unbound to cAu) and cAu-IgG-AF.

Effect of molecular structure on the selective phototoxicity of triarylmethane dyes towards tumor cells.

In response to transmembrane potentials which are negative on the inner side of both the plasma and mitochondrial membranes, cationic dyes displaying appropriate structural features naturally accumulate in the cytosol and inside the mitochondria. Because enhanced mitochondrial membrane potential is a prevalent tumor cell phenotype, a number of cationic dyes preferentially accrue and are retained for longer periods in the mitochondria of tumor cells as compared to normal cells. The opportunities brought about by this phenomenon in chemo- and photochemotherapy of neoplastic diseases is highlighted by the observation that the phototoxic effects associated with some of the cationic photosensitizers known to accumulate in cell mitochondria are much more pronounced in tumor cells than in normal cells. However, the structural determinants of selective phototoxicity towards tumor cells are not well understood, and the lack of a robust model to describe the relationship between molecular structure and tumor selectivity has prevented mitochondrial targeting from becoming a more dependable therapeutic strategy. In this report we describe how the lipophilic/hydrophilic character of a series of cationic triarylmethane dyes affects the selectivity with which these photosensitizers mediate the destruction of tumor cells. Our results indicated that only the more hydrophilic triarylmethanes show tumor selectivity, presumably because these are the only dyes capable of staining energized mitochondria with a high degree of specificity. The partition of the more lipophilic dyes into a variety of extra-mitochondrial subcellular compartments occurs with comparable efficiencies in tumor and in normal cells, and this less specific subcellular localization precludes tumor selectivity from taking place.