Metal-Assisted Protein Quantitation (MAPq): Multiplex Analysis of Protein Expression Using Lanthanide-Modified Antibodies with Detection by Inductively Coupled Plasma Mass Spectrometry.

Understanding the complex relationships between genomics, transcriptomics, and proteomics requires the development of more sensitive and rapid methods of multiplexed protein analysis. This is necessary to understand the relationship between cellular responses to environmental stresses, disease progression, and/or drug treatment; however, most methods are limited by low sensitivity, nonspecificity, and minimal multiplexing capacity. To more fully explore the relationship between multiple cellular pathways, we have developed a novel antibody-based multiplex assay using inductively coupled plasma mass spectrometry (ICP-MS), which we term metal-assisted protein quantitation (MAPq). MAPq utilizes lanthanide-conjugated antibodies to simultaneously quantify up to 35 proteins with low pg/mL sensitivity. This method is especially advantageous for low-abundance proteins, a significant limitation of many multiplex MS methods. We observed a limit of detection of 0.5 pg/mL and a limit of quantitation of 5 pg/mL with virtually no background signal. We applied this method to both cultured cells and mouse tissues to investigate changes in low-abundance nuclear and cytoplasmic proteins following drug or environmental stresses. MAPq was found to be at least 10 times more sensitive than Western blots and could detect quantitative changes in protein expression not readily observed using conventional approaches.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models.

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N²-1-(carboxyethyl)-2'-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

Elevated glucose increases genomic instability by inhibiting nucleotide excision repair.

We investigated potential mechanisms by which elevated glucose may promote genomic instability. Gene expression studies, protein measurements, mass spectroscopic analyses, and functional assays revealed that elevated glucose inhibited the nucleotide excision repair (NER) pathway, promoted DNA strand breaks, and increased levels of the DNA glycation adduct N 2 -(1-carboxyethyl)-2'-deoxyguanosine (CEdG). Glycation stress in NER-competent cells yielded single-strand breaks accompanied by ATR activation, γH2AX induction, and enhanced non-homologous end-joining and homology-directed repair. In NER-deficient cells, glycation stress activated ATM/ATR/H2AX, consistent with double-strand break formation. Elevated glucose inhibited DNA repair by attenuating hypoxia-inducible factor-1α-mediated transcription of NER genes via enhanced 2-ketoglutarate-dependent prolyl hydroxylase (PHD) activity. PHD inhibition enhanced transcription of NER genes and facilitated CEdG repair. These results are consistent with a role for hyperglycemia in promoting genomic instability as a potential mechanism for increasing cancer risk in metabolic disease. Because of the pleiotropic functions of many NER genes beyond DNA repair, these results may have broader implications for cellular pathophysiology.

Metabolic advantages and vulnerabilities in brain metastases.

Metabolic adaptations permit tumor cells to metastasize to and thrive in the brain. Brain metastases continue to present clinical challenges due to rising incidence and resistance to current treatments. Therefore, elucidating altered metabolic pathways in brain metastases may provide new therapeutic targets for the treatment of aggressive disease. Due to the high demand for glucose in the brain, increased glycolytic activity is favored for energy production. Primary tumors that undergo Warburg-like metabolic reprogramming become suited to growth in the brain microenvironment. Indeed, elevated metabolism is a predictor of metastasis in many cancer subtypes. Specifically, metabolic alterations are seen in primary tumors that are associated with the formation of brain metastases, namely breast cancer, lung cancer, and melanoma. Because of this selective pressure, inhibitors of key metabolic factors may reduce tumor cell viability, thus exploiting metabolic pathways for cancer therapeutics. This review summarizes the metabolic advantages and vulnerabilities of brain metastases.

Diagnostic PET Imaging of Mammary Microcalcifications Using 64Cu-DOTA-Alendronate in a Rat Model of Breast Cancer.

The development of improved breast cancer screening methods is hindered by a lack of cancer-specific imaging agents and effective small-animal models to test them. The purpose of this study was to evaluate 64Cu-DOTA-alendronate as a mammary microcalcification-targeting PET imaging agent, using an ideal rat model. Our long-term goal is to develop 64Cu-DOTA-alendronate for the detection and noninvasive differentiation of malignant versus benign breast tumors with PET. Methods: DOTA-alendronate was synthesized, radiolabeled with 64Cu, and administered to normal or tumor-bearing aged, female, retired breeder Sprague-Dawley rats for PET imaging. Mammary tissues were subsequently labeled and imaged with light, confocal, and electron microscopy to verify microcalcification targeting specificity of DOTA-alendronate and elucidate the histologic and ultrastructural characteristics of the microcalcifications in different mammary tumor types. Tumor uptake, biodistribution, and dosimetry studies were performed to evaluate the efficacy and safety of 64Cu-DOTA-alendronate. Results:64Cu-DOTA-alendronate was radiolabeled with a 98% yield. PET imaging using aged, female, retired breeder rats showed specific binding of 64Cu-DOTA-alendronate in mammary glands and mammary tumors. The highest uptake of 64Cu-DOTA-alendronate was in malignant tumors and the lowest uptake in benign tumors and normal mammary tissue. Confocal analysis with carboxyfluorescein-alendronate confirmed the microcalcification binding specificity of alendronate derivatives. Biodistribution studies revealed tissue alendronate concentrations peaking within the first hour, then decreasing over the next 48 h. Our dosimetric analysis demonstrated a 64Cu effective dose within the acceptable range for clinical PET imaging agents and the potential for translation into human patients. Conclusion:64Cu-DOTA-alendronate is a promising PET imaging agent for the sensitive and specific detection of mammary tumors as well as the differentiation of malignant versus benign tumors based on absolute labeling uptake.