Purpose: Autophagy consists of lysosome-dependent degradation of cytoplasmic contents sequestered by autophagic vesicles (AV). The role of autophagy in determining tumor aggressiveness and response to therapy in melanoma was investigated in this study.Experimental Design: Autophagy was measured in tumor biopsies obtained from metastatic melanoma patients enrolled on a phase II trial of temozolomide and sorafenib and correlated to clinical outcome. These results were compared with autophagy measurements in aggressive and indolent melanoma cells grown in two-and three-dimensional (3D) culture and as xenograft tumors. The effects of autophagy inhibition with either hydroxychloroquine or inducible shRNA (short hairpin RNA) against the autophagy gene ATG5 were assessed in three-dimensional spheroids.Results: Patients whose tumors had a high autophagic index were less likely to respond to treatment and had a shorter survival compared with those with a low autophagic index. Differences in autophagy were less evident in aggressive and indolent melanoma cells grown in monolayer culture. In contrast, autophagy was increased in aggressive compared with indolent melanoma xenograft tumors. This difference was recapitulated when aggressive and indolent melanoma cells were grown as spheroids. Autophagy inhibition with either hydroxychloroquine or inducible shRNA against ATG5 resulted in cell death in aggressive melanoma spheroids, and significantly augmented temozolomide-induced cell death.Conclusions: Autophagy is a potential prognostic factor and therapeutic target in melanoma. Three dimensional culture mimics the tumor microenvironment better than monolayer culture and is an appropriate model for studying therapeutic combinations involving autophagy modulators. Autophagy inhibition should be tested clinically in patients with melanoma. Clin Cancer Res; 17(10); 3478-89. Ó2011 AACR.

Key words: magnetic susceptibility; spherical mean value; magnetic resonance; inverse problem; quantitative susceptibility imaging Magnetic susceptibility (simplified as susceptibility hereinafter) is an important physical property of materials for many physical, chemical, and engineering applications. In the past two decades, susceptibility has played an increasingly significant role in biomedical applications such as with biomagnetic susceptometry and MRI. Magnetic susceptibility provides information on the tissue iron concentration that is useful for diagnosis and treatment of a number of diseases such as sickle cell disease, aplastic anemia, thalassemia, hemochromatosis, and Parkinson's disease (1,2). In MRI, the susceptibility effects have essential relevance for imaging contrast and artifact correction, functional brain imaging, molecular imaging, and the measurement of blood oxygenation. Thus, it is highly significant to develop methods that can measure arbitrary susceptibility distributions.Many research efforts have been related to susceptibility quantification with noninvasive MR methods (3-11). A commonly used approach for susceptibility quantification in MRI is to measure the magnetic induction field deviations generated by magnetic susceptibilities on the basis of the frequency or phase of the MR signals (6 -9). General biomedical applications often involve quantifying a susceptibility distribution (r) in a localized region of interest (ROI) within a biological body, which may enclose multiple other regions of distinct susceptibility distributions. In this scenario, the field deviations in the ROI include three contributing components, i.e., the local susceptibility-induced magnetization, the magnet main field inhomogeneity, and the nonlocal demagnetizing fields generated by all the susceptibility distributions within the body and the susceptibility discontinuity at the body-air interface. Since the magnetic susceptibilities of interest are usually in the range of 0.01-100 parts per million (ppm), these three contributions are often comparable in magnitude. The difficulty in separating the three entwined contributions has posed a major challenge for susceptibility quantification on the basis of the induction field measurements.By far, all prior NMR methods for susceptibility quantification are successful, assuming at least one of the following: uniform susceptibility distributions in objects of interest, homogeneous background fields, and certain geometrical shapes for the objects such as spheres, flat disks, and long cylinders. Although several articles have been published related to MR susceptibility imaging (10,11), no true quantitative susceptibility imaging has been achieved for general susceptibility distributions.Here we present a general method of quantitative susceptibility imaging with MRI. We will present a theory from first principles and develop analysis methods to quantify an arbitrary susceptibility distribution inside a localized region on the basis of the magnetic induction field de...

Abstract. Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic ͑aggressive͒ MDA-MB-231 and less metastatic ͑indolent͒ MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide ͑NADH͒ and oxidized flavoproteins ͑Fp͒. The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp/ ͑NADH+ Fp͒. We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.

Noninvasive or minimally invasive prediction of tumor metastatic potential would facilitate individualized cancer management. Studies were performed on a panel of human melanoma xenografts that spanned the full range of metastatic potential measured by an in vivo lung colony assay and an in vitro membrane invasion culture system. Three imaging methods potentially transferable to the clinic [dynamic contrast-enhanced (DCE) MRI, T1-MRI, and low-temperature fluorescence imaging (measurable on biopsy specimens)] distinguished between relatively less metastatic and more metastatic human melanoma xenografts in nude mice. DCE-MRI, analyzed with the shutterspeed relaxometric algorithm and using an arterial input function simultaneously measured in the left ventricle of the mouse heart, yielded a blood transfer rate constant, Ktrans, that measures vascular perfusion/permeability. Ktrans was significantly higher in the core of the least metastatic melanoma (A375P) than in the core of the most metastatic melanoma (C8161). C8161 melanoma had more blood vascular structures but fewer functional blood vessels than A375P melanoma. The A375P melanoma exhibited mean T1 values that were significantly higher than those of C8161 melanoma. Measurements of T1 and T2 relaxation times did not differ significantly between these 2 melanomas. The mitochondrial redox ratio, Fp/(Fp ؉ NADH), where Fp and NADH are the fluorescences of oxidized flavoproteins and reduced pyridine nucleotides, respectively, varied linearly with the in vitro invasive potential of the 5 melanoma cell lines (A375P, A375M, A375P10, A375P5, and C8161). This study shows that a harsh microenvironment may promote melanoma metastasis and provides potential biomarkers of metastatic potential. dynamic contrast enhanced MRI ͉ mitochondrial redox state ͉ T1rho ͉ invasive potential ͉ human melanoma xenografts M elanoma is treated primarily by surgical excision, which is often curative if the tumor is detected in its early stages. However, if recurrence with metastasis occurs, the prognosis is very poor because effective methods for treating systemic disease are not available. Evaluation of the metastatic potential of a melanoma at the time of surgery could determine the aggressiveness of the surgical procedures to be undertaken and the frequency of postsurgical surveillance.Criteria currently available for staging human melanoma malignancies and predicting their metastatic potential include histopathological evaluation, height of the lesion, disease progression to sentinel lymph nodes, and genomic and proteomic approaches currently under development and evaluation (1-5). The objective of this study was to explore a variety of noninvasive and biopsy-based imaging methods that could be used to better distinguish between aggressive and indolent neoplasms and to identify biomarkers of tumor aggressiveness.We chose to study melanoma because of the availability of a panel of human melanoma cell lines and their corresponding xenografts in immunosuppressed mice that span the full rang...