Hari R. Kumar

Three-dimensional neuroblastoma cell culture: Proteomic analysis between monolayer and multicellular tumor spheroids

IntroductionSolid tumors, such as neuroblastoma (NB), are associated with a heterogeneous cell environment. Multicellular tumor spheroid (MCTS) cultures have been shown to better mimic growth characteristics of in vivo solid tumors. Because tumor spheroid growth patterns may be quite different from standard two-dimensional culture systems, we sought to compare the protein expression profiles of two- and three-dimensional in vitro NB cultures, i.e., monolayers and MCTS.Materials and methodsHuman NB cells were grown as both monolayers and spheres. Nuclear and cytosolic proteins were analyzed for differentially secreted proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and selected polypeptides were identified by mass spectrometry (LC-MS/MS).ResultsSeveral metabolic (transketolase, triosephosphate isomerase, pyruvate kinase M1/M2, alpha enolase, and phosphoglycerate mutase-1), cell stress response (heat shock proteins (HSP) 90, 70, and 60; antioxidant, thioredoxin), cell structure (septin 2, adenyl cyclase-associated protein-1), tubulin β-2 chain, actin, translationally controlled tumor protein and cofilin), signal transduction (peptidyl prolyl cis/trans isomerase A), biosynthetic (phosphoserine aminotransferase) and transport (cellular retinoic acid binding protein 1) polypeptides were overexpressed in spheroids. Several protein groups were differentially expressed between NB monolayers and spheroids.ConclusionThe altered proteins among NB spheroids may represent an important link between monolayer cell cultures and in vivo experiments and thus a more ideal in vitro culture system for determining the precise threedimensional microenvironment of NB.

Impact of omphalocele size on associated conditions

PURPOSE Omphalocele is often associated with the presence of other congenital anomalies. Case reports have demonstrated nonclassical associations occurring in smaller omphaloceles. The aim of this study was to determine if omphalocele defect size correlates with the type of anomalies found. METHODS Patient records at a pediatric hospital were retrospectively reviewed for an 8-year period. Data were collected on patient demographics, omphalocele size, and congenital anomalies identified. Size of the abdominal wall defect was determined by either physical examination or operative record of repair. Patient cohorts were designated as those with small (4 cm and less) or large (greater than 4 cm) omphaloceles. RESULTS Fifty-three cases of omphalocele were observed. Twenty-seven cases were classified as small, with 26 classified as large. A predominance of males was noted in the small omphalocele group (78% vs 42%; P = .01). Intestinal anomalies, including Meckels diverticulum and intestinal atresia, were only seen in patients with small omphaloceles. Most cardiac anomalies were associated with large omphaloceles (34.6% vs 3.7%; P = .01). CONCLUSION Small omphalocele size correlates with an increased prevalence of associated gastrointestinal anomalies, a lower prevalence of cardiac anomalies, and a higher predominance of male sex.

Applications of emerging molecular technologies in glioblastoma multiforme

Glioblastoma multimorme (GBM) is the most common primary brain tumor in adults. Median survival from the time of diagnosis is less than a year, with less than 5% of patients surviving 5 years. These tumors are thought to arise through two different pathways. Primary GBMs represent de novo tumors, while secondary GBMs represent the malignant progression of lower-grade astrocytomas. Moreover, despite improvements in deciphering the complex biology of these tumors, the overall prognosis has not changed in the past three decades. The hope for improving the outlook for these glial-based malignancies is centered on the successful clinical application of current high-throughput technologies. For example, the complete sequencing of the human genome has brought both genomics and proteomics to the forefront of cancer research as a powerful approach to systematically identify large volumes of data that can be utilized to study the molecular and cellular basis of oncology. The organization of these data into a comprehensive view of tumor growth and progression translates into a unique opportunity to diagnose and treat cancer patients. In this review, we summarize current genomic and proteomic alterations associated with GBM and how these modalities may ultimately impact treatment and survival.

Proteomic Analysis of Neuroblastoma Microenvironment: Effect of the Host–Tumor Interaction on Disease Progression

BACKGROUND Children with advanced-stage neuroblastoma (NB) traditionally experience poor outcomes. Because early detection of advanced-stage disease may impact survival, finding new targets for early diagnosis is crucial. Evidence suggests the tumor microenvironment may have profound effects on cancer progression. METHODS As little is known concerning the NB-host microenvironment, this study applied proteomic techniques, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to determine protein differences between cell cultured NB and tumors grown in mice for 2, 4, and 5 wk. RESULTS We found an increase in proteins in cultured NB compared with implanted mouse tumors during tumor progression. Additionally, analyzing in vivo tumors to cultured NB, we observed less expressed proteins. However, 16 out of 19 proteins were of mouse origin, thus inferring host-derived factors contributing to tumor growth. CONCLUSION We show that the dynamic relationship between NB and host microenvironment is important for tumor growth and better understanding of this milieu maybe relevant towards finding unique approaches for identifying advanced-stage disease.

Bin1 Is Linked to Metastatic Potential and Chemosensitivity in Neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. At the time of diagnosis, the tumor has metastasized in as many as 7 of 10 cases, and survival in high‐risk patients remains poor. Accurate classification of high‐risk patients is very important since this determines treatment plan, and although a consensus risk classification system has been established for NB, it contains few specific molecular markers that account for aggressive nature and metastatic potential of the tumor. Bin1 expression is reduced in breast, NB, and other cancer types and the reduction correlates with high‐risk clinical features. Here we hypothesize that Bin1 has an inhibitory role in metastasis, and therefore decrease in its expression may be a marker of high‐risk NB.

Proteomic approaches in neuroblastoma: a complementary clinical platform for the future

Neuroblastoma (NB) is one of the most common solid tumors of childhood and displays a remarkable diversity in both biologic characteristics and clinical outcomes. Availability of high-throughput ‘omics technologies and their subsequent application towards oncology has provided insight into the complex pathways of tumor formation and progression. Investigation of NB ‘omics profiles may better define tumor behavior and provide targeted therapy with the goal of improving outcomes in patients with high-risk disease. Utilization of these technologies in NB has already led to advances in classification and risk stratification. The gradual emergence of NB-directed proteomics adds a layer of intricacy to the analysis of biologic organization but may ultimately provide a better comprehension of this complex disease. In this review, we cite specific examples of how NB-directed proteomics has provided information regarding novel biomarkers and possible therapeutic targets. We finish by examining the impact of high-throughput ‘omics in the field of NB and speculate on how these emerging technologies may further be incorporated into the discipline.