Proteomic Analysis of Neuroblastoma Microenvironment: Effect of the Host–Tumor Interaction on Disease Progression Katharyn E. Turner, D.O.,* , † Hari R. Kumar, M.D.,† Derek J. Hoelz, Ph.D.,‡ Xiaoling Zhong, M.D., Ph.D.,* , † Frederick J. Rescorla, M.D.,* , † Robert J. Hickey, Ph.D.,‡ Linda H. Malkas, Ph.D.,‡ and John A. Sandoval, M.D.† ,1 *Section of Pediatric Surgery; †Department of Surgery; and ‡Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine and Riley Hospital for Children, Indianapolis, Indiana Submitted for publication January 8, 2009 Background. Children with advanced-stage neuro- blastoma (NB) traditionally experience poor outcomes. Because early detection of advanced-stage disease may impact survival, finding new targets for early diagno- sis is crucial. Evidence suggests the tumor microenvi- ronment may have profound effects on cancer progression. Methods. As little is known concerning the NB-host microenvironment, this study applied proteomic tech- niques, two-dimensional polyacrylamide gel electro- phoresis (2D PAGE) combined with matrix assisted laser desorption ionization time-of-flight (MALDI- TOF) mass spectrometry to determine protein differ- ences between cell cultured NB and tumors grown in mice for 2, 4, and 5 wk. Results. We found an increase in proteins in cul- tured NB compared with implanted mouse tumors dur- ing tumor progression. Additionally, analyzing in vivo tumors to cultured NB, we observed less expressed pro- teins. However, 16 out of 19 proteins were of mouse or- igin, 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 mi- lieu maybe relevant towards finding unique approaches for identifying advanced-stage disease. Published by Elsevier Inc. All rights reserved. Key Words: neuroblastoma; tumor microenviron- ment; proteomics; cancer progression; murine tumor model. INTRODUCTION Neuroblastoma (NB) is recognized as a malignant solid tumor in infancy with unfortunate outcomes in many children [1]. As tumor heterogeneity contributes to challenges associated with NB treatment, an addi- tional factor that aids in tumor progression and metasta- sis is the role of the host–tumor microenvironment. Multiple lines of evidence suggest complex networks of communication exist in tumor environments estab- lished by cancer cells, fibroblasts, endothelial cells, and immune cells to promote cancer growth and invasion [2–7]. Because of the importance of this element to tu- morigenesis, it is clear that better understanding the in- terplay between NB and the host environment may lead to improvements in the treatment of this deadly disease. To date, NB grown in cell culture has provided a great deal of information regarding this childhood tumor. Nevertheless, limitations to these methods include the inability to model the host–tumor environment ac- curately. For instance, proteomic evidence from our laboratory has shown protein differences when NB was cultured in traditional 2D conditions versus 3D multicellular spheroids [8]. These protein expression differences could reflect the intratumoral environment occurring within NB spheres, thus capturing a better physiologic window of an in vivo tumor as has been suggested using 3D cell culture systems [9, 10]. In order to further the study of the NB–host microenvi- ronment, we evaluated the proteomic differences between in vitro NB cells versus tumors from a pro- gressive in vivo animal model of human NB. By com- paring the protein expression changes between both systems, we sought to better depict the effect the host microenvironment plays in a growing NB tumor. 1 To whom correspondence and reprint requests should be ad- dressed at The Children’s Hospital/University of Colorado at Denver, Section of Pediatric Surgery, 13123 East 16th Avenue, B323, Aurora, CO 80045. E-mail: Sandoval.john@tchden.org. 0022-4804/09 $36.00 Published by Elsevier Inc. All rights reserved. 116 Journal of Surgical Research 156, 116–122 (2009) doi:10.1016/j.jss.2009.02.019