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

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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.

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 additional factor that aids in tumor progression and metastasis is the role of the host–tumor microenvironment. Multiple lines of evidence suggest complex networks of communication exist in tumor environments established by cancer cells, fibroblasts, endothelial cells, and immune cells to promote cancer growth and invasion 2, 3, 4, 5, 6, 7. Because of the importance of this element to tumorigenesis, it is clear that better understanding the interplay 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 accurately. 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 microenvironment, we evaluated the proteomic differences between in vitro NB cells versus tumors from a progressive in vivo animal model of human NB. By comparing the protein expression changes between both systems, we sought to better depict the effect the host microenvironment plays in a growing NB tumor.

Section snippets

Cell Culture and In Vivo Preparation

The SK-N-AS human NB cell line was purchased from American Type Culture Collection (ATCC) in Bethesda, MD. The cells were propagated as a monolayer culture in sterile filtered 45% BioWhittaker Dulbecco's modified Eagle's medium (DMEM from Cambrex Bioscience, Walkersville, MD) with 4.5 g/L glucose and L-glutamine, 45% BioWhittaker HAM'S F-12 with L-glutamine (Cambrex), 10% characterized fetal bovine serum (HyClone, Logan, UT), and 1% antibiotic-antimycotic (Gibco, Carlsbad, CA). Cells were seeded

Growth of NB SK-N-AS Xenograft Tumors

To investigate the significance of the host microenvironment on NB tumor progression, we utilized an in vivo NB nude mouse model. Figure 1A–C show tumors extracted from 2-, 4-, and 5-wk time points. Mean tumor weights were 0.18 ± 0.06 g, 4.28 ± 1.22 g, and 6.77 ± 1.08 g, respectively. We clearly ascertained a progressive increase in tumor growth during the experimental time interval (Fig. 1A–C). Figure 1D shows a 4-wk NB tumor at the time of sacrifice. A large, intra-abdominal mass was readily

Discussion

NB is a lethal pediatric malignancy facing obstacles related to current outcomes of advanced stage disease. As such, strategies aimed at targeting microenvironment factors that foster tumor development, such as anti-angiogenic agents [13], have the potential to become promising adjunctive therapies for high risk NB. Moreover, because a thorough evaluation of the host–tumor relationship may help to increase our knowledge concerning factors mediating NB growth and lead to novel treatment

Acknowledgments

The authors thank the A.N.N.A. Foundation (Indianapolis, IN) for their continuous support of their research.

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