Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth
Erik H. Knelson, … , Stephen G. Marcus, Gerard C. Blobe
Erik H. Knelson, … , Stephen G. Marcus, Gerard C. Blobe
Published July 1, 2014; First published June 17, 2014
Citation Information: J Clin Invest. 2014;124(7):3016-3031. https://doi.org/10.1172/JCI74270.
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Research Article Oncology

Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth

Abstract

Neuroblastoma prognosis is dependent on both the differentiation state and stromal content of the tumor. Neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here, we identified critical growth-limiting components of the differentiating stroma secretome and designed a potential therapeutic strategy based on their central mechanism of action. We demonstrated that expression of heparan sulfate proteoglycans (HSPGs), including TβRIII, GPC1, GPC3, SDC3, and SDC4, is low in neuroblasts and high in the Schwannian stroma. Evaluation of neuroblastoma patient microarray data revealed an association between TGFBR3, GPC1, and SDC3 expression and improved prognosis. Treatment of neuroblastoma cell lines with soluble HSPGs promoted neuroblast differentiation via FGFR1 and ERK phosphorylation, leading to upregulation of the transcription factor inhibitor of DNA binding 1 (ID1). HSPGs also enhanced FGF2-dependent differentiation, and the anticoagulant heparin had a similar effect, leading to decreased neuroblast proliferation. Dissection of individual sulfation sites identified 2-O, 3-O-desulfated heparin (ODSH) as a differentiating agent, and treatment of orthotopic xenograft models with ODSH suppressed tumor growth and metastasis without anticoagulation. These studies support heparan sulfate signaling intermediates as prognostic and therapeutic neuroblastoma biomarkers and demonstrate that tumor stroma biology can inform the design of targeted molecular therapeutics.

Authors

Erik H. Knelson, Angela L. Gaviglio, Jasmine C. Nee, Mark D. Starr, Andrew B. Nixon, Stephen G. Marcus, Gerard C. Blobe

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Figure 1

HSPG expression is localized to the stroma, and high expression is associated with improved patient prognosis.

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HSPG expression is localized to the stroma, and high expression is assoc...
(A) Microarray dataset analysis (GSE7529) for HSPG expression in neuroblastic tumors based on stromal status (n = 8 stroma-rich, n = 11 stroma-poor). *P < 0.05; **P < 0.01; ****P < 0.0001 by Mann-Whitney U test. (B) IHC for TβRIII, SDC3, and SDC4 in the stroma of neuroblastoma tumor samples. HSPGs are labeled with red/pink stain, with methyl green nuclear counterstaining. Original magnification, ×60 (TβRIII); ×40 (SDC3 and SDC4); scale bars: 50 μM. Percentages shown indicate background-subtracted quantification of red channel densitometry in early-stage specimens relative to stage 4 specimens. (C) TGF-β binding and crosslinking with TβRIII pulldown for surface and sTβRIII in the indicated neuroblastoma cell lines, SHEP cells, and S16 Schwann cells. Western blots for GPC1, GPC3, SDC3, and SDC4 expression. Densitometry for HSPGs normalized to β-actin is shown as the percentage of control. (D) Analysis of event-free survival split by TGFBR3, then SDC3, then GPC1 expression in the Obertheur dataset (denoted as I.) compared with stratification by MYCN status. NA, nonamplified. Low HSPG expression group: 12 of 33 MYCN amplified. High HSPG expression group: 1 of 33 MYCN amplified. (E) Serum ELISA for TβRIII, SDC3, and GPC1 using neuroblastoma patient samples (n = 60). Survival analysis split by TβRIII, then SDC3, then GPC1 soluble protein levels (blue: top 13%, red: bottom 13%), or by TβRIII, then SDC3 (blue: top 25%, red: bottom 25%). The differences between the survival curves were not statistically different by log-rank test.