Pigment epithelium-derived factor (PEDF) in neuroblastoma: a multifunctional mediator of Schwann cell antitumor activity
Susan E. Crawford1,4,
,
Veronica Stellmach1,
Mark Ranalli5,*,
Xuemei Huang1,
Lijun Huang1,
Olga Volpert2,
George H. De Vries6,7,
Lisa P. Abramson8 and
Noël Bouck3,4
1 Department of Pathology,
2 Department of Urology,
3 Department of Microbiology-Immunology and
4 The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, IL 60611, USA
5 Department of Pediatrics, Division of Hematology-Oncology, Children’s Memorial Hospital, Chicago, IL 60614, USA
6 Research Service, Hines VA Hospital, Hines, IL 60141, USA
7 Department of Cell Biology, Neurobiology and Anatomy, Loyola University, Maywood, IL 60153, USA
8 Department of Surgery, Children’s Memorial Hospital, Chicago, IL 60614, USA
* Present address: Department of Pediatrics, Division of Hematology/Oncology, Columbus Children’s Hospital, 700 Children’s Drive, Columbus, OH, USA
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Fig. 1. PEDF expression increased with increasing tumor differentiation. Tumors displaying different levels of differentiation were stained with an antibody raised to peptides derived from PEDF. (A) Undifferentiated tumor, arrows indicate weakly staining endothelial cells. (B) A neuroblastoma with some diffentiated cells, arrow indicates a mature ganglion cell. (C) A ganglioneuroblastoma, a tumor with many differentiated cells that stained most strongly, arrow indicates Schwann cells. Magnification, 10x (A,C); 20x (B).
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Fig. 2. Schwann cells secrete PEDF. Media conditioned by human Schwann cells (20 µg protein, lane 2) and rat Schwann cells (20 µg protein, lane 3) were subjected to immunoblot analysis using an affinity purified antibody raised to PEDF-derived peptides. Recombinant human PEDF (20 ng, lane 1) was used as a positive control. M, molecular weight markers and their migration distance.
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Fig. 3. Cells from stromal and tumor compartments of neuroblastoma cells differed in their angiogenic phenotype. (A) Media conditioned by rat Schwann cells or by cultured neuroblastoma tumor cells growing as more differentiated S-type or less differentiated N-type, or by tumor cell line SK-N-BE(2) were assayed for their ability to induce capillary endothelial cell migration. Media were tested alone, or in the presence of an inducer (bFGF was the purified inducer added to the Schwann cell media; VEGF was the purified inducer added the S-type cell media), or in the presence of antibodies to neutralize PEDF or VEGF. The Student’s t test was used to compare data *P<0.009 with that obtained from the corresponding media tested alone. Error bars indicate s.e.m. (B) Controls for the activities of antibodies and proteins were performed by the indicated additions to DMEM/0.1% BSA.
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Fig. 4. PEDF protected Schwann cells from apoptosis. Rat Schwann cells grown in serum free media with or without PEDF for 5 days then evaluated for apoptosis using TUNEL staining. (A) Percentage of apoptotic Schwann cells cultured without or with 1 nM PEDF. 100% represents 75 cells counted. (B-C) Representative TUNEL staining of Schwann cells cultured without (B) or with PEDF (C).
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Fig. 5. PEDF was the major tumor cell differentiation factor in human Schwann cell conditioned media. SK-N-BE(2) cells grown in (A) media alone, (B) media plus Schwann cell conditioned media; (C) media plus Schwann cell conditioned media and anti-PEDF antibody; (D) quantification of differentiation elicited by Schwann cell media, where 100% represents 55 out of 117 cells counted (open circles, media alone; closed circles, media plus anti-PEDF antibody); (E) purified rPEDF at the indicated concentrations were added to the growth media of SH-SY5Y neuroblastoma cells and differentiation quantified. 100% represents 150 differentiated cells/254 cells counted.
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Fig. 6. PEDF triggered differentiation in neuroblastoma tumors in vivo. A total of 2 ug PEDF/day for 4 days were injected into subcutaneous SK-N-BE(2) tumors raised in nude mice. Tumors were excised, fixed and stained for neurofilament protein. Vehicle treated tumors (A,B) and rPEDF treated tumors (C,D) are shown at low power (A,C) and high power (B,D). The asterisk indicates an injection site.
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Fig. 7. Model of proposed autocrine and paracrine activities of PEDF in neuroblastoma tumors. The tumor and stromal compartments of neuroblastomas are composed of variable mixtures of cells capable of producing and/or responding to PEDF in a cell type-specific manner.
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