Cross-talk between Schwann cells and neuroblasts influences the biology of neuroblastoma xenografts

Abstract

Neuroblastoma (NB) tumors with abundant schwannian stroma have a differentiated phenotype, low vascularity, and are associated with a favorable prognosis. These observations suggest that cross-talk between Schwann cells and neuroblasts may influence tumor biology. To test this hypothesis, we developed a novel NB xenograft model with infiltrating mouse Schwann cells. Human SMS-KCNR NB cells were injected intrafascicularly (sciatic nerve-engrafted NB, n = 19) or outside the sciatic nerve (control, n = 12). Xenografts were harvested 4 to 12 weeks after tumor cell inoculation for histological studies. Schwann cells were immunostained with S-100 and species-specific p75(NGFR), major histocompatibility complex, and human leukocyte antigen antibodies. The number of proliferating cells, infiltrating Schwann cells, apoptotic cells, differentiated neuroblasts, and blood vessels in the sciatic nerve-engrafted NB tumors were compared to controls. Significantly more Schwann cells were detected in the sciatic nerve-engrafted NB xenografts than controls (P < 0.001). The infiltrating Schwann cells were S-100-positive and reacted with anti-mouse major histocompatibility complex class Ib and p75(NGFR) but not anti-human p75(NGFR) and human leukocyte antigen class I antibodies. The sciatic nerve-engrafted tumors also had lower numbers of proliferating neuroblasts, higher numbers of differentiated neuroblasts and apoptotic cells, and decreased vascular density compared to controls. Our results indicate that infiltrating Schwann cells of mouse origin are capable of promoting human neuroblast differentiation, inducing apoptosis, and inhibiting proliferation and angiogenesis in vivo.

Figure 1

Representative histological sections of tumors engrafted inside versus outside the sciatic nerve. A and B: H&E-stained sections of SMS-KCNR NB cells engrafted inside versus outside the sciatic nerve. C–F: S-100-immunostained serial sections adjacent to the respective H&E sections demonstrating NB cells encased by the sciatic nerve and Schwann cells infiltrated into the tumors. Schwann cells are highlighted by anti-S-100 antibody in C and E. Control xenografts with SMS-KCNR NB cells engrafted outside the sciatic nerve are shown in D and F. Significantly more S-100-positive Schwann cells were present in the tumor engrafted inside the sciatic nerve (arrows in E) compared to controls (F). G and H: NB cells of tumors engrafted inside the sciatic nerve were larger with abundant eosinophilic cytoplasm (arrows in G) compared to control tumors (H). I and J: Serial sections from the same tumor stained with GAP-43 show higher levels of GAP-43 expression in the NB cells engrafted in the sciatic nerve than in the controls.

Figure 2

Representative photographs of tumors engrafted inside versus outside the sciatic nerve stained by double immunofluorescence. A–C: Human schwannoma serves as a negative control for anti-mouse p75^NGFR antibody. Schwann cells in the human schwannoma are positive for S-100 (A) but negative for anti-mouse p75^NGFR (B). A merged image is shown in C. D–I: Representative photographs taken from serial sections of a tumor engrafted inside the sciatic nerve that is stained by double immunofluorescence using S-100 (D) and p75^NGFR anti-mouse antibodies (E). A merged image is shown in F. High-power views demonstrate that S-100-positive Schwann cells also react with anti-mouse p75^NGFR antibody and the two antibodies are co-localized (arrowheads in G–I). J–L: Human schwannoma serves as a positive control for anti-human p75^NGFR antibody. S-100-positive Schwann cells (J) were positive for anti-human p75^NGFR (K). L: A merged image shows dual positivity in the Schwann cells. M–R: The S-100-positive Schwann cells do not react with anti-human p75^NGFR.

Figure 3

A–H: Representative photographs taken from anti-mouse MHC class Ib and anti-human HLA class I-immunostained histological sections from sciatic nerve-engrafted tumors and control tissues from human and mouse. Schwann cells in the human schwannoma were negative (A) and the tubular epithelial cells of mouse kidney were positive for the anti-mouse MHC class Ib antibody (B). S-100-positive infiltrating Schwann cells (D) in the sciatic nerve-engrafted tumor were positive for the anti-mouse MHC antibody (C). Tissues from mouse tail (E) and S-100-positive infiltrating Schwann cells (H) in the sciatic nerve-engrafted tumor (G) were negative but epithelial cells of human kidney glomerulus were positive (F) for the anti-human HLA class I antibody. Original magnifications, ×400.

Figure 4

Representative photographs of MIB-1 (Ki-67)-immunostained tumors engrafted inside versus outside the sciatic nerve. A–D: Proliferating cells identified by MIB-1 antibody in control versus sciatic nerve-engrafted NB cells. E: The mean number of MIB-1 (Ki-67)-positive cells (brown cells) per 10 HPFs of the sciatic nerve-engrafted tumors and controls are shown in the bar graph. Representative photographs of a TUNEL assay of tumors engrafted inside versus outside the sciatic nerve. F–I: Apoptotic cells identified by TUNEL analysis (green cells) in control versus sciatic nerve-engrafted NB cells. J: The mean number of apoptotic cells per 10 HPFs of the sciatic nerve-engrafted tumors and controls are shown in the bar graph. Original magnifications, ×400.

Figure 5

Representative photographs taken from CD31-immunostained slides of tumors engrafted outside (A and C) versus inside the sciatic nerve (B and D). Arrows in D point to smaller vessels seen in the sciatic nerve-engrafted tumor. E: The MVDs of the sciatic nerve-engrafted tumors and controls are shown in the bar graph.