Tissue microenvironment modulates CXCR4 expression and tumor metastasis in neuroblastoma

Abstract

Neuroblastoma (NB) is derived from intrinsic migratory neural crest cells and has a high potential for distant metastasis. Growing evidence has implicated chemokine receptors, especially CXCR4, which normally control immune and inflammatory cell migration, as having important roles in tumor progression. In this study, we investigated the expression of CXCR4 in eight different NB cell lines and found that CXCR4 expression is dynamically regulated in NB and can be modulated by different tissue stromata. In addition, we demonstrate that IL-5 and IFN-gamma are released from stromal cells and act as differential mediators for CXCR4 expression. We also overexpressed CXCR4 in two NB cell lines, NUB-7 and SK-N-BE(2), and studied the role of CXCR4 in NB metastasis both in vitro and in vivo. In vitro transwell invasion assay showed that CXCR4 overexpression promoted NB cell migration preferentially toward a bone marrow stromal cell-conditioned medium. Using an in vivo xenograft model, CXCR4-overexpressing cells showed an increased incidence of metastasis, most notably bone marrow metastasis. Our studies reveal critical roles for CXCR4 in NB metastasis and provide insights into the regulatory mechanism of chemokine receptors in NB and the importance of the tissue microenvironment in modulating tumor cell behavior.

Figure 1

Expression of chemokines and chemokine receptors in NB cell lines. (A) RT-PCR showing different profiles of the chemokine/receptor system in eight NB cell lines [NUB-7, SK-N-BE(2), NBL-S, LAN-5, IMR-32, GOTO, SK-N-SH, and SK-N-BE(1)] and one primary NB tumor sample (NB12). GAPDH was used as internal control. (B) CXCR4 expression shown by Western blot analysis in five NB cell lines [NUB-7, SK-N-BE(2), NBL-S, LAN-5, and IMR-32] relative to the α-tubulin loading control.

Figure 2

Chemokine receptors induced by stromal cells. Stromal cells were isolated from different mouse organs: adrenal gland, liver, bone marrow, bone, and lung. (A) Expression of CXCR4 is affected by SCM. NUB-7 cells were incubated with conditioned media from different stromal cells for 48 hours under normal culture conditions. Expression of CXCR4 was studied by RT-PCR. (B) Western blot analysis showing the distinct effects of stromal cells on CXCR4 expression in NUB-7 cells. HeLa cell lysate was used as positive control. (C) Immunofluorescence staining of CXCR4 in liver SCM-induced NUB-7 cells and nontreated NUB-7 cells. Permeabilized CXCR4-overexpressing and control NUB-7 cells were incubated with anti-CXCR4 monoclonal antibody followed by Cy3-labeled (red) secondary antibody. Immunoreactive CXCR4 was located in the cytosol and membrane of those cells. (D) Regulation of CXCR4 expression by different SCM in two other NB cell lines, LAN-5 and NBL-S. Expression of CXCR4 was studied by RT-PCR.

Figure 3

(A) Cytokine antibody array with SCM from the adrenal gland, liver, and lung. Twenty-two cytokines were screened in three SCM. A conditioned medium was obtained as described above. α-MEM supplied with 10% BS was used as negative control. Array was performed with the ChemiArray system according to the manufacturer's protocol. (B) The map of mouse antibody array I can be used to identify individual cytokines. (C) CXCR4 expression is regulated by recombinant IL-5 and IFN-γ in NUB-7 cells. NUB-7 cells were cultured for 48 hours with different doses of mouse recombinant IL-5 and IFN-γ (0, 0.1, and 1 ng/ml). CXCR4 expression was assessed by Western blot analysis.

Figure 4

Overexpression of CXCR4 in NB cell lines. For CXCR4 overexpression, both NUB-7 and SK-N-BE(2) cells were infected by CXCR4-pBABE. Empty pBABE vector was used as control. (A) RT-PCR showing the expression level of CXCR4 after CXCR4 overexpression in the NUB-7 cell line. (B) Western blot analysis confirmed the overexpression of CXCR4 in both NUB-7 and SK-N-BE(2) transfectants. Blots were scanned and analyzed using ImageJ software (The National Institutes of Health [NIH], Bethesda, MD). (C) Effect of CXCR4 overexpression on NUB-7 cell proliferation. Cell proliferation was measured with Alamar Blue assay after 4 days. All experiments were carried out in triplicate. (D and E) Effect of CXCR4 expression on NB cell adhesion. Cell adhesion assay was performed using NUB-7 cells and control cells (C). NUB-7 cells were also treated with neutralizing antibody to CXCR4 (5 µg/ml) or specific CXCR4 inhibitor AMD3100 (1 µM) for 10 minutes at normal culture conditions. Cell adhesion assay was performed as described in the Materials and Methods section. (F) Effect of CXCR4 expression on NB cell invasiveness. Transwell cell invasion assay was performed using liver or bone marrow SCM as chemoattractant. Invaded cells on the bottom of the insert membrane were fixed with 70% ethanol and stained with hematoxylin. For quantification, the average number of migrating cells per field was assessed by counting 10 random fields under a microscope (original magnification, x250). Results are representative of three separate experiments. *P < .05, compared with each control. (G) Release of SDF-1α from stromal cells. Liver and adrenal gland SCM were obtain as described above. The measurement of SDF-1α was carried out using ELISA kits (R&D Systems), according to the manufacturer's instructions. α-MEM supplemented with 10% FBS was used as negative control.

Figure 5

CXCR4 expression regulates NB metastasis in a mouse xenograft model. (A) Both CXCR4-overexpressing (NUB-7-CXCR4) and control NUB-7 cells (NUB-7-pBABE) were injected intravenously to NOD/SCID mice at a dose of 1 x 10⁶ cells. Mice were euthanized after 3 weeks. Femurs were decalcified, and processed tissue sections were stained with hematoxylin-eosin. Disseminated metastasis was observed in the liver, kidney, and lung (detailed in Table 2). Extensive bone marrow metastasis was observed after CXCR4 overexpression. Photomicrographs of representative bone marrow sections are shown (original magnification, x200). (B) CXCR4-overexpressing SK-N-BE(2) cells (1 x 10⁶; SK-N-BE(2)-CXCR4) and control SK-N-BE(2) cells (SK-N-BE(2)-PBABE) were injected subcutaneously, with four mice in each group. After sacrificing the mice at 3 weeks, images of primary tumor, liver metastasis, and kidney metastasis were taken under bright field. Figures shown are representative of tissues from two groups. Liver and kidney metastases could only be observed in the CXCR4-overexpressing group pointed by arrows. (C) In the subcutaneous model, the CXCR4-overexpressing group (SK-N-BE(2)-CXCR4) also showed bone marrow metastasis from femur sections (original magnification, x200).