The chemokine receptor CXCR4 strongly promotes neuroblastoma primary tumour and metastatic growth, but not invasion

Abstract

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.

Figure 1. CXCR4 cell surface expression.

IGR-NB8 and IGR-N91, two well characterized NB cell lines, and transduced cell lines/clones were analyzed for their cell surface CXCR4 expression by flow cytometry (FACS). Percent positive cells is indicated as well as the mean fluorescent intensity (brackets) of the CXCR4 staining. The NB cell line SH-SY5Y constitutively expressed high levels of CXCR4 as previously described . The prostate cancer cell line PC3 showed comparable levels of CXCR4 expression upon transduction as NB8-CXCR4 and N91-CXCR4 clones.

Figure 2. In vivo orthotopic tumour growth and metastasis.

Tumour growth measured in mice engrafted with NB8-E6 and NB8-CXCR4-C3 (a), and N91-E2 and N91-CXCR4-14 (c) The mean tumour volume ± S.D. as measured by echo-doppler at indicated days after implantation is shown. The tumour take expressed as number of mice with tumour/total mice and percent mice with tumours is indicated. (b) and (d) Detection of metastases in mice engrafted with either NB8-E6/NB8-CXCR4-C3 or N91-E2/N91-CXCR4-14 cells. Macroscopic liver metastases were detected by gross examination. Micrometastases were detected in lung, bone marrow, muscle and blood by GFP-PCR. Bars represent percentages of mice with macroscopic liver metastases or GFP-PCR positive signals in indicated organs per tumour bearing animal.

Figure 3. Effect of functional CXCR4 overexpression in NB cell lines.

(a) In vitro growth of NB8-E6, NB8-CXCR4-C3, -B2, and -E9 cells, in 10% (black squares) or 2% serum (open squares). (statistical analysis by two-way ANOVA and Bonferroni correction) A representative of three independent experiments is shown. (b) In vitro growth of NB8-E6 and NB8-CXCR4-C3 cells in the presence of 100 ng/ml CXCL12 (black squares) or in medium alone (open squares). (c) Western blot analysis of ERK1/2 phosphorylation in response to CXCL12 in CXCR4-transduced cells.

Figure 4. NB8 cell migration and invasion in vitro.

(a) Migration of NB8-E6, N91-E2, and CXCR4-overexpressing clones (NB8-CXCR4-C3, -B2, -E9, N91-CXCR4-14) towards CXCL12, in absence or presence of the CXCR4 blocker (TN14003 inhibitor). Results of a representative experiment are shown and are expressed as mean number of cells in one field. Five fields were counted for each result. The experiment was performed in triplicates and repeated three times. Error bars indicate S.D. (b) Invasion of SH-SY5Y, NB8 and N91 control and CXCR4 transduced clones through Matrigel®-coated membranes in N2 supplemented serum-free medium alone, with or without CXCL12. CXCL12-mediated invading cells (invaded cell in the absence of CXCL12 have been subtracted) -in a typical field are shown. The mean number of invading cells in 5 fields is given. Error bars indicate S.D. (* indicates p<0.05, Student's t-test)

Figure 5. CXCL12 production in mouse tissues and NB tumours.

The production of CXCL12 was measured by ELISA in normal mouse tissues, as well as in the different clones, and primary tumours. Results of triplicates are expressed as pg of CXCL12 per mg of extracted protein. One representative of three independent experiment is shown. Experiments were performed in triplicates.

Figure 6

(a) FACS profiles of shRNA-mediated cell surface CXCR4 silencing in N91-shRNA-CS1 and N91-shRNA-CS2 clones and N91-pAB303 control cells. Percent positive cells is indicated as well as the mean fluorescent intensity (brackets) of the CXCR4 staining. (b) In vitro growth capacities of N91-shRNA-CS1, N91-shRNA-CS2 clones and N91-pAB303 control cells. (c) In vivo growth capacities of IGR-N91 cells were CXCR4 has been knocked down by shRNA. Comparison of tumour volumes after 44 days when mice were sacrificed. * indicates p<0.05 (Mann-Whitney test).