Expression and functional heterogeneity of chemokine receptors CXCR4 and CXCR7 in primary patient-derived glioblastoma cells

Abstract

Glioblastoma (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of these tumors indicates a need to identify new therapeutic targets. Therapy resistance of GBMs is attributed to heterogeneity of the glioblastoma due to genetic alterations and functional subpopulations. Chemokine receptors CXCR4 and CXCR7 play important roles in progression of various cancers although the specific functions of the CXCL12-CXCR4-CXCR7 axis in GBM are less characterized. In this study we examined the expression and function of CXCR4 and CXCR7 in four primary patient-derived GBM cell lines of the proliferative subclass, investigating their roles in in vitro growth, migration, sphere and tube formation. CXCR4 and CXCR7 cell surface expression was heterogeneous both between and within each cell line examined, which was not reflected by RT-PCR analysis. Variable percentages of CXCR4+CXCR7- (CXCR4 single positive), CXCR4-CXCR7+ (CXCR7 single positive), CXCR4+CXCR7+ (double positive), and CXCR4-CXCR7- (double negative) subpopulations were evident across the lines examined. A subpopulation of slow cell cycling cells was enriched in CXCR4 and CXCR7. CXCR4+, CXCR7+, and CXCR4+/CXCR7+ subpopulations were able to initiate intracranial tumors in vivo. CXCL12 stimulated in vitro cell growth, migration, sphere formation and tube formation in some lines and, depending on the response, the effects were mediated by either CXCR4 or CXCR7. Collectively, our results indicate a high level of heterogeneity in both the surface expression and functions of CXCR4 and CXCR7 in primary human GBM cells of the proliferative subclass. Should targeting of CXCR4 and CXCR7 provide clinical benefits to GBM patients, a personalized treatment approach should be considered given the differential expression and functions of these receptors in GBM.

Figure 1. CXCL11, CXCR4, and CXCR7 expression by primary patient-derived GBM cells.

(A) RT-PCR analysis detected CXCL11, CXCR4, and CXCR7 mRNAs in L0, L1, L2, and S2 cells. Actin was used as a control. Panels are representative from three independent experiments. (B) CXCR4 and CXCR7 are heterogeneously expressed on the cell surface of primary GBM cells as determined by flow cytometry analysis. Representative pictures are shown. (C) CCR3 and CXCR3 are co-expressed on the cell surface of primary GBM cells as determined by flow cytometry analysis. Representative pictures are shown.

Figure 2. Increased CXCR3, CXCR4, and CXCR7 in slow cycling GBM cells.

Flow cytometry identified significantly higher percentages of CXCR4-, CXCR7- and CXCR3-expressing cells in slow cycling subpopulations of GBM cells when compared to the overall population, with the exception of CXCR4-expressing cells in the overall population of S2 that is more abundant than in the slow cycling population. Filled bar: overall population. Open bar: slow cycling population. *P<0.05; **P<0.01.

Figure 3. CXCL12 stimulated sphere formation in vitro and CXCR4+, CXCR7+, and CXCR4+/CXCR7+ cells generate tumors in vivo.

(A) CXCL12 concentration-response assessment indicated that CXCL12 promoted in vitro sphere formation of L0 cells (0.3, 1, 3, 10, 30 nM, P<0.05). CCX733 (100 nM) suppressed CXCL12-regulated (10 nM) sphere formation while AMD3100 (1 µM) had no effect. All conditions contained 0.1% DMSO. ***P<0.001. (B) Representative sections from tumors derived from L0 sub-populations. The various sub-populations indicated in the figures were implanted intracranially into NSG mice. Shown are representative sections subjected to anti-human Nestin immunohistochemistry. Note that all sub-populations are capable of forming tumors in vivo.

Figure 4. CXCL12 promoted cell growth through both CXCR4 and CXCR7.

(A) CXCL12 concentration-response assessment showed that CXCL12 significantly enhanced cell growth in L0 and L1 cells. *P<0.05; **P<0.01. Representative results of three individual experiments performed in triplicate are shown. (B) AMD3100 (1 µM), CCX733 (100 nM), and CCX771 (100 nM) significantly blocked CXCL12 (10 nM) induced cell growth in L0 and L1 cells. CCX704 (100 nM) had no effect. All conditions contained 0.1% DMSO. *P<0.05; **P<0.01. Representative results of three individual experiments performed in triplicate are shown.

Figure 5. Differential impact of TMZ on the distribution of CXCR4- and CXCR7-expressing apoptotic and non-apoptotic subpopulations.

(A) GBM lines were treated with TMZ as described in Methods. Apoptotic subpopulations, determined by positive ApoStat staining, were detected in TMZ treated samples in all cell lines. Histograms are representative from three independent experiments. (B) Heterogeneity in CXCR4- and CXCR7-expressing subpopulations in the apoptotic and non-apoptotic subpopulations after TMZ treatment, as determined by flow cytometry analysis. Bar graphs represent the average of three independent experiments. Data were analyzed with Graphpad Prism 5 software.

Figure 6. CXCL12 stimulated GBM cell migration in L0 and S2 cells is mediated by CXCR4 and CXCR7.

(A) CXCL12 concentration-response assessment showed that CXCL12 significantly enhanced cell migration in L0 and S2 cells (0.3, 1, 3, 10, 30 nM). *P<0.05; **P<0.01. Representative results of three individual experiments performed in triplicate are shown. (B) AMD3100 (1 µM), CCX733 (100 nM), and CCX771 (100 nM) significantly blocked CXCL12 (3 nM) induced cell migration in L0 and S2 cells. *P<0.05; **P<0.01. CCX704 did not alter CXCL12 induced cell migration. All conditions contained 0.1% DMSO. Representative results of three individual experiments performed in triplicate are shown.

Figure 7. CXCL12 stimulation of L0 tube formation in vitro is mediated by CXCR4.

(A) Representative images of control and CXCL12 (20 nM) treated GBM L0 cells. (B) CXCL12 (20 nM) significantly increased total tube length, total tube area, and total branch points of L0 cells. AMD3100 (1 µM) inhibited CXCL12 stimulation of tube formation of L0 cells. CXCR7 inhibitors did not block the stimulation of CXCL12. All conditions contained 0.1% DMSO. *P<0.05; **P<0.01. Representative results of three individual experiments performed in triplicate are shown.