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Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis

MERAV DARASH-YAHANA^*, ELI PIKARSKY, RINAT ABRAMOVITCH^*,, EVELYNE ZEIRA^*, BOAZ PAL^*, REBEKAH KARPLUS^*, KATIA BEIDER^*, SHANI AVNIEL^*, SHAFIKA KASEM, EITHAN GALUN^* and AMNON PELED^*,1

^* Goldyne Savad Institute of Gene Therapy,
Department of Pathology and
MRI Lab, HBRC, Hadassah Hebrew University Medical Center, Jerusalem, Israel

¹Correspondence: Goldyne Savad Institute of Gene Therapy, P.O.B. 12000, Jerusalem 91120, Israel. E-mail: peled{at}hadassah.org.il

SPECIFIC AIMS

Recent reports suggest a crucial role for high levels of CXCR4 expression in the metastatic process, however these studies do not distinguish between the role of CXCR4 in the arrest of tumor cells on endothelium, extravasation into target tissue, or initial survival, growth, vascularization, and invasiveness within target tissue. The purpose of this study was to examine the role of high expression levels of chemokine receptor CXCR4 on prostate tumor growth in vitro and in vivo. We examined the expression pattern of CXCR4 and CXCL12 in prostate cancer cell lines, human prostate cancer xenografts, and in primary human prostate cancers. We next studied the effect of overexpressing CXCR4 on in vivo tumor growth, angiogenesis, and metastasis, and its effect on in vitro proliferation and migration of human hormone refractory prostate cancer cell lines.

PRINCIPAL FINDINGS

1. CXCR4 overexpression correlates with prostate cancer metastasis
Immunohistochemical staining showed CXCR4 expression in almost all primary human prostate tumor biopsies, but not in normal human prostate tissue. Immunohistochemical analysis revealed that 26 of 116 prostate tumors were strongly positive (22%) for CXCR4 staining. In two studies, incidence of prostate tumors expressing high levels of CXCR4 was higher in patients who developed metastasis. These results are strongly supported by the work of Sun YX. et al., which reported that in clinical samples from a large cohort (n=600) of prostate patients, expression of CXCR4 correlated with increased malignancy. The importance of increased CXCR4 expression to the metastatic phenotype of prostate tumors was not addressed in this study.

2. Overexpression of CXCR4 accelerates prostate cell proliferation and VEGF secretion
Low cell surface expression levels of CXCR4 were observed in PC3, DU145, LNCaP and 22Rv1 prostate cancer cell lines. To study in vitro and in vivo roles of CXCR4 in prostate cancer development and metastasis, we generated PC3 and 22Rv1 prostate cancer cell lines that express high levels of CXCR4. For in vivo monitoring of tumor growth and metastases, PC3 cells were also stably transduced with a retrovirus carrying the luciferase (luc) gene (PC3L). In vitro, CXCL12 stimulated (in a dose-dependent manner) proliferation of PC3LG-CXCR4 and 22Rv1G-CXCR4 only. CXCL12 distinctly up-regulated secretion of VEGF in PC3LG-CXCR4 tumor cells. In vitro, we found no effect of CXCL12 on migration of PC3 and 22Rv1 cells overexpressing CXCR4.

3. Overexpression of CXCR4 accelerates prostate tumor growth
To investigate the role of CXCR4 in prostate tumor development in vivo, PC3LG, PC3LG-CXCR4, 22Rv1G, and 22Rv1G-CXCR4 cells were injected subcutaneously into NOD/SCID mice. Mice injected with cells expressing high levels of CXCR4 developed larger tumors with increased tumor burden vs. control tumors (Fig. 1 A, B). Neutralizing interactions of CXCL12/CXCR4 in vivo with CXCR4 specific antibodies inhibited CXCR4-dependent tumor growth of PC3LG-CXCR4 cells (Fig. 1C ). The effect of overexpression of CXCR4 on growth and metastasis was further tested using luc gene technology with a cooled charged coupled device (CCCD) camera. In vitro PC3LG-CXCR4 and PC3LG cells both expressing the luciferase gene showed similar levels ofrelated light units (Fig. 1D ). An increased difference in tumor size between tumors derived from PC3LG-CXCR4 vs. PC3LG cells was detected over time (*P<0.01, Fig. 1E, F ). CCCD images of tumors derived from PC3LG-CXCR4 cells and PC3LG cells were taken on days 15, and 107 following injection of tumor cells (Fig. 1F ). MRI analysis further demonstrated increased tumor growth in PC3 and 22Rv1 prostate tumor cells overexpressing CXCR4. Both MRI and morphologic analyses of H&E-stained tissue sections taken from all PC3 tumors overexpressing CXCR4 showed invasion of the tumor into adjacent striated muscle tissue. These results suggest a role for CXCR4 in growth, invasion, and spreading of prostate tumors within the target organ.

View larger version (34K): [in this window] [in a new window]   Figure 1. Overexpression of CXCR4 increases size and weight of tumors in vivo. A, B) PC3LG, PC3LG-CXCR4, 22Rv1G, and 22Rv1G-CXCR4 cells (5x106/mice) were injected SC into NOD/SCID mice. After 90 days, tumor-size (cm3) and weight (g) were measured. For the first set of experiments (black bars), each mean value represents a minimum of 5 mice +/– SE from 3 different experiments (*P<0.01). C) Effects of neutralizing antibodies to CXCR4 on PC3LG-CXCR4 and PC3LG injected tumors was tested. D) PC3LG-CXCR4 and PC3LG cells have similar levels of luc activity in vitro as measured by related light units. E, F) Disease progression of PC3LG or PC3LG-CXCR4 tumors was monitored on days 15–107 using a CCCD camera for localization and intensity of light emission following injection of luciferin. Data shown (E) represent the average of 3 experiments, five mice in each group ± SE (*P<0.01). A representative mouse in each experimental group (F) is shown.

4. Overexpression of CXCR4 accelerates prostate tumor vascularization
The finding that tumors that do not overexpress CXCR4 grow slowly, developing a necrotic core when reaching a diameter of 10mm³, while tumors that overexpress CXCR4 continue to grow beyond this point, suggests that CXCR4 may facilitate angiogenesis in tumors. We studied this hypothesis by comparing the number of blood vessels in histological sections as well as by using an in vivo intratumoral vessel functionality MRI-based assay. Macroscopic assessment of tumors overexpressing CXCR4 on day 90 revealed increased vascularization. To analyze blood vessel density in the tumors, we performed immunohistochemical staining using an antibody against Factor VIII. After staining, the average number of blood vessels per high power field was counted (excluding necrotic areas). Factor VIII staining on tissue sections collected on day 30 showed a 4.5-fold increase in the number of blood vessels in tumors overexpressing CXCR4 (8.9±1) vs. control (1.67±0.7) tumors (Fig. 2A , *P<0.01). An overall tendency for reduced blood vessel numbers in response to anti-CXCR4 treatment was shown in both PC3LG-CXCR4 and PC3LG groups.

View larger version (22K): [in this window] [in a new window]   Figure 2. Overexpression of CXCR4 promotes tumor vascularization in vivo. A) NOD/SCID mice were implanted subcutaneously with transduced PC3LG or PC3LG-CXCR4 cells. Microvessel density (Factor VIII) staining was performed on paraffin-embedded tissue sections derived from CXCR4 and control tumors. Data shown represent mean value of counted blood vessels of stained tumor sections from days 30 and 60 (five mice in each group ± SE, *P<0.01). B) MRI analysis of vessel functionality (VF). Color-coded VF maps were derived and overlaid (|VF|>*0.01, see color scale) on original coronal images. A representative image of VF maps from control and CXCR4 mice on day 73 is shown. C) Mean ± SD values of VF were calculated from the region of interest containing the whole tumor, and were normalized to contra-lateral muscle using 3 mice per group and 4 slices per mouse. VF fold values for CXCR4 tumors were higher during the entire experiment (*P<0.01, **P<0.05).

To complement data obtained on blood vessel density, vessel functionality (VF) was measured by MRI to study oxygen delivery efficiency into the tumor mass. Functionality of the vasculature was derived from GE images acquired during inhalation of air-CO₂ and carbogen (95% oxygen+5% CO₂). MRI analysis showed that VF values for PC3LG-CXCR4 tumors were significantly higher during the entire experiment (Fig. 2B ). VF maps showed an increase in vessel functionality in tumors produced by PC3 cells overexpressing CXCR4 vs. control (Fig. 2C ). While functional vessels were observed at the center of tumors overexpressing CXCR4, no functional vessels were observed at the center of control tumors (Fig. 2B ). Mean ± SD values of VF were calculated from a region of interest containing the whole tumor, and normalized to contra-lateral muscle (Fig. 2C , P<0.01). These results suggest that high expression of CXCR4 results in early neovascularization of tumors while in control tumors development of necrosis was mediated by poor perfusion.

5. Overexpression of CXCR4 accelerates prostate tumor metastasis
Metastatic potential of tumors overexpressing CXCR4 as opposed to control tumors was also examined. This was done by analyzing whole body CCCD images of mice expressing the luc gene and collecting tissue samples from lungs, lymph nodes (LN) and bones. Bone metastasis was not observed for CXCR4 or control mice, and metastases to LN and lungs were detectable. We found that mice implanted with prostate cells overexpressing CXCR4 (PC3LG-CXCR4) showed an accelerated pattern of metastasis to LN and lungs. The LN metastatic pattern of mice implanted with cells overexpressing CXCR4 was apparently more widely spread, as seen in whole body CCCD images. These results suggest a role for high levels of CXCR4 in prostate cancer metastasis.

CONCLUSIONS AND SIGNIFICANCE

Prostate cancer is unique among epithelial malignancies because it preferentially metastasizes to bone marrow (BM) and LN. It was recently reported that treatment of SCID mice with anti-CXCR4 neutralizing mAbs following intravenous or orthotopic injection of human breast cancer tumor cells significantly inhibited LN and lung metastases. In clear cell renal carcinoma, an association of strong CXCR4 expression with poor tumor-specific survival was also recently reported. While these data suggest a crucial role for CXCR4 in the metastatic process, they do not distinguish between the role of chemokine receptors in arrest of tumor cells on the endothelium, extravasation into target tissue, or initial survival, growth, vascularization, and invasiveness within target tissue.

What then is the role-played by high expression levels of CXCR4 in prostate cancer? Most recent studies of expression of CXCR4 in malignant tissues suggest that this receptor is important in recruitment of circulating neoplastic cells to distant sites ("homing" hypothesis). This would presumably be due to improved interaction with BM endothelial cells (Fig. 3 A, homing). Our findings support a role for CXCR4 in prostate cancer cells spreading within target tissue rather than improving interaction with endothelial cells. Increased CXCR4 expression may enhance tumor metastasis through at least two additional mechanisms, enhanced proliferation by activating the MAP/Erk kinase pathway and accelerated angiogenesis by increasing VEGF secretion (Fig. 3C , growth). These mechanisms may act at primary sites as well as at distant sites throughout the lifespan of the tumor. In respect to metastasis, proliferative and angiogenic mechanisms would not alter frequency of metastatic seeding, but would rather affect the ability of a micrometastasis to develop into a clinically relevant lesion.

View larger version (29K): [in this window] [in a new window]   Figure 3. A model for the role of high expression levels of CXCR4 in prostate tumor metastasis. CXCR4 can facilitate interaction between tumor cells and endothelial cells by activating 1) rolling; 2) integrin function; 3) arrest; and 4) transendothelial migration of tumor cells (A-homing). Following transendothelial migration, CXCL12 can direct intratissue localization of tumor cells (B-invasion). At the site of metastasis, tumor growth and survival as well as angiogenesis depends on ERK activation and increased VEGF secretion CXCR4 (C-growth).

In conclusion, high levels of chemokine receptor CXCR4 confer a more aggressive behavior on prostate cancer cells. CXCR4 may act not only as a homing receptor, but also as a positive regulator of tumor growth and angiogenesis, indicating the possibility of using CXCR4 as a future therapeutic interventional target.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0935fje; doi: 10.1096/fj.03-0935fje

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