Abstract
The ability of CCL2 to influence prostate cancer tumorigenesis and metastasis may occur through two distinct mechanisms: 1) a direct effect on tumor cell growth and function, and 2) an indirect effect on the tumor microenvironment by the regulation of macrophage mobilization and infiltration into the tumor bed. We have previously demonstrated that CCL2 exerts a direct effect on prostate cancer epithelial cells by the regulation of their growth, invasion, and migration, resulting in enhanced tumorigenesis and metastasis. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis by regulating monocyte/macrophage infiltration into the tumor microenvironment and by stimulating a phenotypic change within these immune cells to promote tumor growth (tumor-associated macrophages). VCaP prostate cancer cells were subcutaneously injected in male SCID mice and monitored for tumor volume, CD68+ macrophage infiltration, and microvascular density. Systemic administration of anti-CCL2 neutralizing antibodies (CNTO888 and C1142) significantly retarded tumor growth and attenuated CD68+ macrophage infiltration, which was accompanied by a significant decrease in microvascular density. These data suggest that CCL2 contributes to prostate cancer growth through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor.
Keywords: Monocyte chemoattractant protein 1, prostate cancer, chemokine, tumor-associated macrophage, angiogenesis
Introduction
The mechanisms by which elevated levels of chemokines and upregulation of chemokine receptors influence cancer development and metastasis remain unclear. Increasing evidence suggests that chemokines secreted from stromal elements (e.g., bone marrow endothelial cells) directly stimulate tumor cells that express (or overexpress) chemokine receptors and regulate tumor cell proliferation and migration [1]. Alternatively, tumor cells can secrete elevated levels of chemokines to induce a paracrine/autocrine response on tumor growth. Dysregulation of chemokines and chemokine receptors has been demonstrated to alter tumor development and progression through enhanced proliferation, increased invasiveness, increased angiogenesis, and recruitment of immune cells that promote tumor growth (e.g., tumorassociated macrophages or TAMs). Monocyte chemoattractant protein 1 (CCL2) is a member of the cytokine/chemokine superfamily and is known to promote the migration of monocytes and macrophages to sites of inflammation. Recently, CCL2 has been shown to be an active mediator of the tumorigenesis and metastasis of several solid tumors, including a role in regulating the migration and proliferation of breast cancer, multiple myeloma, and, recently, prostate cancer [2–6].
Lu et al. [7] examined CCL2 expression in 83 patients with prostate cancer and determined that CCL2 expression correlated with advanced stage and that prostate cancer cells produced CCL2 in vitro, which mediated proliferation and invasion in an autocrine/paracrine manner. We have previously reported the ability of CCL2 to influence prostate cancer tumorigenesis and metastasis through a direct promotional effect on tumor cell proliferation and migration [6]. We have also demonstrated that prostate cancer cells in vitro and in human cancer tissues exhibit an upregulation of the CCL2 receptor CCR2. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor. Using anti-human (CNTO888)-specific and anti-mouse (C1142)-specific neutralizing antibodies to CCL2, we demonstrate inhibition of prostate tumor growth and migration in vivo through direct effects on prostate cancer cells and blocking of TAM infiltration into the tumors (indirect effects).
Materials and Methods
Materials
Human recombinant CCL2 was obtained from Chemicon International (Temecula, CA); anti-phospho AktSer473, anti-Akt, anti-phospho p44/p42, and anti-total p44/p42 were obtained from Cell Signaling (Beverly, MA); and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO).
Description of CNTO888 and C1142, and Control Antibodies
CNTO888 is a human IgG1κ antibody that neutralizes human CCL2 (Centocor, Inc., Malvern, PA). C1142 is a rat/mouse chimeric antibody that neutralizes mouse CCL2/JE. CNTO888 and C1142 do not cross-react with or neutralize mouse CCL2/JE or human CCL2, respectively (data not shown). Clinical-grade human IgG (huIgG) served as a negative control for CNTO888, whereas C1322 rat/mouse chimeric nonspecific antibody (Centocor, Inc.) served as a negative control for C1142.
Cell Culture
VCaP cells are a human prostate cancer cell line derived from vertebral bone metastasis [8]. VCaP cells were maintained in Dulbecco's modified Eagle's medium 1640 + 10% fetal calf serum (Invitrogen, Carlsbad, CA). Cells were passaged by trypsinization using 1 x trypsin + EDTA (Invitrogen) and resuspended in appropriate growth media.
Xenograft Model of Tumorigenesis
Xenograft tumors were established as previously described [9]. Briefly, male SCID mice (5–6 weeks of age) were injected subcutaneously in the flank with 1 x 106 VCaP cells in 200 µl of Matrigel (BD Biosciences, Inc., San Jose, CA). Tumor volumes were calculated by caliper measurements performed weekly to monitor and track tumor growth (tumor volume = LWW x 0.56). Mice were separated into one of four groups (n = 5 per group): 1) huIgG; 2) C1322 control mouse antibody; 3) anti-CCL2 (CNTO888); and 4) anti-CCL2/JE (C1142). Mice were treated with 2 mg/kg antibody, twice weekly, by intraperitoneal injection beginning on day 28 and for the remainder of the study.
Histology
Xenograft tumors were harvested and placed in fresh 10% formalin. Tumors were paraffin-embedded, and 5-µm sections were cut and placed on glass slides. Hematoxylin-eosin staining was performed according to the manufacturer's instructions (Sigma, Inc., St.Louis, MO). Identificationof neovascularization was accomplished by labeling with an anti-CD31 antibody, and macrophage infiltration was identified using an anti-CD68 antibody. Tissue sections were incubated for 10 minutes in citrate buffer (pH 6.0) and microwaved. Sections were incubated with anti-CD31 (1:50; DakoCytomation, Inc., Carpinteria, CA) or anti-CD68 (1:1600; DakoCytomation, Inc.) for 30 minutes and detected with LSAB + detection/DAB (3,3∼-diaminobenzidine; Sigma, Inc.) for 5 minutes. Slides were dipped in hematoxylin for 1 second as a counterstain.
Endothelial Tube Formation Assay
In vitro tube formation was performed as previously described [10]. Growth factor-reduced Matrigel was diluted with cold serum-free medium to a concentration of 10 mg/ml. Fifty microliters of the solution was added to each well of a 96-well plate and allowed to form a gel at 37°C for 30 minutes. Human dermal microvacular endothelial cells (HDMVECs; 150,000 cells/ml) in VCaP conditioned media (VCaP CM) were added to each well and incubated overnight at 37°C in 5% CO2. Either control antibodies (huIgG or C1322; 30 µg/ml) or anti-CCL2 antibodies (CNTO888 and/or C1142; 30 µg/ml) were added to the conditioned media. Under these conditions, endothelial cells will form delicate networks of tubes that are detectable within 2 to 3 hours and are fully developed after 8 to 12 hours. After overnight incubation, the wells were washed, and the Matrigel and its endothelial tubes were fixed with 3% paraformaldehyde. Tube formation was quantified by counting the number of sprouts that developed per objective field (x 100), and assays were performed in triplicate from three independent experiments.
Macrophage Migration
Human recombinant CCL2 was used as a chemoattractant in the lower chamber of a modified Boyden chamber. Cells were harvested by 0.5 µM EDTA release and resuspended in serum-free media at 5 x 104 cells/ml. A total of 2.5 x 104 cells was added to the upper chamber of the transwell insert and incubated for 24 hours at 37jC and 5% CO2 atmosphere. At the end of the incubation period, the cells were fixed with 4% formaldehyde in phosphate-buffered saline for 5 minutes. Nonadherent cells were removed from inserts with cotton-tipped swab. Cells that had migrated to the underside of the inserts were stained with 0.5% crystal violet for 5 minutes and rinsed thoroughly with tap water. The inserts were allowed to dry, and the cells were counted using an inverted microscope.
Immunoblot Analysis
Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 µM okadaic acid, and 1 µg/ml aprotinin, leupeptin, and pepstatin). Proteins were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in 0.1% Tween in Tris-buffered saline (TBS) for 1 hour at room temperature and then were incubated overnight at 4°C with primary antibodies. Membranes were washed thrice before incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) for 1 hour at room temperature. Protein expression was visualized by ECL chemiluminescence (Promega, Madison, WI) and quantitated using Image J software (NCI, Bethesda, MD).
Statistical Analysis
Data were analyzed with GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). One-way analysis of variance was used with Bonferroni's post hoc analysis for comparison between multiple groups. Student's t test was used for comparison between two groups. Significance was defined as P < .05.
Results
To determine the role of CCL2 in prostate cancer growth in vivo, we implanted VCaP xenografts in male SCID mice (n = 5) and monitored tumor growth by caliper measurement and calculation of tumor volume. Twenty-eight days post-implantation, tumors were approximately 200 mm3, and mice were divided into therapy groups: 1) huIgG control antibody; 2) anti-human CCL2 antibody CNTO888; 3) mouse control antibody C1322; and 4) anti-mouse CCL2/JE antibody C1142. Mice were treated with antibodies at 2 mg/kg, twice weekly, by intraperitoneal injection. Antibodies were delivered for 3 weeks, and animals were sacrificed on day 50. Tumor volume measurements revealed a 55% reduction in tumor growth on day 50 by the administration of a neutralizing anti-mouse CCL2/JE antibody (C1142) compared to the administration of C1322 (Figure 1A). Similarly, administration of a neutralizing anti-human CCL2 antibody (CNTO888) resulted in a 42% reduction in tumor volume on day 50 compared to the administration of huIgG control antibody (Figure 1B). These data suggest that inhibition of either tumor-derived human CCL2 or stromal mouse-derived CCL2/JE can significantly delay the growth of these tumors.
Figure 1.
Inhibition of tumor growth by neutralization of CCL2. Tumor volume was monitored weekly by caliper measurements in mice receiving C1142 versus C1322 (A), and huIgG versus CNTO888 (B). Antibody treatment was initiated on day 28 (Tx) and administered at 2 mg/kg, i.p., twice weekly, for the duration of the experiment. Immunohistochemical analysis of xenograft tumors displayed normal prostate adenocarcinoma morphology by hematoxylin-eosin staining in mice treated with C1322 (C), C1142 (D), huIgG (G), or CNTO 888 (H). Neovascularization for each individual tumor (n = 5 per group; four fields per tumor) was visualized by immunohistochemical staining of CD31, and representative sections are presented (E, F, I, and J). (K) The ability of CCL2 inhibition to reduce blood vessel formation was analyzed using an in vitro tube formation assay. Tube formation using HDMVECs was induced by VCaP CM, and the number of sprouts per x 100 objective field is displayed. The graph represents the mean of three independent experiments, each performed in triplicate (mean ± SD; *P < .01).
To understand possible mechanisms by which antibodies could delay tumor growth, xenograft tumors were collected for histologic analysis and quantification of microvascular density. Sections were stained for CD31 (PECAM, a marker of vascular endothelium). Inhibition of CCL2 with either anti-human CCL2 or anti-mouse CCL2/JE neutralizing antibodies decreased the amount of angiogenesis as identified by a decrease in CD31 staining compared to isotype controls [Figure 1, E and F (anti-mouse), and I and J (anti-human)]. To further elucidate the role of CCL2 inhibition in blood vessel formation, we applied an in vitro tube formation assay as previously described [10]. Human dermal microvascular endothelial cells grown in VCaP CM in Matrigel formed a capillary-like network of tubes (Figure 1K). Administration of either CNTO888 or C1142 (30 µg/ml) to VCaP CM significantly reduced the number of capillary-like tubes that formed compared to isotype control antibody-treated cells (Figure 1K).
Here we assessed the proliferative status of VCaP xenografts by immunohistochemical staining for Ki67 (a marker of proliferation) and contrasted the effects on proliferation with apoptosis by staining similar sections with an apoptosis stain (ApopTag; Millipore, Billerica, MA) (Figure 2). Qualitative analysis revealed that inhibition of host-derived CCL2 resulted in a significant decrease in Ki67 staining and a modest induction of apoptosis (Figure 2, A–F). Inhibition of tumor-derived CCL2 did not demonstrate a significant induction of apoptosis (Figure 2, J and K) and a mild decrease in proliferation, as evidenced by a slight decrease in Ki67 staining (Figure 2, L and M). The modest effect of anti-CCL2 therapy on apoptosis remains unclear; however, inhibition of host-derived CCL2 significantly attenuated proliferation. This decrease in Ki67 staining was accompanied by a decrease in phosphorylated Akt and phosphorylated p44/p42 MAPK (Figure 3, A–H). As a mechanism of induced proliferation, VCaP cells were stimulated with increasing concentrations of human CCL2 in vitro and demonstrated a dose-dependent activation of Akt (Figure 4A) and p70 S6 kinase, a downstream target of Akt that is known to be important in cellular proliferation (Figure 4B). Furthermore, we assessed the inhibition of CCL2 using the anti-human CCL2 antibody CNTO888 (30 µg/ml) to attenuate the in vitro proliferation of VCaP cells. Administration of anti-human CCL2 antibodies reduced the proliferation of VCaP cells over a 96-hour period (Figure 4C).
Figure 2.
Inhibition of mouse CCL2 reduces proliferation in VCaP xenografts. Apoptosis was visualized by immunohistochemical staining with ApopTag (A–D, H–K). Proliferation was visualized by immunohistochemical staining for Ki67 (E–F, L–M).
Figure 3.
Inhibition of mouse CCL2 attenuates Akt and MAPK p44/p42 activity. Tumor specimens were collected and stained with anti-phospho Akt (A and B; E and F) or anti-phospho p44/p42 (C and D; G and H) to visualize the intratumoral activity of these signaling pathways.
Figure 4.
CCL2 stimulates Akt and p70S6 kinase activation in VCaP cells. CCL2 induces Akt phosphorylation (A) and p70S6 kinase phosphorylation (B) in a dose-dependent fashion. (C) VCaP cells (8 x 105 cells/well) were treated with hrCCL2 (100 ng/ml) for 24 to 72 hours in the presence or in the absence of CNTO888 (30 µg/ml). The proliferation of VCaP cells was assessed by WST-1 assay (□ control; ◆ 30 µg/ml CNTO888; ▴ 100 ng/ml hrCCL2; ▾ 100 ng/ml hrCCL2 + 30 µg/ml CNTO888).
CCL2 is known to promote monocyte/macrophage infiltration into tissues, and the role of TAMs in prostate cancer biology has demonstrated a direct role in the regulation of tumor growth and angiogenesis [6,7]. We assessed macrophage infiltrates in xenograft tumors by immunohistochemistry. Macrophages were identified by CD68+ (lysosomal glycoprotein, a marker of monocytes and macrophages) staining. Inhibition of CCL2 reduced monocyte/macrophage infiltration, as evidenced by a lack of CD68+ staining in treated tumors compared to control tumors [Figure 5, A and C (antihuman), and B and D (anti-mouse)]. Macrophage infiltration was quantified by manual counting of CD68+ cells per x 100 objective field. Inhibition of either human or mouse CCL2 demonstrated a significant decrease in the number of CD68+ cells present within VCaP xenografts (huIgG: 85.33 ± 12.10; CNTO888: 9.00 ± 7.94; C1322: 131.00 ± 19.08; C1142: 13.67 ± 3.06; mean ± SD, P < .0001) (Figure 5E). Inhibition of macrophage migration using CNTO888 was confirmed in an in vitro migration assay (Figure 6). VCaP CM induced a significant increase in the number of migrating human U937 premonocytic cells (a premacrophage cell line). Preincubation of U937 cells with CNTO888 (30 µg/ml) significantly attenuated the migratory effect induced by VCaP CM (SFM: 33 ± 69; VCAP CM: 805 ± 28; huIgG: 756 ± 136; CNTO888: 225 ± 111; P < .001).
Figure 5.
Inhibition of CCL2 reduced the number of infiltrating macrophages in VCaP xenografts. Macrophage infiltration was visualized by immunohistochemical staining for CD68 (A–D). (E) The number of infiltrating macrophages was determined by counting the total number of macrophages in four independent fields per section (n = 3 tumors per group were analyzed for CD68). The data are presented as mean ± SD (*P < .01).
Figure 6.
Inhibition of tumor-derived CCL2 reduces the number of migrating U937 monocytes. U937 cell migration was measured in response to VCaP CM. The total number of migrating U937 cells was calculated by manually counting four independent fields (migration assays were performed in triplicate). The data are presented as mean ± SD (*P < .001).
Discussion
We continue to gather increasing evidence demonstrating an important role for CCL2 in prostate cancer tumorigenesis. As a result of in vitro studies demonstrating that PCa cells are stimulated to proliferate and migrate by CCL2, we sought to determine the effects of CCL2 inhibition in vivo on tumor growth and macrophage infiltration. The data presented here describe the use of CCL2 neutralizing antibodies that specifically recognize either the human CCL2 (CNTO888) or the mouse homolog CCL2/JE (C1142) in an in vivo xenograft model of prostate cancer. Neutralizing antibodies that specifically inhibit human versus mouse CCL2 lend a powerful tool for clearly dissecting the role of tumor-derived CCL2 (human CCL2) and host/microenvironment-derived CCL2 (mouse CCL2/JE). Tumor volume measurements revealed a 42% reduction of tumor growth on day 50 by the administration of a neutralizing anti-human CCL2 (CNTO888) antibody compared to human IgG control (Figure 1). Similarly, the administration of a neutralizing anti-mouse CCL2/JE (C1142) antibody resulted in a 55% reduction in tumor volume on day 50 compared to the administration of mouse control antibody (Figure 1). These data suggest that CCL2 is produced directly by tumor cells and from stromal cells of the microenvironment, both of which influence tumor growth in vivo. These data do not indicate whether the growth benefit of CCL2 is a reflection of direct tumor cell stimulation by CCL2 or by CCL2-mediated changes in the microenvironment that impact tumor growth. Inhibition of CCL2 with either anti-human CCL2 (CNTO888) or anti-mouse CCL2/JE (C1142) neutralizing antibodies decreased the amount of angiogenesis, as identified by a decrease in CD31 staining compared to relevant control groups (Figure 1). Furthermore, inhibition of CCL2 attenuated monocyte/macrophage infiltration as evidenced by a lack of CD68+ staining compared to controls (Figure 2). Although these studies were performed in a small sample size (n = 5 per group) and differences were observed between the two control antibody groups, these differences were not significant, and a significant decrease in macrophage infiltration was quantified in both the anti-mouse CCL2/JE (C1142) and the anti-human CCL2 (CNTO888) treatment groups. These data suggest that inhibition of CCL2, both tumor-derived and host-derived, attenuates the growth of prostate cancer xenografts potentially by a dual mechanism: 1) a direct effect on tumor cell survival and proliferation, and 2) changes in the microenvironment that provide tumor cell advantage (e.g., angiogenesis and infiltration of macrophages).
The pronounced decrease in tumor growth and the inhibition of histologic Ki67 staining by anti-mouse CCL2 antibody C1142 suggest that the predominant mechanism of CCL2-regulated tumor growth is the regulation of immune cell infiltration (e.g., TAMs). TAMs are known to produce elevated levels of mitogenic stimuli (e.g., VEGF) that directly stimulate prostate cancer cell growth and induce neovascularization of neoplastic tissues. Interestingly, CCL2 has been demonstrated to play an important role in osteoclast activation in prostate cancer [11]. Osteoclasts are terminally differentiated cells formed from the fusion of monocyte/macrophage lineage cells. The data presented here support the role of CCL2 in regulating monocyte lineage cells in the development of prostate cancer and demonstrates that inhibition of CCL2 changes the host-tumor microenvironment by inhibiting the infiltration of immune cells that promote tumor development.
The relationship between cancer and inflammation has been recognized since the 1860s when Virchow observed leukocytes in neoplastic tissues [12]. Proinflammatory cytokines and chemokines, produced by cancer cells, normal cells of the tumor microenvironment, and invading inflammatory cells, have been shown to facilitate tumor growth, invasion, and metastasis by producing chemotactic factors, inducing the production of angiogenic factors, and contributing to the maintenance of a chronic state of inflammation through self-activating feedback loops [12–14]. These cytokines and chemokines can contribute to the morbidity and mortality of patients, resulting in the activation of multiple signaling pathways that lead to clinical syndromes such as cachexia and coagulopathy [15].
Traditionally, cancer therapy has been directed at the development of cytotoxic agents and, more recently, at the development of “targeted” therapies that inhibit the dysregulation or overexpression of a particular oncogene or signal transduction pathway. However, cancer is the result of a complex interplay between a growing tumor and local and systemic host responses to the presence of malignancy. The potential exists not only to treat cancer cells themselves but also to decrease the morbidity and mortality of cancer by targeting factors produced by the cancer and the host. CCL2 presents such a target. Therapeutics disrupting the effects of CCL2 are being tested in multiple inflammatory diseases [6]. Targeting the direct and indirect effects of CCL2 has great potential for development in the treatment of prostate cancer and other malignancies.
Acknowledgements
The authors thank Karen Giles for manuscript preparation.
Footnotes
This research was supported, in part, by National Institutes of Health Grant PO1 CA093900-01 (R.D.L. and K.J.P.).
Dr. Loberg was supported by the University of Michigan Prostate SPORE Career Development Award P50 CA69568-06A. Dr. Pienta is an American Cancer Society clinical research professor.
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