MET tyrosine kinase receptor expression and amplification as prognostic biomarkers of survival in gastroesophageal adenocarcinoma.

BACKGROUND: MET gene amplification and Met protein overexpression may be associated with a poor prognosis. The MET/Met status is typically determined with fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Targeted proteomics uses mass spectrometry-based selected reaction monitoring (SRM) to accurately quantitate Met expression. FISH, IHC, and SRM analyses were compared to characterize the prognostic value of MET/Met in gastroesophageal adenocarcinoma (GEC). METHODS: Samples from 447 GEC patients were analyzed for MET gene amplification (FISH) and Met protein expression (IHC and SRM). Cox proportional hazards models and Kaplan-Meier estimates were applied to explore relations between Met, overall survival (OS), and clinical/pathological characteristics. Spearman's rank coefficient was used to assess the correlation between parameters. RESULTS: Patients with MET-amplified tumors had worse OS when: the MET/centromere enumeration probe for chromosome 7 FISH ratio was ≥ 2 (hazard ratio [HR], 3.13; 95% confidence interval [CI], 1.84-5.33), the MET gene copy number was ≥5 (HR, 2.51; 95% CI, 1.45-4.34), or ≥ 10% of the cells had ≥15 copies (HR, 4.28; 95% CI, 2.18-8.39). Similar observations were made with Met protein overexpression by IHC (≥1 + intensity in ≥ 25% of the tumor cell membrane: HR, 1.39; 95% CI, 1.04-1.86) or SRM (≥400 amol/µg: HR, 1.76; 95% CI, 1.06-2.90). A significant correlation was observed between MET FISH/Met IHC, MET FISH/Met SRM, and Met IHC/Met SRM; only MET FISH and Met SRM were independent negative prognostic biomarkers in multivariate analyses. CONCLUSIONS: MET amplification and overexpression, assessed by multiple methods, were associated with a worse prognosis in univariate analyses. However, only MET amplification by FISH and Met expression by SRM were independent prognostic biomarkers. Compared with IHC, SRM may provide an added benefit for informed decisions about Met-targeted therapy. Cancer 2017;123:1061-70. © 2016 American Cancer Society.

The evolving biology and treatment of prostate cancer.

Since the effectiveness of androgen deprivation for treatment of advanced prostate cancer was first demonstrated, prevention strategies and medical therapies for prostate cancer have been based on understanding the biologic underpinnings of the disease. Prostate cancer treatment is one of the best examples of a systematic therapeutic approach to target not only the cancer cells themselves, but the microenvironment in which they are proliferating. As the population ages and prostate cancer prevalence increases, challenges remain in the diagnosis of clinically relevant prostate cancer as well as the management of the metastatic and androgen-independent metastatic disease states.

Targeting CCL2 with systemic delivery of neutralizing antibodies induces prostate cancer tumor regression in vivo.

The identification of novel tumor-interactive chemokines and the associated insights into the molecular and cellular basis of tumor-microenvironment interactions have continued to stimulate the development of targeted cancer therapeutics. Recently, we have identified monocyte chemoattractant protein 1 (MCP-1; CCL2) as a prominent regulator of prostate cancer growth and metastasis. Using neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), we show that treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated PC-3Luc-mediated overall tumor burden in our in vivo model of prostate cancer metastasis by 96% at 5 weeks postintracardiac injection. Anti-CCL2 inhibition was not as effective as docetaxel (40 mg/kg, every week for 3 weeks) as a single agent, but inhibition of CCL2 in combination with docetaxel significantly reduced overall tumor burden compared with docetaxel alone, and induced tumor regression relative to initial tumor burden. These data suggest an interaction between tumor-derived chemokines and host-derived chemokines acting in cooperation to promote tumor cell survival, proliferation, and metastasis.

Phase I Study of AMG 337, a Highly Selective Small-molecule MET Inhibitor, in Patients with Advanced Solid Tumors.

PURPOSE: This first-in-human, open-label phase I study evaluated AMG 337, an oral, highly selective small-molecule inhibitor of MET in advanced solid tumors.Patients and Methods: Patients enrolled into dose-escalation cohorts received AMG 337 up to 400 mg once daily or up to 250 mg twice daily, following a modified 3+3+3 design. Dose expansion was conducted in MET-amplified patients at the maximum tolerated dose (MTD). Primary endpoints included assessment of adverse events (AEs), establishment of the MTD, and pharmacokinetics; clinical response was a secondary endpoint. RESULTS: The safety analysis set included 111 patients who received ≥1 dose of AMG 337. Thirteen patients had ≥1 AE qualifying as dose-limiting toxicity. The MTD was determined to be 300 mg once daily; the MTD for twice-daily dosing was not reached. Most frequent treatment-related AEs were headache (63%) and nausea (31%). Grade ≥3 treatment-related AEs occurred in 23 patients (21%), most commonly headache (n = 6) and fatigue (n = 5). Maximum plasma concentration occurred at 3.0 hours following 300-mg once-daily dosing, indicating AMG 337 absorption soon after treatment. Objective response rate was 9.9% (11/111; 95% CI, 5.1%-17.0%) in all patients and 29.6% (8/27; 95% CI, 13.8%-50.2%) in MET-amplified patients; median (range) duration of response was 202 (51-1,430+) days in all patients and 197 (64-1,430+) days in MET-amplified patients. CONCLUSIONS: Oral AMG 337 was tolerated with manageable toxicities, with an MTD and recommended phase II dose of 300 mg once daily. The promising response rate observed in patients with heavily pretreated MET-amplified tumors warrants further investigation.See related commentary by Ma, p. 2375.

CCL2 induces prostate cancer transendothelial cell migration via activation of the small GTPase Rac.

Nearly 85% of the men who will die of prostate cancer (PCa) have skeletal metastases present. The ability of PCa cells to interact with the microenvironment determines the success of the tumor cell to form metastatic lesions. The ability to bind to human bone marrow endothelial (HBME) cells and undergo transendothelial cell migration are key steps in allowing the PCa cell to extravasate from the bone microvasculature and invade the bone stroma. We have previously demonstrated that monoctyte chemoattractant protein 1 (MCP-1; CCL2) is expressed by HBME cells and promotes PCa proliferation and migration. In the current study, we demonstrate that the CCL2 stimulation of PCa cells activates the small GTPase, Rac through the actin-associated protein PCNT1. Activation of Rac GTPase is accompanied by morphologic changes and the ability of the cells to undergo diapedesis through HBME cells. These data suggest a role for HBME-secreted CCL2 in promoting PCa cell extravasation into the bone microenvironment.

Annexin II/annexin II receptor axis regulates adhesion, migration, homing, and growth of prostate cancer.

One of the most life-threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short-term and long-term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest that annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche.

The Role of Minimal Residual Disease Testing in Myeloma Treatment Selection and Drug Development: Current Value and Future Applications.

Treatment of myeloma has benefited from the introduction of more effective and better tolerated agents, improvements in supportive care, better understanding of disease biology, revision of diagnostic criteria, and new sensitive and specific tools for disease prognostication and management. Assessment of minimal residual disease (MRD) in response to therapy is one of these tools, as longer progression-free survival (PFS) is seen consistently among patients who have achieved MRD negativity. Current therapies lead to unprecedented frequency and depth of response, and next-generation flow and sequencing methods to measure MRD in bone marrow are in use and being developed with sensitivities in the range of 10-5 to 10-6 cells. These technologies may be combined with functional imaging to detect MRD outside of bone marrow. Moreover, immune profiling methods are being developed to better understand the immune environment in myeloma and response to immunomodulatory agents while methods for molecular profiling of myeloma cells and circulating DNA in blood are also emerging. With the continued development and standardization of these methodologies, MRD has high potential for use in gaining new drug approvals in myeloma. The FDA has outlined two pathways by which MRD could be qualified as a surrogate endpoint for clinical studies directed at obtaining accelerated approval for new myeloma drugs. Most importantly, better understanding of MRD should also contribute to better treatment monitoring. Potentially, MRD status could be used as a prognostic factor for making treatment decisions and for informing timing of therapeutic interventions. Clin Cancer Res; 23(15); 3980-93. ©2017 AACR.

Pathogenesis and treatment of prostate cancer bone metastases: targeting the lethal phenotype.

Traditionally, prostate cancer treatment, as well as all cancer treatment, has been designed to target the tumor cell directly via various hormonal and chemotherapeutic agents. Recently, the realization that cancer cells exist in complex microenvironments that are essential for the tumorigenic and metastatic potential of the cancer cells is starting the redefine the paradigm for cancer therapy. The propensity of prostate cancer cells to metastasize to bone is leading to the design of novel therapies targeting both the cancer cell as well as the bone microenvironment. Tumor cells in the bone interact with the extracellular matrix, stromal cells, osteoblasts, osteoclasts, and endothelial cells to promote tumor-cell survival and proliferation leading to a lethal phenotype that includes increased morbidity and mortality for patients with advanced prostate cancer. Several strategies are being developed that target these complex tumor cell-microenvironment interactions and target the signal transduction pathways of other cells important to the development of metastases, including the osteoclasts, osteoblasts, and endothelial cells of the bone microenvironment. Current and new therapies in metastatic prostate cancer will comprise a multitargeted approach aimed at both the tumor cell and the tumor microenvironment. Here, we review the current therapeutic strategies for targeting the prostate cancer-bone microenvironment and several single- and multiagent targeted approaches to the treatment of advanced prostate cancer that are under development.

Development of the VCaP androgen-independent model of prostate cancer.

Prostate epithelial cell growth is dependent on the presence of androgens, and transition of prostate cancer to an androgen-independent phenotype results in a highly aggressive, currently incurable cancer. We have developed a new preclinical model of androgen-independent prostate cancer derived from the VCaP prostate cancer epithelial cell line. VCaP cells were subcutaneously implanted and serially passaged in castrated male severe combined immunodeficient mice. Androgen independence was confirmed by WST-1 (a tetrazolium salt) cell proliferation assay in the absence or presence of dihydrotesterone (1-100 nM). VCaP androgen-sensitive cells responded dose dependently to dihydrotesterone, whereas VCaP androgen-independent cells did not alter their proliferation in response to dihydrotesterone. Gene expression of androgen receptor, B-cell lymphoma-2, prostate cancer antigen 3, prostate acid phosphatase, 6 transmembrane epithelial antigen of the prostate, and survivin was determined by polymerase chain reaction amplification. B-cell lymphoma-2 expression was up regulated in the VCaP androgen-independent lines compared to the VCaP androgen-sensitive, suggesting a possible mechanism of androgen independence. Furthermore, tumor-associated angiogenesis was assessed by immunofluorescence confocal microscopy of CD31. VCaP androgen-independent tumors showed enhanced angiogenesis compared to VCaP androgen-sensitive tumors. These results illustrate the development of a novel model of prostate cancer androgen independence and provide a new system to study angiogenesis and the transition to androgen independence.

GLUT4 facilitative glucose transporter specifically and differentially contributes to agonist-induced vascular reactivity in mouse aorta.

OBJECTIVE: We hypothesized that GLUT4 is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs), and GLUT4 is necessary for agonist-induced VSMC contraction. METHODS AND RESULTS: Glucose deprivation and indinavir, a GLUT4 antagonist, were used to assess the role of GLUT4 and non-GLUT4 transporters in vascular reactivity. In isolated endothelium-denuded mouse aorta, approximately 50% of basal glucose uptake was GLUT4-dependent. Norepinephrine-mediated contractions were dependent on both GLUT4 and non-GLUT4 transporters, serotonin (5-HT)-mediated contractions were mainly GLUT4-dependent, and prostaglandin (PG) F(2alpha)-mediated contractions were dependent on non-GLUT4 transporters, whereas indinavir had no effect in GLUT4 knockout vessels. We also observed a 46% decrease in GLUT4 expression in aortas from angiotensin II hypertensive mice. Indinavir caused a less profound attenuation of maximal 5-HT-mediated contraction in these vessels, corresponding to the lower GLUT4 levels in the hypertensive aortas. Finally, and somewhat surprisingly, chronic GLUT4 knockout was associated with increased vascular reactivity compared with that in wild-type animals, suggesting that chronic absence or reduction of GLUT4 expression in VSMCs leads to opposite effects observed with acute inhibition of GLUT4. CONCLUSIONS: Thus, we conclude that GLUT4 is constitutively expressed in large arteries and likely participates in basal glucose uptake. In addition, GLUT4, as well as other non-GLUT4 facilitative glucose transporters, are necessary for agonist-induced contraction, but each transporter participates in VSMC contraction selectively, depending on the agonist, and changes in GLUT4 expression may account for some of the functional changes associated with vascular diseases like hypertension.

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

BACKGROUND: Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. METHOD: We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. RESULTS: Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. CONCLUSION: Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

Differential expression analysis of MIM (MTSS1) splice variants and a functional role of MIM in prostate cancer cell biology.

We previously identified MIM-A (missing in metastasis, MTSS1) by differential display techniques as missing in invasive, metastatic bladder cancer cell lines and suggested that MIM-A is a novel putative metastasis suppressor gene. Characterization of the MIM gene revealed a WH2 (Wiskott-Aldrich syndrome protein homology 2) domain in the C-terminus that is known to bind actin monomers and regulate organization of the actin cytoskeleton. Here, we further describe two alternatively splice variants of MIM-A, called MIM(12del) and MIM-B, which share > 50% amino acid sequence homology with MIM-A in the C-terminal domain. We show that expression of all three transcripts is down-regulated in prostate cancer cell lines and tumor samples from patients. In addition, we generated stably-transfected PC-3 cells overexpressing MIM-A to evaluate the importance of MIM-A in prostate cancer biology. The initial experiments show that expression of MIM decreased the number of actin filaments and was associated with a decrease in the G:F actin ratio. Overexpression of MIM-A had no effect on PC-3 cell adhesion to extracellular matrices, as well as no effect on PC-3 motility. Further, overexpression of MIM-A reduced the rate of PC-3 cell proliferation. These results support the hypothesis that MIM-A is an actin-binding protein and implicate a role of MIM-A in the regulation of cellular proliferation. These data suggest that the reduction of MIM-A gene expression in prostate cancer and other cancers may contribute to tumor growth and development, as well as metastasis.

Detection and isolation of circulating tumor cells in urologic cancers: a review.

The American Cancer Society has estimated that in 2003, there will be approximately 239,600 new cases of urologic cancer diagnosed and 54,600 urologic cancer-related deaths in the United States. To date, the majority of research and therapy design have focused on the microenvironment of the primary tumor site, as well as the microenvironment of the metastatic or secondary (target) tumor site. Little attention has been placed on the interactions of the circulating tumor cells and the microenvironment of the circulation (i.e., the third microenvironment). The purpose of this review is to present the methods for the detection and isolation of circulating tumor cells and to discuss the importance of circulating tumor cells in the biology and treatment of urologic cancers.

Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow.

HSC homing, quiescence, and self-renewal depend on the bone marrow HSC niche. A large proportion of solid tumor metastases are bone metastases, known to usurp HSC homing pathways to establish footholds in the bone marrow. However, it is not clear whether tumors target the HSC niche during metastasis. Here we have shown in a mouse model of metastasis that human prostate cancer (PCa) cells directly compete with HSCs for occupancy of the mouse HSC niche. Importantly, increasing the niche size promoted metastasis, whereas decreasing the niche size compromised dissemination. Furthermore, disseminated PCa cells could be mobilized out of the niche and back into the circulation using HSC mobilization protocols. Finally, once in the niche, tumor cells reduced HSC numbers by driving their terminal differentiation. These data provide what we believe to be the first evidence that the HSC niche serves as a direct target for PCa during dissemination and plays a central role in bone metastases. Our work may lead to better understanding of the molecular events involved in bone metastases and new therapeutic avenues for an incurable disease.

GAS6/AXL axis regulates prostate cancer invasion, proliferation, and survival in the bone marrow niche.

Our recent studies have shown that annexin II, expressed on the cell surface of osteoblasts, plays an important role in the adhesion of hematopoietic stem cells (HSCs) to the endosteal niche. Similarly, prostate cancer (PCa) cells express the annexin II receptor and seem to use the stem cell niche for homing to the bone marrow. The role of the niche is thought to be the induction and sustenance of HSC dormancy. If metastatic PCa cells occupy a similar or the same ecological niche as HSCs, then it is likely that the initial role of the HSC niche will be to induce dormancy in metastatic cells. In this study, we demonstrate that the binding of PCa to annexin II induces the expression of the growth arrest-specific 6 (GAS6) receptors AXL, Sky, and Mer, which, in the hematopoietic system, induce dormancy. In addition, GAS6 produced by osteoblasts prevents PCa proliferation and protects PCa from chemotherapy-induced apoptosis. Our results suggest that the activation of GAS6 receptors on PCa in the bone marrow environment may play a critical role as a molecular switch, establishing metastatic tumor cell dormancy.

Co-inoculation of prostate cancer cells with U937 enhances tumor growth and angiogenesis in vivo.

Tumor-associated macrophages (TAMs) have been implicated in promoting tumor growth and development. Here we present evidence that demonstrates that co-inoculation of male athymic nude mice with PC-3 prostate cancer cells and U937 promonocytic cells enhances tumor growth and increases tumor angiogenesis. Male athymic nude mice were co-inoculated with PC-3 and U937 cells (control or IL-4 stimulated) and tumor growth was monitored over time. Immunohistochemical analysis of tumor specimens was performed for proliferation markers (e.g., Ki67) and the effects of IL-4 stimulation on U937 cells were analyzed for chemokine expression. The presence of U937 cells increased the rate of tumor growth in vivo and stimulated increased microvascular density within the tumor bed. Stimulation of U937 cells with IL-4 resulted in a significant increase in several pro-angiogenic and pro-tumor chemokines (e.g., CCL2). Co-inoculation increases prostate cancer growth via upregulation of chemokines that induce angiogenesis within the tumor.

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression.

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.

An in vivo mouse model for human prostate cancer metastasis.

We developed a sensitive real-time polymerase chain reaction (QPCR) assay that allows us to track early lodging/homing events in vivo. We used this technology to develop a metastasis assay of human prostate cancer (PCa) growth in severe combined immunodeficient mice. For this purpose, marked human PCa cell lines were implanted subcutaneously or in the prostate (orthotopically) of severe combined immunodeficient mice as models of primary tumors. Mice were then sacrificed at various time points, and distant tissues were investigated for the presence of metastatic cells. At 3 weeks, a number of tissues were recovered and evaluated by QPCR for the presence of metastatic cells. The data demonstrate that several PCa cell lines are able to spread from the primary lesion and take up residence in distant sites. If the primary tumors were resected at 3 weeks, in several cases, metastatic lesions were identified over the course of 9 months. We propose that this new model may be particularly useful in exploring the molecular events in early metastasis, identifying the metastatic niche, and studying issues pertaining to dormancy.

The lethal phenotype of cancer: the molecular basis of death due to malignancy.

The last decade has seen an explosion in knowledge of the molecular basis and treatment of cancer. The molecular events that define the lethal phenotype of various cancers--the genetic and cellular alterations that lead to a cancer with a poor or incurable prognosis--are being defined. While these studies describe the cellular events of the lethal phenotype of cancer in detail, how these events result in the common clinical syndromes that kill the majority of cancer patients is not well understood. It is clear that the central step that makes most cancers incurable is metastasis. Understanding the traits that a cancer acquires to successfully grow and metastasize to distant sites gives insight into how tumors produce multiple factors that result in multiple different clinical syndromes that are lethal for the patient.

CCL2 as an important mediator of prostate cancer growth in vivo through the regulation of macrophage infiltration.

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.