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Antivascular therapy has emerged as a rational strategy to improve the treatment of androgen-independent prostate cancer owing to the necessity of establishing a vascular network for the growth and progression of the primary and metastatic tumor. We determined whether recombinant human apolipoprotein(a) kringle V, rhLK8, produces therapeutic efficacy in an orthotopic human prostate cancer animal model. Fifty thousand androgen-independent human prostate cancer cells (PC-3MM2) were injected into the prostate of nude mice. After 3 days, these mice were randomized to receive the vehicle solution (intraperitoneally [i.p.], daily), paclitaxel (8 mg/kg i.p., weekly), rhLK8 (50 mg/kg i.p., daily), or a combination of paclitaxel and rhLK8 for 4 weeks. Treatment with paclitaxel or rhLK8 alone did not show significant therapeutic effects on tumor incidence or on tumor size compared with the control group. The combination of rhLK8 and paclitaxel significantly reduced tumor size and incidence of lymph node metastasis. Significant reduction in microvessel density and cellular proliferation and induction of apoptosis of tumor cells, and tumor-associated endothelial cells, were also achieved. Similarly, PC-3MM2 tumors growing in the tibia showed significant suppression of tumor growth and lymph node metastasis by the combination treatment with rhLK8 and paclitaxel. The integrity of the bone was significantly preserved, and apoptosis of tumor cells and tumor-associated endothelial cells was increased. In conclusion, these results suggest that targeting the tumor microenvironment with the antivascular effect of rhLK8 combined with conventional cytotoxic chemotherapy could be a new and effective approach in the treatment of androgen-independent prostate cancer and their metastases.

To test the hypothesis that tumor-associated macrophages (TAMs) enhance the growth and metastasis of human prostate cancer in the bone, we evaluated the effects of decreasing interleukin-6 (IL-6) production by tumor cells and TAMs in a mouse model of bone metastasis. Human PC-3MM2 cells that produce IL-6 were transfected with lentivirus containing IL-6 small hairpin RNA (shRNA) or nonspecific RNA and injected into the tibias of nude mice treated intraperitoneally every 5days for 5weeks with phosphate-buffered saline (PBS), liposomes containing PBS, or liposomes containing clodronate (to decrease the number of macrophages). Transfection of PC-3MM2 cells with IL-6 shRNA significantly decreased cellular expression of IL-6 and the number of TAMs and osteoclasts in bone tumors, which correlated with significant decreases in tumor size, bone lysis, and incidence of lymph node metastasis. Treatment of mice with clodronate liposomes significantly decreased the number of TAMs and osteoclasts in the bone tumors, the expression of IL-6 in the PC3-MM2 cells, and the production of tumor necrosis factor (TNF)-α by TAMs. These findings correlated with a significant decrease in tumor size, bone lysis, and lymph node metastasis. Knocking down IL-6 in tumor cells and decreasing TAMs was associated with the lowest incidences of bone tumors and lymph node metastasis. These results suggest that TAMs enhance the growth of prostate cancer cells in the bone., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In vivo animal experiments are essential to current prostate cancer research, and are particularly critical to studying interactions between tumor cells and their microenvironment. Numerous pre-clinical animal models of prostate cancer are currently available, including transgenic mouse models and human prostate cancer xenograft mouse models. In contrast to transgenic mouse models producing more heterogeneous cohorts of tumors, xenograft mouse models provide more controlled approaches. This unit describes the detailed procedures necessary to establish several distinct pre-clinical mouse models of human prostate cancer, including an orthotopic prostate xenograft model, an orthotopic bone metastasis model, an experimental metastasis model of intra-cardiac injection, and a vossicle model of tumor-bone interaction.

The antivascular function of the platelet-derived growth factor receptor (PDGFR) inhibitor imatinib combined with paclitaxel has been demonstrated by invasive immunohistochemistry. The purpose of this study was to 1) noninvasively monitor the response to anti-PDGFR treatment, and 2) understand the underlying mechanism of this response. Thus, response to treatment was studied in a prostate cancer bone metastasis model using macromolecular (biotin-bovine serum albumin [BSA]-Gd-diethylene triamine pentaacetic acid [GdDTPA]) dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Human prostate cancer (PC-3MM2) bone metastases that caused osteolysis and grew in neighboring muscle showed a high blood-volume fraction (fBV) and vascular permeability (PS) at the tumor periphery compared to muscle tissue and intraosseous tumor. Imatinib alone or with paclitaxel significantly reduced PS by 35% (one-tailed paired t-test, P = 0.045) and 40% (P = 0.0003), respectively, whereas paclitaxel alone or no treatment had no effect. Based on changes in PS, we hypothesized that imatinib interferes with the signaling pathway of vascular endothelial growth factor (VEGF). This mechanism was verified by immunohistochemistry. It demonstrated reduced activation of both PDGFR-beta and VEGF receptor 2 (VEGFR2) in imatinib-treated mice. Our study therefore demonstrates that macromolecular DCE-MRI can be used to detect early vascular effects associated with response to therapy targeted to PDGFR, and provides insight into the role played by VEGF in anti-PDGFR therapy., ((c) 2008 Wiley-Liss, Inc.)

Aberrant expression and/or activity of members of the Src family of nonreceptor protein tyrosine kinases (SFK) are commonly observed in progressive stages of human tumors. In prostate cancer, two SFKs (Src and Lyn) have been specifically implicated in tumor growth and progression. However, there are no data in preclinical models demonstrating potential efficacy of Src inhibitors against prostate cancer growth and/or metastasis. In this study, we used the small molecule SFK/Abl kinase inhibitor dasatinib, currently in clinical trials for solid tumors, to examine in vitro and in vivo effects of inhibiting SFKs in prostate tumor cells. In vitro, dasatinib inhibits both Src and Lyn activity, resulting in decreased cellular proliferation, migration, and invasion. In orthotopic nude mouse models, dasatinib treatment effectively inhibits expression of activated SFKs, resulting in inhibition of both tumor growth and development of lymph node metastases in both androgen-sensitive and androgen-resistant tumors. In primary tumors, SFK inhibition leads to decreased cellular proliferation (determined by immunohistochemistry for proliferating cell nuclear antigen). In vitro, small interfering RNA (siRNA)-mediated inhibition of Lyn affects cellular proliferation; siRNA inhibition of Src affects primarily cellular migration. Therefore, we conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer and that Src and Lyn activities affect different cellular functions required for prostate tumor growth and progression.

Purpose: To further assess preclinical and early clinical evidence that imatinib mesylate, a platelet-derived growth factor receptor (PDGFR) inhibitor, modulates taxane activity in prostate cancer and bone metastases, a randomized study was conducted., Experimental Design: Men with progressive castration-resistant prostate cancer with bone metastases (n = 144) were planned for equal randomization to i.v. 30 mg/m(2) docetaxel on days 1, 8, 15, and 22 every 42 days with 600 mg imatinib daily or placebo, for an improvement in median progression-free survival from 4.5 to 7.5 months (two-sided alpha = 0.05 and beta = 0.20). Secondary end points included differential toxicity and bone turnover markers, tumor phosphorylated PDGFR (p-PDGFR) expression, and modulation of p-PDGFR in peripheral blood leukocytes., Results: Accrual was halted early because of adverse gastrointestinal events. Among 116 evaluable men (57 docetaxel + imatinib; 59 docetaxel + placebo), respective median times to progression were 4.2 months (95% confidence interval, 3.1-7.5) and 4.2 months (95% confidence interval, 3.0-6.8; P = 0.58, log-rank test). Excess grade 3 toxicities (n = 23) in the docetaxel + imatinib group were principally fatigue and gastrointestinal. Tumor p-PDGFR expression was observed in 12 of 14 (86%) evaluable bone specimens. In peripheral blood leukocytes, p-PDGFR reduction was more likely in docetaxel + imatinib-treated patients compared with docetaxel + placebo (P < 0.0001), as were reductions in urine N-telopeptides (P = 0.004) but not serum bone-specific alkaline phosphatase (P = 0.099)., Conclusions: These clinical and translational results question the value of PDGFR inhibition with taxane chemotherapy in prostate cancer bone metastases and are at variance with the preclinical studies. This discordance requires explanation.

Background: Inhibiting epidermal growth factor receptor (EGF-R) and vascular endothelial growth factor receptor (VEGF-R) activation with AEE788 can decrease prostate cancer (CaP) growth/progression. We determined whether tumor cells or tumor-associated endothelial cells were the primary target by treating multidrug-resistant (MDR) CaP growing in the prostate of nude mice., Methods: MDR human CaP cells with 30-fold increased taxane-resistance were implanted into nude mouse prostates. After 2 weeks, mice were randomized to control, paclitaxel, AEE788, and AEE788/paclitaxel for 10 weeks. Mice were necropsied and tumors stained., Results: AEE788 or AEE788 plus paclitaxel significantly reduced tumor incidence and tumor weight, and eradicated lymph node metastasis. Inhibiting VEGF-R and EGF-R phosphorylation induced apoptosis of tumor-associated endothelial cells causing a second apoptotic wave of surrounding tumor cells., Conclusion: Inhibiting VEGF-R and EGF-R activation on tumor-associated endothelial cells with AEE788 combined with paclitaxel can bypass CaP cell resistance and prevent lymph node metastasis.

Background: Inhibiting phosphorylation of platelet-derived growth factor receptor (PDGFR) by treatment with the PDGFR kinase inhibitor imatinib and the chemotherapeutic agent paclitaxel reduces the incidence and size of human prostate cancer bone lesions in nude mice. Because tumor cells and tumor-associated endothelial cells express activated PDGFR, the primary target for imatinib has been unclear., Methods: We selected multidrug-resistant human PC-3MM2 prostate cancer cells (termed PC-3MM2-MDR cells) by culturing them in increasing concentrations of paclitaxel. PC-3MM2-MDR cells were implanted into one tibia of 80 nude mice. Two weeks later, the mice were randomly assigned to receive distilled water (control group), paclitaxel, imatinib, or imatinib plus paclitaxel for 10 weeks (20 mice per group). Tumor incidence and weight, bone structure preservation and osteolysis, and the incidence of lymph node metastasis were determined. The phosphorylation status of PDGFR on tumor cells and tumor-associated endothelial cells and levels of apoptosis were examined with immunohistochemical analyses. Microvessel density was assessed as the number of cells expressing CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1). All statistical tests were two-sided., Results: PC-3MM2-MDR cells were resistant to paclitaxel and imatinib in vitro. Treatment of implanted mice with imatinib plus paclitaxel led to statistically significant decreases in bone tumor incidence (control = 19 mice with tumors of 19 mice total; imatinib plus paclitaxel = four of 18 mice; P < .001), median tumor weight (control = 1.3 g, interquartile range [IQR] = 1.0-1.9; imatinib plus paclitaxel = 0.1 g, IQR = 0-0.3; P < .001), bone lysis, and the incidence of lymph node metastasis (control = 19 of 19 mice total; imatinib plus paclitaxel = three of 18 mice; P < .001). Treatment with imatinib alone had similar effects, and imatinib treatment also inhibited phosphorylation of PDGFR on tumor cells and tumor-associated endothelial cells and increased the level of apoptosis of endothelial cells, but not tumor cells. Treatment with imatinib and more so with imatinib and paclitaxel decreased mean vessel density (three CD31/PECAM-1-positive cells, 95% confidence interval [CI] = 0 to 9; and control group = 38 CD31/PECAM-1-positive cells, 95% CI = 17 to 59) (P < .001), which was followed by apoptosis of tumor cells., Conclusion: Tumor-associated endothelial cells, rather than tumor cells themselves, appear to be the target for imatinib in prostate cancer bone metastasis.

Prostate cancer cells metastasize to the bone where their interaction with osteoclasts and osteoblasts can lead to alterations in the structure of the bone. We determined whether the systemic administration of the bisphosphonate, zoledronate, could prevent bone lysis and halt the proliferation of human prostate cancer cells injected into the tibia of nude mice. Zoledronate did not affect the in vitro proliferation of human prostate cancer PC-3MM2 cells. The in vivo administration of zoledronate produced significant bone preservation but did not inhibit the progressive growth of PC-3MM2 cells. The systemic administration of STI571 (imatinib mesylate, Gleevec), an inhibitor of phosphorylation of the platelet-derived growth factor receptor, in combination with paclitaxel, produced apoptosis of tumor cells and bone- and tumor-associated endothelial cells. The systemic administration of zoledronate with STI571 and paclitaxel produced a significant preservation of bone structure, a decrease in tumor incidence and weight, and a decrease in incidence of lymph node metastasis. This therapeutic activity was correlated with inhibition of osteoclast function, inhibition of tumor cell proliferation, and induction of apoptosis in tumor-associated endothelial cells and tumor cells. Cancer is a heterogeneous disease that requires multimodality therapy. The present data recommend the combination of a bisphosphonate agent with protein tyrosine kinase inhibitor and an anticycling drug for the treatment of prostate cancer bone metastasis.

Purpose: To study the platelet-derived growth factor receptor (PDGFR) inhibitor imatinib mesylate in androgen-independent prostate cancer (AIPC), alone and in combination with docetaxel, we designed a modular phase I trial. Our goals were to (1) evaluate the toxicity and maximum-tolerated dose of docetaxel with imatinib, and (2) evaluate the decline of prostate-specific antigen (PSA) induced by imatinib alone, and imatinib and docetaxel., Patients and Methods: Twenty-eight men with AIPC and bone metastases were enrolled to receive imatinib 600 mg daily lead-in for 30 days, then imatinib 600 mg daily and one of six possible doses of docetaxel weekly for 4 weeks every 6 weeks., Results: During the imatinib lead-in module, one dose-limiting toxicity (DLT) event was observed, while two (7%) of 28 had PSA decline (both < 50%). With imatinib and docetaxel, cycle 1 DLT was found in three of 12 patients at docetaxel 30 mg/m(2), in three of four patients at docetaxel 45 mg/m(2), and in five of six patients at docetaxel 35 mg/m(2). DLTs (n = 40 total events) were principally fatigue (35%) and nausea (20%). Eight (38%) of 21 had PSA decline greater than 50%, and six (29%) of 21 had PSA decline less than 50%. Serial PSA declines beyond 18 months were observed. PDGFR-expressing tumor declined on serial bone marrow biopsies with combination therapy alone., Conclusion: With imatinib 600 mg daily, the maximum-tolerated dose of docetaxel was determined to be 30 mg/m(2) weekly for 4 weeks every 6 weeks. Long-term responses were observed. The role of imatinib in modulating outcomes to docetaxel in AIPC is being tested in a randomized phase II trial.

Once prostate cancer metastasizes to bone, conventional chemotherapy is largely ineffective. We hypothesized that inhibition of phosphorylation of the epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) expressed on tumor cells and tumor-associated endothelial cells, which is associated with tumor progression, in combination with paclitaxel would inhibit experimental prostate cancer bone metastasis and preserve bone structure. We tested this hypothesis in nude mice, using human PC-3MM2 prostate cancer cells. PC-3MM2 cells growing adjacent to bone tissue and endothelial cells within these lesions expressed phosphorylated EGF-R and PDGF-R alpha and -beta on their surfaces. The percentage of positive endothelial cells and the intensity of receptor expression directly correlated with proximity to bone tissue. Oral administration of PKI166 inhibited the phosphorylation of EGF-R but not PDGF-R, whereas oral administration of STI571 inhibited the phosphorylation of PDGF-R but not EGF-R. Combination therapy using oral PKI166 and STI571 with i.p. injections of paclitaxel induced a high level of apoptosis in tumor vascular endothelial cells and tumor cells in parallel with inhibition of tumor growth in the bone, preservation of bone structure, and reduction of lymph node metastasis. Collectively, these data demonstrate that blockade of phosphorylation of EGF-R and PDGF-R coupled with administration of paclitaxel significantly suppresses experimental human prostate cancer bone metastasis.

Purpose: The expression of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, frequently increases during prostate tumor progression. However, whether reduced c-Met expression inhibits tumor growth and metastasis has not been ascertained., Experimental Design: c-Met expression was reduced by infection of an adenovirus expressing a c-Met ribozyme into the highly metastatic human prostate cancer cell line PC3-LN4. In vitro, effects on c-Met, Akt, and extracellular signal-regulated kinase 1/2 expression and phosphorylation, Src expression and activity, and vascular endothelial growth factor expression were determined, as were effects on cell migration and invasion. Prostate tumor formation and metastasis to regional lymph nodes in nude mice were examined after both ex vivo and in vivo infection of cells., Results: Infection of PC3-LN4 cells with the Ad-c-Met-expressing ribozyme decreased steady-state c-Met levels, decreased Src kinase activity, decreased vascular endothelial growth factor expression, and decreased migration and invasion versus the pU1 (control) virus. Significant inhibition of tumorigenicity (histologically confirmed tumors in only 1 of 10 mice) and consequent lymph node metastasis were observed upon ex vivo infection of Ad-c-Met. Similarly, gene therapy experiments led to complete inhibition of tumor growth in 7 of 8 mice., Conclusions: Reduction in c-Met expression substantially inhibits both tumor growth and lymph node metastasis of PC3-LN4 cells in orthotopic nude mouse models. Therefore, targeting the c-Met signaling pathways may be important in controlling tumor growth and metastasis in human prostate cancers.

Background: Expression of platelet-derived growth factor (PDGF) and activation (by autophosphorylation) of its receptor (PDGF-R), a tyrosine kinase, are associated with the growth of metastatic prostate tumor cells in the bone parenchyma. The tyrosine kinase inhibitor STI571 blocks the PDGF signaling pathway by inhibiting PDGF-R autophosphorylation. We examined the effects of STI571, given alone or with paclitaxel (Taxol), on tumor growth in a mouse model of prostate cancer metastasis., Methods: Human prostate cancer PC-3MM2 cells were injected into the tibias of male nude mice. Three days later the mice (20 per group) were randomly assigned to 5 weeks of treatment with oral and injected water (control), daily oral STI571, weekly injected paclitaxel, or STI571 plus paclitaxel. Lesions in bone and the surrounding muscles were then harvested and analyzed by histology, western blotting (for PDGF-R phosphorylation), immunohistochemistry (for expression of proangiogenic molecules), and double immunofluorescence (to identify endothelial cells and apoptotic tumor cells). Growth of bone lesions was monitored by digital radiography. Bone lesions from control mice were used to establish short-term cell cultures for analysis of PDGF-R phosphorylation. All statistical tests were two-sided., Results: PC-3MM2 cells cultured from bone lesions and treated in vitro with STI571 had less phosphorylated PDGF-R than untreated cells. In control mice, bone lesions expressed high levels of PDGF and activated (i.e., phosphorylated) PDGF-R, whereas lesions in the adjacent musculature did not. Activated PDGF-R was present on the surface of endothelial cells within the bone lesions but not in endothelial cells of uninjected bone. Mice treated with STI571 or STI571 plus paclitaxel had a lower tumor incidence, smaller tumors, and less bone lysis and lymph node metastasis than mice treated with water or paclitaxel alone (P<.001 for all). Mice treated with STI571 or STI571 plus paclitaxel had less phosphorylated PDGF-R on tumor cells and tumor-associated endothelial cells, less tumor cell proliferation, statistically significantly more apoptotic tumor cells (all P<.001), and fewer tumor-associated endothelial cells (P<.001) than control mice., Conclusions: Endothelial cells appear to express phosphorylated PDGF-R when they are exposed to tumor cells that express PDGF. Using STI571 to inhibit PDGF-R phosphorylation may, especially in combination with paclitaxel, produce substantial therapeutic effects against prostate cancer bone metastasis.

The high rate of prostate cancer mortality invariably reflects the inability to control the spread of the disease. The urokinase-type plasminogen activator and its receptor (u-PAR) contribute to prostate cancer metastases by promoting extracellular matrix degradation and growth factor activation. The current study was undertaken to determine the efficacy of a urokinase-derived peptide (A6) in reducing the lymph node metastases of prostate cancer using a model in which prostatic tumors established in nude mice from orthotopically implanted PC-3 LN4 prostate cancer cells disseminate to the lymph nodes. As a first step in evaluating the in vivo effectiveness of A6, we determined its effect on in vitro invasiveness. In vitro, A6 reduced the invasiveness of PC-3 LN4 cells through a Matrigel-coated filter without affecting growth rate. A first in vivo survival experiment showed that all A6-treated mice were alive after 57 days, and half of them tumor-free, whereas all control mice receiving vehicle had died. In a second experiment with a larger tumor inoculum and a longer delay until treatment, whereas 71% of control mice and 83% of mice treated with a scrambled peptide developed lymph node metastases, only 22 to 25% of A6-treated mice had positive lymph nodes. Further, lymph node volume, reflective of tumor burden at the secondary site, was diminished 70% in A6-treated mice. In conclusion, we provide definitive evidence that a peptide spanning the connecting region of urokinase suppresses metastases and, as a single modality, prolongs the life span of prostate tumor-bearing mice.

We evaluated whether treatment of orthotopic human prostate cancer in nude mice with pegylated IFN-alpha-2b (PEG-IFN-alpha-2b) and docetaxel could represent a two-compartment targeting of primary tumor (tumor cells and tumor-associated endothelial cells) and inhibition of regional lymph node metastasis. The antiangiogenic properties of IFN were combined with the cytotoxic properties of docetaxel, resulting in apoptosis of both tumor cells and endothelium and hence significant inhibition of primary tumor growth. We first determined the optimal biological dose of PEG-IFN-alpha-2b (70,000 IU/week) necessary to down-regulate the expression of basic fibroblast growth factor, matrix metalloprotease-9, and matrix metalloprotease-2. The therapeutic dose of docetaxel (10 mg/kg/week) was determined by efficacy and minimal body weight loss. Therapy beginning 3 days after orthotopic implantation of PC3-MM2 prostate cancer cells reduced tumor weight by 37% in mice treated with PEG-IFN-alpha-2b, by 60% in mice treated with docetaxel, and by 83% in those given both drugs. PEG-IFN-alpha-2b also induced apoptosis of tumor-associated endothelial cells and hence a significant decrease in microvessel density. Our data indicate that the combination of PEG-IFN-alpha and docetaxel inhibits neoplastic angiogenesis by inducing a decrease in the local production of proangiogenic molecules by tumor cells, resulting in increased apoptosis of tumor-associated endothelial cells.

Purpose: Vascular endothelial growth factor (VEGF), which is produced by tumor cells, is a potent endothelial cell mitogen. The aim of the present study was to evaluate the response of orthotopic prostate cancer xenografts and prostate cancer bone metastasis to anti-VEGF receptor (flk-1) antibody (DC101) treatment., Experimental Design: Orthotopic prostate cancer models (PC-3M-MM2 and LNCaP-LN3 prostate carcinoma cells) and a prostate cancer bone metastasis model (PC-3M-MM2) were used for these experiments. Early and established tumors were treated with saline, paclitaxel, DC101, or a DC101-plus-paclitaxel combination for 5 weeks (PC-3M-MM2) and 12 weeks (LNCaP-LN3). At the end of therapy, tumors were removed and weighed. Apoptosis, tumor cell proliferation, and angiogenesis- and metastasis-related gene expression were evaluated using immunohistochemistry, in situ hybridization, and terminal deoxynucleotidyl transferase-ediated nick end labeling (TUNEL)., Results: After treatment of early tumors (PC-3M-MM2), median prostate tumor weights (+/-SE) were 1230 +/- 210 mg in untreated controls, 482 +/- 121 mg in mice treated with paclitaxel (P = 0.009), 148 +/- 27 mg in mice treated with DC101 (P < 0.001), and 48 +/- 10 mg in mice treated with the combination of DC101 and paclitaxel (P < 0.001). Lymph node metastasis occurred in 7 of the 9 control mice, 5 of the 9 paclitaxel-treated animals, 5 of the 12 DC101-treated animals, and 2 of the 11 animals in the combination therapy group. Treatment with DC101 alone or in combination with paclitaxel reduced tumor-induced neovascularity measured by microvessel density and tumor cell proliferation (by proliferating cell nuclear antigen) and enhanced apoptosis (measured by TUNEL) in tumor cells and endothelial cells compared with controls. In the tibial prostate cancer metastasis model, significant inhibition of tumor growth was observed. In the LNCaP-LN3 orthotopic prostate cancer model, tumors occurred in 7 of the 10 control mice, 4 of the 10 paclitaxel-treated animals, 5 of the 10 DC101-treated animals, and 2 of the 11 animals in the combination therapy group (P < 0.05). The efficacy of DC101 was much greater in the treatment of early tumors, which suggests that tumor burden may be a critical factor in determining the response to DC101. In vitro and in vivo analysis of endothelial cell function showed reduced matrix metalloproteinase type 9 production in endothelial cells treated with DC101., Conclusions: This study confirms the principle of tumor growth inhibition by targeting angiogenesis within tumors and supports the use of anti-VEGF receptor agents.