Research Article Suppression of Prostate Cancer Nodal and Systemic Metastasis by Blockade of the Lymphangiogenic Axis Jeremy B. Burton,1 Saul J. Priceman,1 James L. Sung,1 Ebba Brakenhielm,2,4 Dong Sung An,3 BronislawPytowski, 5 Kari Alitalo,6 and Lily Wu1,2 1Department of Molecular and Medical Pharmacology, 2Department of Urology, 3Department of Medicine, and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California; 4Rouen Medico- Pharmacological University, Rouen, France; 5Department of Cell Biology, ImClone Systems, New York, New York; and 6Molecular Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland Abstract progresses, systemic metastasis to bone and liver ultimately lead to patient morbidity and mortality. Current treatments include Lymph node involvement denotes a poor outcome for patients radical prostatectomy, usually with pelvic lymphadenectomy for with prostate cancer. Our group, along with others, has shown lymph node assessment, followed by radiation or hormone therapy that initial tumor cell dissemination to regional lymph nodes (4). There are currently no effective treatments for recurrent or via lymphatics also promotes systemic metastasis in mouse metastatic disease, highlighting the importance of alternative models. The aim of this study was to investigate the efficacy of strategies for early intervention. suppressive therapies targeting either the angiogenic or Angiogenesis is essential for the growth of solid cancers beyond lymphangiogenic axis in inhibiting regional lymph node and 2 mm, which is the limit of nutrient diffusion (5). This process also systemic metastasis in subcutaneous and orthotopic prostate clearly contributes to metastasis of most solid cancers. Vascular tumor xenografts. Both androgen-dependent and more endothelial growth factor-A (VEGF-A) signaling through its aggressive androgen-independent prostate tumors were used receptor VEGFR-2 is critical for the development and maintenance in our investigations. Interestingly, we observed that the of tumor blood vasculature (6, 7). Inhibition of VEGF signaling, by threshold for dissemination is lower in the vascular-rich targeting either the ligand or the receptor, suppresses both tumor prostatic microenvironment compared with subcutaneously growth and metastasis and is currently being tested in clinical grafted tumors. Both vascular endothelial growth factor-C trials as single agents and in combination with chemotherapy or (VEGF-C) ligand trap (sVEGFR-3) and antibody directed radiation therapy (6, 8, 9). More recently, lymphangiogenesis has against VEGFR-3 (mF4-31C1) significantly reduced tumor received much attention as an important mediator of tumor cell lymphangiogenesis and metastasis to regional lymph nodes dissemination. VEGF-C and VEGF-D, the major lymphangiogenic and distal vital organs without influencing tumor growth. ligands for the receptor VEGFR-3, induce proliferation of lymphatic Conversely, angiogenic blockade by short hairpin RNA against endothelial cells and sprouting of lymphatic vessels (10, 11). VEGF or anti–VEGFR-2 antibody (DC101) reduced tumor VEGFR-3–mediated lymphangiogenesis also potently influences blood vessel density, significantly delayed tumor growth, and lymph node metastasis in various tumor models (12–14). Recent reduced systemic metastasis, although it was ineffective in studies by our group and others have also provided evidence for the reducing lymphangiogenesis or nodal metastasis. Collectively, direct contribution of VEGF to lymphangiogenesis, in addition to these data clarify the utility of vascular therapeutics in its principal functions in angiogenesis (15–17). Overall, targeting prostate tumor growth and metastasis, particularly in the of the VEGFR-2 and VEGFR-3 signaling pathways are promising context of the prostate microenvironment. Our findings therapies for the treatment of solid cancers. highlight the importance of lymphangiogenic therapies in In prostate cancer, the expression of VEGF-C and VEGFR-3 the control of regional lymph node and systemic metastasis. has been shown to be highly associated with regional lymph [Cancer Res 2008;68(19):7828–37] node metastasis (18–21). Our previous studies have correlated the levels of tumor-derived VEGF-C with the extent of tumor Introduction lymphatics and subsequent lymph node and lung metastases in Prostate cancer is the most common cancer among men and xenograft models of human prostate cancer (22). The precise second in cancer-related deaths in the United States (1). Whereas contributions of intratumoral and peritumoral lymphatics to monitoring serum PSA and histopathology (Gleason grade) are lymph node metastasis have been extensively debated and useful in clinical assessment, pelvic lymph node metastasis remains require further investigation (23). Recent reports have highlighted the most significant indicator of patient prognosis and determi- that lymphogenous spread can augment systemic metastasis (22, nant of therapeutic aggressiveness (2, 3). As prostate carcinoma 24). Although angiogenesis and lymphangiogenesis are critical mediators of the metastatic process, the distinct contributions of each axis to nodal and systemic metastasis of prostate cancer remain unclear. Note: Supplementary data for this article are available at Cancer Research Online In the current study, we used VEGF or VEGF-C pathway-specific (http://cancerres.aacrjournals.org/). J.B. Burton and S.J. Priceman contributed equally to this work. therapies to decipher their roles in lymph node and lung metastasis Requests for reprints: Lily Wu, Department of Urology, MRL 2210, Box 951738, of prostate cancer. Using overexpression and short hairpin RNA University of California, Los Angeles, CA 90095-1738. Phone: 310-825-8511; Fax: 310- (shRNA) silencing of these growth factors, we show that VEGF-C 206-5343; E-mail: [email protected]. I2008 American Association for Cancer Research. and, to a lesser extent, VEGF are required for lymph node and doi:10.1158/0008-5472.CAN-08-1488 subsequent lung metastasis. Furthermore, the findings from using Cancer Res 2008; 68: (19). October 1, 2008 7828 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Inhibiting Prostate Cancer Nodal Metastasis specific inhibitors of the VEGFR-2 and VEGFR-3 axes indicate that, Before implanting cells, LAPC-9 supernatants were collected and analyzed in prostate cancer, angiogenesis plays a critical role in prostate by ELISA and Western blot to ensure equivalent expression levels. ex vivo  5 tumor growth and systemic metastasis, but targeting the VEGFR-2 Noninvasive and imaging. CWR22Rv-1 cells (5 10 ) were axis alone does not significantly reduce tumor lymphangiogenesis implanted subcutaneously above the right shoulder of immunodeficient SCID/beige male mice (Taconic). Tumor size was measured regularly using or nodal metastasis. However, targeting the lymphangiogenic axis digital calipers and by noninvasive optical imaging as follows. After significantly reduces both lymph node and systemic metastasis in administration of Renilla luciferase substrate, coelenterazine (1 mg/kg i.v.), our model, without significantly influencing primary tumor growth. anaesthetized mice (i.p. injection of a 4:1 mixture of ketamine and xylazine) Consequently, we believe that combination treatments targeting were imaged as previously described (22). Primary tumors were grown to both vascular axes in conjunction with conventional therapy may the ethical limit of 1.5 cm in diameter, at which time the animals were offer the best protection against recurrent, disseminated disease in sacrificed. prostate cancer patients with a poor prognosis. For orthotopic implants, transduced CWR22Rv-1 cells (1  105)or LAPC-9 cells (2.5  105) suspended in Matrigel were implanted in the surgically exposed prostate region of SCID/beige male mice, as previously Materials and Methods described (26). Cells (in 10 AL/lobe) were implanted at the base of the Tumor cells. The androgen-independent, androgen-responsive exposed seminal vesicles in each dorsolateral lobe. Incisions were closed CWR22Rv-1 tumor cell line (kind gift from Dr. David Agus, Cedars-Sinai with vicryl sutures (Novartis) and tumor growth was monitored optically Medical Center) was maintained in vitro in RPMI containing 10% fetal over the course of the next 3 wk. bovine serum (FBS) and 1% penicillin/streptomycin. The androgen- Micro-computed tomography contrast imaging. Tumor-bearing mice dependent human prostate cancer cell line LAPC-9 was a kind gift from were anesthetized and Fenesta vascular contrast (Alerion) agent was Dr. Charles Sawyers (Memorial Sloan Kettering Cancer Center). LAPC-9 injected i.v. into the tail vein and imaged after 1 h. One representative xenografts were maintained in vivo and manipulated ex vivo, as previously animal from the control and experimental group was used for vascular described (22). contrast computed tomography (CT) imaging. Mice were imaged with a Lentiviral production and tumor cell transduction. For all in vivo micro-CT scanner (MicroCAT II, Siemens Preclinical Solutions) over 7 min studies, cells were transduced using lentivirus carrying cytomegalovirus using 70 kVp, 500-ms exposures, and 360j rotation to create images with (CMV) promoter-driven Renilla