Stromal VEGF-C And C-C Chemokine Receptor 7 Mediate Prostate Cancer Metastasis To Sentinel Lymph Node

BackgroundMetastasis is the main cause of cancer mortality, including prostate cancer. Recent reports suggested a direct role of the lymphatic system in the tumor spread not only to loco-regional lymph nodes but also to distant organs. Preliminary findings from our group suggest that a preferred route of prostate cancer dissemination is via lymphatic vessels to the regional lymph nodes (LN), which in turn provide a reservoir for subsequent spread to distal vital organs. Vascular Endothelial Growth Factor sub-family member C, VEGF-C, has been demonstrated to play a dominant role in lymphatic vessels formation and thus facilitating tumor cells metastasizing to lymph nodes. Chemokine-receptor axis has been suggested helping drain tumor cells escaping to certain organs.ObjectivesOur first aim is to build a lymphatic metastasis model of prostate cancer by modulating lymphangiogenesis in the stroma. The secondary aim is to demonstrate the CCR7/CCL21 chemokine-receptor axis as the guidance signal in lymph node metastasis of prostate cancer.Methods1. In order to determine the role of stromal cell secreted VEGF-C in prostate tumor dissemination, we generated a recombinant human prostate xenograft model composed of Renilla luciferase (RL)-expressing LAPC-9 human prostate tumor cells accompanied with murine NIH 3T3 stromal cells that overexpressing human VEGF-C (hVEGF-C). The genetic modulation of both the tumor and stromal cells was accomplished by lentiviral vectors, and the lentivirus expresses only the Gfp gene was used as control. The LAPC-9/3T3 tumor-stroma combined xenografts model was established subcutaneously in severe combined immunodeficiency (SCID) mouse. Optical CCD imaging of RL was employed to monitor tumor growth and dissemination in vivo and ex vivo. Expression and secretion of chemokines and receptors by primary tumor and tumor metastases were determined by quantitative real-time polymerase chain reaction (real time RT-PCR), immunohistochemical staining and western blot assay, respectively.2. To refine our model system, we employed prostate cancer cell lines CWR22Rvl (CWR) and LNCaP as in vitro models, which were marked as highly expressing human CCR7 with CMV-CCR7-IRES-GFP lentiviruses. An adeno-CCL21 vector (Ad-CCL21) was used to generate the medium containing CCL21 for chemokine-receptor interaction study. The assays of gene profiles and protein production alterations were carried out using real time RT-PCR and western blot. Chemotaxis assay was performed to test the chemotactic activity of CCR7+prostate cancer cells.Results1. The LAPC-9/3T3-hVEGF-C tumors exhibited abundant peritumeral lymphatic vessels promoting regional LN metastases and lung metastasis with detectable luciferase signals, at an incidence of 4/6 animals in the cohort. Conversely, the LAPC-9/3T3-GFP control tumors displayed negligible metastatic potential in 0/6 mice. The expression of a selected set of metastasis-related genes between these 2 tumor samples were analyzed and compared by real time RT-PCR. We observed a consistent elevation of hVegfr-3, hMmp9, Ccr7 and Ccl21in the nodal metastatic lesion, and Cxcr4 in lung metastasis lesion. Moreover, we isolated and expanded the primary and LN metastatic lesions and propagated as subcutaneous tumors in SCID mice to profile their gene expression. We observed a consistent up-regulation of C-C chemokine receptor 7 (CCR7) (11.62 folds, P=0.0007) and its cognate ligand CCL21 (11.37 folds, P=0.001) at the gene expression level in the LN lesions over primary tumors, which were confirmed by immunohistochemical staining of LN and tumor sections. Furthermore, the disrupted single tumor cell derived from the LN metastases with higher expression of CCR7 responded more robustly to chemotactic attraction of CCL21 in an in vitro migration assay as compared to control primary tumor cells lacking CCR7 upregulation (P= 0.000009).2. The gene modified CWR and LNCaP cells chemotactically responded to Ad-CCL21 arose conditioned medium (CCL21-CM), and the chemotaxis pattern could be impaired by the anti-CCL21 antibody. The CCL21-CM also caused the activation of prosurvival PI3K/Akt signaling pathway and NF-κB.ConclusionsOur findings suggest that, stromal VEGF-C expression induced lymphangiogenesis and functional lymphatics that provide an escape route for the prostate tumor cells. And the nodal enrichment possibly induced the metastasis to lung by the upregulation of CXCR4 in prostate tumor cells. Moreover, the CCR7/CCL21 axis upregulated in the prostate tumor cells likely provided a chemotactic guidance aiding the tumor cells to navigate to regional lymph nodes through the lymphatics.