Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy.

Tumor-infiltrating myeloid cells (TIMs) support tumor growth by promoting angiogenesis and suppressing antitumor immune responses. CSF-1 receptor (CSF1R) signaling is important for the recruitment of CD11b(+)F4/80(+) tumor-associated macrophages (TAMs) and contributes to myeloid cell-mediated angiogenesis. However, the impact of the CSF1R signaling pathway on other TIM subsets, including CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs), is unknown. Tumor-infiltrating MDSCs have also been shown to contribute to tumor angiogenesis and have recently been implicated in tumor resistance to antiangiogenic therapy, yet their precise involvement in these processes is not well understood. Here, we use the selective pharmacologic inhibitor of CSF1R signaling, GW2580, to demonstrate that CSF-1 regulates the tumor recruitment of CD11b(+)Gr-1(lo)Ly6C(hi) mononuclear MDSCs. Targeting these TIM subsets inhibits tumor angiogenesis associated with reduced expression of proangiogenic and immunosuppressive genes. Combination therapy using GW2580 with an anti-VEGFR-2 antibody synergistically suppresses tumor growth and severely impairs tumor angiogenesis along with reverting at least one TIM-mediated antiangiogenic compensatory mechanism involving MMP-9. These data highlight the importance of CSF1R signaling in the recruitment and function of distinct TIM subsets, including MDSCs, and validate the benefits of targeting CSF1R signaling in combination with antiangiogenic drugs for the treatment of solid cancers.

TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/beta-catenin signaling.

Mutations in the genes encoding the Wnt receptor Frizzled-4 (FZD4), coreceptor LRP5, or the ligand Norrin disrupt retinal vascular development and cause ophthalmic diseases. Although Norrin is structurally unrelated to Wnts, it binds FZD4 and activates the canonical Wnt pathway. Here we show that the tetraspanin Tspan12 is expressed in the retinal vasculature, and loss of Tspan12 phenocopies defects seen in Fzd4, Lrp5, and Norrin mutant mice. In addition, Tspan12 genetically interacts with Norrin or Lrp5. Overexpressed TSPAN12 associates with the Norrin-receptor complex and significantly increases Norrin/beta-catenin but not Wnt/beta-catenin signaling, whereas Tspan12 siRNA abolishes transcriptional responses to Norrin but not Wnt3A in retinal endothelial cells. Signaling defects caused by Norrin or FZD4 mutations that are predicted to impair receptor multimerization are rescued by overexpression of TSPAN12. Our data indicate that Norrin multimers and TSPAN12 cooperatively promote multimerization of FZD4 and its associated proteins to elicit physiological levels of signaling.

A potent, imaging adenoviral vector driven by the cancer-selective mucin-1 promoter that targets breast cancer metastasis.

PURPOSE: With breast cancer, early detection and proper staging are critical, and will often influence both the treatment regimen and the therapeutic outcome for those affected with this disease. Improvements in these areas will play a profound role in reducing mortality from breast cancer. EXPERIMENTAL DESIGN: In this work we developed a breast cancer-targeted serotype 5 adenoviral vector, utilizing the tumor-specific mucin-1 promoter in combination with the two-step transcriptional amplification system, a system used to augment the activity of weak tissue-specific promoters. RESULTS: We showed the strong specificity of this tumor-selective adenovirus to express the luciferase optical imaging gene, leading to diagnostic signals that enabled detection of sentinel lymph node metastasis of breast cancer. Furthermore, we were able to target hepatic metastases following systemic administration of this mucin-1 selective virus. CONCLUSIONS: Collectively, we showed that the amplified mucin-1 promoter-driven vector is able to deliver to and selectively express a desirable transgene in metastatic lesions of breast tumors. This work has strong clinical relevance to current diagnostic staging approaches, and could add to targeted therapeutic strategies to advance the fight against breast cancer.

Suppression of prostate cancer nodal and systemic metastasis by blockade of the lymphangiogenic axis.

Lymph node involvement denotes a poor outcome for patients with prostate cancer. Our group, along with others, has shown that initial tumor cell dissemination to regional lymph nodes via lymphatics also promotes systemic metastasis in mouse models. The aim of this study was to investigate the efficacy of suppressive therapies targeting either the angiogenic or lymphangiogenic axis in inhibiting regional lymph node and systemic metastasis in subcutaneous and orthotopic prostate tumor xenografts. Both androgen-dependent and more aggressive androgen-independent prostate tumors were used in our investigations. Interestingly, we observed that the threshold for dissemination is lower in the vascular-rich prostatic microenvironment compared with subcutaneously grafted tumors. Both vascular endothelial growth factor-C (VEGF-C) ligand trap (sVEGFR-3) and antibody directed against VEGFR-3 (mF4-31C1) significantly reduced tumor lymphangiogenesis and metastasis to regional lymph nodes and distal vital organs without influencing tumor growth. Conversely, angiogenic blockade by short hairpin RNA against VEGF or anti-VEGFR-2 antibody (DC101) reduced tumor blood vessel density, significantly delayed tumor growth, and reduced systemic metastasis, although it was ineffective in reducing lymphangiogenesis or nodal metastasis. Collectively, these data clarify the utility of vascular therapeutics in prostate tumor growth and metastasis, particularly in the context of the prostate microenvironment. Our findings highlight the importance of lymphangiogenic therapies in the control of regional lymph node and systemic metastasis.

Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer.

The accurate assessment of nodal involvement in prostate cancer is crucial to planning treatment, yet there is a shortage of noninvasive imaging techniques capable of visualizing nodal lesions directly. This study demonstrates the feasibility of using recombinant human adenoviral vectors to detect nodal metastases in a human prostate cancer model. This was achieved by the prostate-restricted expression of optical and positron emission tomography (PET) imaging reporter genes by the viral vector coupled with the innate lymphotropic properties of adenovirus. We show that peritumoral administration of these vectors results in the direct detection of reporter gene expression in metastatic lesions within sentinel lymph nodes. Notably, this approach parallels the current lymphoscintigraphy method but enables the direct PET visualization of sentinel lymph node metastases, eliminating the need for invasive lymphadenectomy. These findings may lead to more effective diagnostic and therapeutic strategies for individuals with advanced-stage prostate cancer.

Modulating metastasis by a lymphangiogenic switch in prostate cancer.

Prostate cancer dissemination is difficult to detect in the clinic, and few treatment options exist for patients with advanced-stage disease. Our aim was to investigate the role of tumor lymphangiogenesis during metastasis. Further, we implemented a noninvasive molecular imaging technique to facilitate the assessment of the metastatic process. The metastatic potentials of several human prostate cancer xenograft models, LAPC-4, LAPC-9, PC3 and CWR22Rv-1 were compared. The cells were labeled with luciferase, a bioluminescence imaging reporter gene, to enable optical imaging. After tumor implantation the animals were examined weekly during several months for the appearance of metastases. Metastatic lesions were confirmed by immunohistochemistry. Additionally, the angiogenic and lymphangiogenic profiles of the tumors were characterized. To confirm the role of lymphangiogenesis in mediating metastasis, the low-metastatic LAPC-9 tumor cells were engineered to overexpress VEGF-C, and the development of metastases was evaluated. Our results show CWR22Rv-1 and PC3 tumor cell lines to be more metastatic than LAPC-4, which in turn disseminates more readily than LAPC-9. The difference in metastatic potential correlated with the endogenous production levels of lymphangiogenic growth factor VEGF-C and the presence of tumor lymphatics. In agreement, induced overexpression of VEGF-C in LAPC-9 enhanced tumor lymphangiogenesis leading to the development of metastatic lesions. Taken together, our studies, based on a molecular imaging approach for semiquantitative detection of micrometastases, point to an important role of tumor lymphatics in the metastatic process of human prostate cancer. In particular, VEGF-C seems to play a key role in prostate cancer metastasis.

Vascular endothelial growth factor-a promotes peritumoral lymphangiogenesis and lymphatic metastasis.

Metastases are commonly found in the lymphatic system. The molecular mechanism of lymphatic metastasis is, however, poorly understood. Here we report that vascular endothelial growth factor (VEGF)-A stimulated lymphangiogenesis in vivo and that overexpression of VEGF-A in murine T241 fibrosarcomas induced the growth of peritumoral lymphatic vessels, which occasionally penetrated into the tumor tissue. As a result of peritumoral lymphangiogenesis, metastases in lymph nodes of mice were detected. VEGF-A-overexpressing tumors contained high numbers of infiltrating inflammatory cells such as macrophages, which are known to express VEGF receptor (VEGFR)-1. It seemed that in the mouse cornea, VEGF-A stimulated lymphangiogenesis through a VEGF-C/-D/VEGFR-3-independent pathway as a VEGFR-3 antagonist selectively inhibited VEGF-C-induced, but not VEGF-A-induced, lymphangiogenesis. Our data show that VEGF-A contributes to lymphatic mestastasis. Thus, blockage of VEGF-A-induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.