Jeremy B. Burton

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.

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.

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.

Micro-PET/CT Monitoring of Herpes Thymidine Kinase Suicide Gene Therapy in a Prostate Cancer Xenograft: The Advantage of a Cell-specific Transcriptional Targeting Approach

Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.

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.

Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer.

Functional Molecular Imaging of Prostate Cancer Lymph Node Metastases: Adenovirus-Mediated Lymph Node Detection

Like other solid epithelial tumors, nodal involvement in prostate cancer signifies a poor clinical outcome. Yet, the lack of standard pelvic lymph node (LN)-sampling procedures and specific noninvasive imaging approaches to accurately assess nodal involvement is hampering the management decision. This chapter will discuss the debates in the clinical community on LN dissection and the development of various imaging modalities for prostate cancer. We will also describe a novel approach of using recombinant human adenoviral vectors (Ads) to directly map nodal metastases in experimental models of human prostate cancer. By exploiting the innate lymphotrophic properties of adenovirus and the prostate-restricted expression of imaging reporter genes incorporated into the viral vector, we were able to produce bioluminescent or positron emission tomography (PET) signals that correlate with the presence of metastatic lesions in the draining LNs. Significantly, this approach enables the direct PET visualization of sentinel lymph node (SLN) metastases, without the need for lymphadenectomy. We believe that developing and implementing more effective diagnostic imaging modalities to assess nodal status could improve the outcomes for patients with advanced stage prostate cancer.

The development of a potent, multifunctional gene delivery vector driven by the cancer-selective mucin-1 promoter to detect and treat breast cancer metastasis.

Abstract #6022 With breast cancer, early detection and proper staging are critical, influencing both the treatment regimen and therapeutic outcome for a patient. Improvements in these areas will play a profound role in reducing mortality due to this disease. We therefore aim to develop a targeted adenoviral based gene therapy vector that can be used in the detection and staging of metastatic breast cancer. We did this through application of the two-step transcriptional amplification system (TSTA). This system was developed in our lab, and has shown the ability to greatly augment the activity of weak promoters, while still maintaining tissue specificity.
 We incorporated the tumor specific promoter Mucin1 into the TSTA system to successfully target breast tumor models. Studies carried out in vitro have demonstrated that this MUC1 TSTA vector is able to achieve efficiency of up to 250 fold over basal promoter levels, without loss of specificity. Moreover, when incorporated into an adenoviral vector to express firefly luciferase (AdMUC1-TSTA-fl), it exhibits higher reporter gene expression than a AdCMV-fl virus at the same multiplicity of infection (MOI).
 Biodistribution studies have shown that, depending upon the initial route of administration, this system holds great potential for use in the detection of lymph node metastais, as well as distant metastases. This vector has shown low off target expression following intravenous injection into non-tumor bearing mice. Through use of in vivo xenograft models of breast cancer, we have shown the ability to target and detect both lymph node metastais, as well as liver metastasis using our AdMUC1 TSTA-fl reporter virus in combination with in vivo optical imaging. Based on these results, we believe that this system holds strong promise for clinical application.
 A critical aspect of this project will be the incorporation of cytotoxic genes for therapeutic studies. At the same time, the ability to express imaging reporter genes for bioluminescent imaging or PET (positron emission tomography) is highly desirable, allowing for efficient, non-invasive monitoring of pharmacokinetics of transgene expression in real time. To address this issue, we developed a MUC-1 driven TSTA amplification scheme able to simultaneously express two separate transgenes, the firefly luciferase and the herpes simplex virus thymidine kinase (HSV-tk sr39tk) gene. This bi-directional reporter TSTA construct has shown strong efficiency, and specificity, and is currently being evaluated for use in therapeutic application of breast tumors in vivo . This approach holds great promise for the treatment and diagnosis of metastatic breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6022.