The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.

Activating anaplastic lymphoma kinase (ALK) mutations were recently detected in most familial and 10% of sporadic neuroblastomas. However, the role of mutated ALK in tumorigenesis remains elusive. We demonstrate that targeted expression of the most frequent and aggressive variant, ALK^F1174L, is tumorigenic in mice. Tumors resembled human neuroblastomas in morphology, metastasis pattern, gene expression, and the presence of neurosecretory vesicles as well as synaptic structures. This ALK-driven neuroblastoma mouse model precisely recapitulated the genetic spectrum of the disease. Chromosomal aberrations were syntenic to those in human neuroblastoma, including 17q gain and MYCN oncogene amplification. Targeted ALK^F1174L and MYCN coexpression revealed a strong synergism in inducing neuroblastoma with minimal chromosomal aberrations, suggesting that fewer secondary hits are required for tumor induction if both oncoproteins are targeted. Treatment of ALK^F1174L transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALK^F1174L activity. We conclude that an activating mutation within the ALK kinase domain is sufficient to induce neuroblastoma development, and ALK inhibitors show promise for treating human neuroblastomas harboring ALK mutations.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen–specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody‐derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re‐directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell‐mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti‐tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti‐tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.

Extensive oncologic experience argues that the most efficacious applications of antiangiogenic agents rely upon a combination with cytotoxic drugs. Yet there remains a lack of clarity about how to optimize scheduling for such drug combinations. Prudent antiangiogenic therapy might transiently normalize blood vessels to improve tumor oxygenation and drug exposure. Using [¹⁵O]H₂O positron emission tomography imaging in a preclinical mouse model of non–small cell lung cancer, we observed that short-term treatment with the vascular endothelial growth factor receptor/platelet-derived growth factor receptor inhibitor PTK787 licensed a transient window of improved tumor blood flow. The improvement observed was associated with a reduced leakiness from tumor vessels, consistent with induction of a vascular normalization process. Initiation of a cytotoxic treatment in this window of tumor vessel normalization resulted in increased efficacy, as illustrated by improved outcomes of erlotinib administration after initial PTK787 treatment. Notably, intermittent PTK787 treatment also facilitated long-term tumor regression. In summary, our findings offer strong evidence that short-term antiangiogenic therapy can promote a transient vessel normalization process that improves the delivery and efficacy of a targeted cytotoxic drug. Cancer Res; 74(10); 2816–24. ©2014 AACR.