Early in cancer growth, metastases and dendritic cells (DCs) traffic to draining lymph nodes (DLN). In lung cancer and other carcinomas, immune-suppression dominates a tumor microenvironment characterized by immature DCs, weak T-effector responses, proliferation of T-regulatory cells (Tregs), and over-production of immuno-suppressive cytokines. This heavily represses anti-tumor immunity. A high-impact area of new scientific discovery is immunotherapy. We find dramatic clinical responses in some advanced-stage cancer patients by blocking suppression of effector T cells. Only a fraction (<25%) of such patients, however, respond with durable remissions. Preliminary work shows that a unique class of glycans known as heparan sulfate (HS) drives subversive DC traffic and DC immaturity. While we have inhibited such DC traffic by targeting lymphatic endothelial HS, new work suggests that DC-specific HS alterations may modulate both pathologic chemokine- dependent DC traffic as well as DC maturation and function. New studies also show that these properties may favorably impact anti-tumor T cell functions, with inhibition of tumor growth and progression. Herein we target such glycans in tumor and cell-based studies, while studying immune-function and molecular mechanisms. This proposal addresses the hypothesis that targeting HS glycans on the surface of DCs in lung cancer through genetic means and novel inhibitors will inhibit DLN colonization by tolerogenic DCs and improve anti- tumor immunity. Reduced immune-tolerance and improved tumor-antigen responses by more mature DCs, with improved T cell induction will result in a novel endogenous anti-tumor state. To test this, we propose to: (1) Characterize tumor growth and anti-tumor immunity in model antigen- as well as spontaneous lung carcinoma models in mice bearing DC-glycan alterations. We will assess how a DC-targeted mutation in a key sulfating HS biosynthetic enzyme (Ndst1) affects T cell immunity in tumors and thoracic DLN of mice with orthotopic Ovalbumin-expressing Lewis lung carcinomas (LLC-Ova), including Ova-specific immunity and effects on tumor growth. Immunity and tumor growth in a KRAS transgenic mutant model will also be studied. (2) Study anti-tumor DC and T cell functions in ex-vivo preparations from lung carcinoma bearing mice with DC-specific alterations in HS biosynthesis. DC maturation, antigen presentation, and the capacity of DCs from LLC-Ova tumors grown in DC-targeted HS mutants to activate Ova-sensitized T cells will be examined, as will tumor-cytolytic capacity of CD8+ T cells isolated from DLNs of tumor-bearing mutant vs control mice. Studies will also include the effects of mutation on DLN colonization by plasmacytoid DCs and their functional capacity. (3) Assess chemokine-receptor interactions and signaling mechanisms in the setting of DC-targeted alterations in HS biosynthesis; and the effects of novel HS inhibitors on tumor growth and immunity. We will study how HS mutation affects DC-surface binding of lymphatic chemokines CCL21, CXCL12, or CX3CL1 to cognate receptors. The effect of HS mutation on DC migration and maturation signaling in response to chemokine will be assessed, including the activation of pDCs through novel TLR-dependent mechanisms. Finally, we will study how novel HS inhibitors affect signaling by tumor DCs as well as lung carcinoma growth and immunity. This work examines novel mechanisms whereby altering trafficking and maturation of tumor DCs may effectively re-program endogenous anti-tumor immunity. The genetic target validation and rational introduction of glycan-targeting inhibitors may guide new therapies to achieve immune eradication of human lung cancer.
In cancer, chemokines drive the transit of tumor cells and pathologic cell traffic that includes migration of tolerance-promoting dendritic cells (DCs) from the tumor to draining lymph nodes (DLNs). In lung cancer, such tumor-infiltrating DCs (TIDCs) induce immune-suppression. Initial work shows that genetically targeting certain complex sugars (glycans) specifically on DCs may slow lymphatic TIDC traffic and induce maturation of TIDCs, which importantly correlates with carcinoma growth inhibition and an anti-tumor immune profile. While this strategy appears to effectively ?re-program? immunity against the tumor, our understanding of mechanisms and pre-clinical targeting approaches in lung cancer models remains limited. Herein, we employ novel genetic methods and inhibitors to interfere with such glycans in TIDCs, while examining for anti-tumor immunologic shifts in the tumor and DLNs. This study examines novel mechanisms and validates key genetic targets to induce anti-tumor immunity and tumor regression in lung cancer, with an eye to therapeutic translation. !
