Searching for the Molecular Basis of Non-Inflamed Tumors

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Immunobiology

First Advisor

Chen, Lieping

Abstract

The emergence and unprecedented success of immune checkpoint blockade therapies have tremendously advanced the clinical strategies for cancer treatment. Despite the exceptional performance of immunotherapies in various types of solid tumors and hematologic malignancies, it has been recognized that the majority of cancer patients still receive limited benefits from current therapeutic approaches. Response to immunotherapies relies predominantly on inflammation, especially intra-tumoral T cells, and CD8+ T-cell infiltration is one of the most favorable prognostic markers for various cancer types. However, the lack of inflammation, particularly T-cell infiltration into the immunosuppressive tumor microenvironment, is widely observed in patients and correlated with primary and acquired resistance to immunotherapies. Certain types of cancer, such as pancreatic cancer, have been largely characterized as immunologically “cold”, and barely any clinical response to current immunotherapies could be observed in those patients. Hence, deeper understanding of the molecular mechanisms which control the exclusion and infiltration of T cells is urgently needed to facilitate the design and development of turning “cold” tumor “hot” as a therapeutic approach to improve patient survival. Through bioinformatics analyses of TCGA pancreatic cancer patient data, Slit Guidance Ligand 2 (SLIT2) was identified as the top candidate associated with impaired CD8+ T-cell infiltration and worse clinical outcome, leading to the central hypothesis that elevated SLIT2 expression in the tumor microenvironment dictates an immune phenotype of T-cell exclusion. Genetic perturbation of tumoral SLIT2 expression led to significantly elevated T-cell infiltration into the tumor core and robust tumor growth control by anti-tumor immunity in vivo. Functional normalization of the tumor infiltrated lymphocytes was also observed in the absence of tumoral SLIT2. Mechanistically, we demonstrated that recombinant purified SLIT2 protein can directly act on T cells to antagonize chemotactic signals. Functional epitope mapping illustrated that SLIT2’s inhibitory activity on T cells is largely conferred by the third leucine-rich repeat (LRR) domain of its N-terminal fragment, while the binding site of its canonical receptor ROBO1 is functionally dispensable. Soluble ROBO1 extracellular fragment failed to neutralize SLIT2’s inhibitory effect on T-cell chemotaxis, and T-cell surface ROBO1 expression could not be detected. Monoclonal SLIT2 antibody 6F9 restored T-cell chemotaxis suppressed by SLIT2 fragment without ROBO1 binding site, and single agent 6F9 administration led to robust SLIT2-expressing tumor growth control. Together, this work illustrated the profound impact of SLIT2 on T cell-mediated anti-tumor immunity, and implicated that the effect of SLIT2 on T-cell exclusion could be mediated via a non-ROBO1 receptor. My study presented in this thesis supports SLIT2 as an actionable target for cancer immune normalization therapy.

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