Clinically Relevant Approaches for Targeting Triple-Negative Breast Cancer Stem Cells While Enhancing Anti-Tumor Immunity

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype characterized by high rates of therapeutic resistance, disease recurrence, and poor survival outcomes. Although chemotherapy is the current standard of care, recent advances in immunotherapy have provided only modest clinical benefit. These limitations are driven in part by cancer stem cells (CSCs) that promote treatment resistance, tumor heterogeneity, and immune evasion. This thesis investigates three translational approaches aimed at targeting heterogenous CSC populations while enhancing anti-tumor immunity in clinically relevant TNBC models. Using in silico molecular docking, I first identified the over-the-counter flavonoid supplements quercetin and luteolin as potential inhibitors of the immune-suppressing ectoenzyme CD73, and the CSC-associated YAP and Wnt signaling pathways. In combination with the chemotherapeutic agent paclitaxel, quercetin and luteolin antagonized the enrichment of CSCs and reduced CD73 in human TNBC cell lines, patient-derived tumor fragments, and syngeneic immune-competent mouse models. Next, I showed that combining the chemotherapeutic agent doxorubicin with the clinically approved ALDH inhibitor disulfiram broadly upregulated PD-L1 expression on CSCs, sensitizing these otherwise resistant populations to the anti-PD-L1 atezolizumab, irrespective of baseline PD-L1 status. Mechanistically, CSC-associated STAT3 signaling was identified as a potential mediator of this phenotypic shift, providing a rationale for leveraging PD-L1 intrinsic signaling vulnerabilities across heterogenous CSC populations. Lastly, I explored the repurposing of pathogenic bacterial effector proteins OspG from Shigella flexneri and SseL from Salmonella enterica to preferentially inhibit CSC-associated NF-κB signaling without inducing the acquired tolerance commonly observed with preclinical NF-κB inhibitors. Co-expression of OspG and SseL significantly suppressed tumor growth in an orthotopic TNBC mouse model, supporting their potential as novel therapeutic candidates for overcoming CSC-driven treatment resistance. Together, these studies concurrently address the challenges of CSC heterogeneity and immune evasion in TNBC. By showing the therapeutic potential of rationally designed combination strategies at clinically achievable dosage, this thesis establishes a translational foundation for the development of more effective, durable, and accessible therapeutic approaches for TNBC.