Abstract C126: <i>Lactobacillus</i> -Derived Exosomes Target Ferroptosis and Cancer Stemness to Overcome Therapeutic Resistance in Head and Neck Cancer, with Preferential Efficacy in HPV-Positive Tumors

Abstract Background: Therapeutic failure in Head and Neck Squamous Cell Carcinoma (HNSCC) is driven by a subpopulation of cancer stem cells (CSCs) that exhibit profound chemo- and radioresistance, leading to tumor recurrence. The microbiome has emerged as a novel reservoir for anti-cancer therapeutics, yet the specific effectors remain poorly defined. We hypothesized that exosomes derived from the commensal bacterium Lactobacillus rhamnosus (LREs) could overcome HNSCC resistance by targeting both tumor bulk and the CSC niche. Methods: LREs were isolated from L. rhamnosus cultures and functionally characterized. To investigate their multi-modal anti-tumor activity, we employed a comprehensive suite of preclinical models, including therapy-resistant HNSCC cell lines, patient-derived organoids, and Polyploid Giant Cancer Cells (PGCCs)—a known source of CSCs. Mechanistic pathways were interrogated using RNA-sequencing and molecular assays for cell death, stemness, and epithelial-mesenchymal transition (EMT). In vivo efficacy was validated in murine xenograft and zebrafish models, which were also used to assess differential activity in HPV-positive versus HPV-negative HNSCC. Results: LREs induced potent cytotoxicity in HNSCC cells, including radioresistant subtypes, and demonstrated strong synergy with cisplatin. Mechanistic investigation revealed that LREs concurrently induced both apoptosis and ferroptosis. Crucially, LREs targeted the drivers of tumor recurrence, significantly downregulating CSC markers (SOX2, NANOG, CD44) and reversing the EMT phenotype, evidenced by E-cadherin upregulation and Vimentin downregulation. These potent anti-tumor effects were validated in vivo, where LREs suppressed tumor growth in xenograft models. Strikingly, in zebrafish avatar models, LREs exhibited significantly more pronounced anti-tumor activity in HPV-positive HNSCC, irrespective of p53 mutational status, suggesting a defined therapeutic window. Conclusion: Our findings establish L. rhamnosus-derived exosomes as a first-in-class, microbiota-based therapeutic that dismantles HNSCC resistance and stemness by co-activating dual cell death pathways. The pronounced efficacy in HPV-positive tumors nominates LREs as a promising precision therapy for this distinct HNSCC subtype, offering a novel strategy to combat therapeutic failure and prevent tumor recurrence. Citation Format: Jae Won Chang, Seung-Nam Jung, Mi Ae Lim, Sicong Zheng Zheng, Doh Hoon Kim, Ji Won Kim, Bon Seok Koo. Lactobacillus-Derived Exosomes Target Ferroptosis and Cancer Stemness to Overcome Therapeutic Resistance in Head and Neck Cancer, with Preferential Efficacy in HPV-Positive Tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C126.