Abstract 3516: Reversing extracellular vesicle induced tumor immune suppression at the sentinel lymph node: Role of secretory autophagy and mitophagy

Abstract

Abstract The goal is to reverse cancer immune evasion at the level of the sentinel lymph node (SLN). Reduced expansion of CD8+ T cells and other innate immune effector cells in the cancer draining SLN is associated with progression and resistance to checkpoint inhibitors. Cancer-derived extracellular vesicles (EVs) that are PD-L1+ suppress immune recognition at the level of the SLN. The experimental goal is to remodel the SLN to overcome cancer EV-associated immune suppression and induce immune rejection of the tumor. We developed three methodologies for this project: a) Collection of draining lymph fluid to characterize EVs shed by 4T1 syngeneic breast tumors growing in the mammary fat pad. b) Chromatographic separation and characterization of the repertoire of EVs shed by tumors into the tumor microenvironment interstitial space using western blotting, mass spectrometry, and electron microscopy. c) Nanoparticle (NP) delivery of purified populations of EVs to the tumor draining SLN in combination with cytokine chemoattractants for innate immune cells recruitment. We characterized the in vivo interstitial fluid (IF) content of a GFP-4T1 syngeneic murine cancer model to study resident IF EVs transit to the draining lymph node. GFP labeling confirmed the IF EV tumor cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; including PINK1 and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial pinching), and VPS35, SEC22b, and Rab33b (vacuolar sorting). SLN immune cell populations could be massively remodeled by introducing hydrogel NPs which have a controlled release of T-cell and dendritic cell chemoattractant to the subcapsular sinus. NPs were successfully used to deliver concentrated packages of EVs subpopulations to the SLN. Introduction of the large CD81-/VEGF+/PD-L1- EV subpopulation (amphisome characteristics) to the SLN augmented tumor growth, angiogenesis, and metastasis, even when cytokine induction was used to remodel the SLN. In marked contrast, introduction of the CD81+/PD-L1+ EV subpopulation (containing mitophagy components) to the SLN in combination with NP release of chemoattractants, induced immune rejection of the syngeneic breast cancer, reducing tumor growth, and blocking metastasis. These findings demonstrate that different populations of EVs have opposite effects on cancer immune evasion at the level of the SLN and that EV-mediated immune suppression can be reversed by SLN remodeling to augment dendritic and CD8+ T cells. Citation Format: Marissa Howard, James Erickson, Amanda Haymond, Alessandra Luchini, Fatah Kashanchi, Lance Liotta. Reversing extracellular vesicle induced tumor immune suppression at the sentinel lymph node: Role of secretory autophagy and mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3516.