Opinion: Ligand identity ambiguity in LNP-induced metastasis promotion via TLR9-MyD88/cGAS-STING-dependent NETosis – Endogenous mtDNA or exogenous bacterial PAMPs?

Wang and colleagues recently reported that lipid nanoparticle (LNP) administration promotes experimental pulmonary tumor metastasis through TLR9-MyD88- and cGASSTING-mediated neutrophil extracellular trap (NET) formation, invoking mitochondrial DNA (mtDNA) released from necrotic muscle cells as the activating ligand. This Opinion challenges a fundamental assumption underlying that interpretation: because TLR9 recognizes unmethylated CpG dinucleotides shared equally by mtDNA and bacterial DNA, and cGAS detects double-stranded DNA in a sequence-independent manner, the genetic rescue experiments are equally consistent with bacterial nucleic acid or lipopolysaccharide (LPS) contamination of the LNP preparations. Standard endotoxin detection methods, including the Limulus amebocyte lysate assay, are demonstrably unreliable in lipid matrices, where LPS lipid A inserts stably into hydrophobic domains and evades detection. Total lipid A quantification by EndoQuant (acid hydrolysis/HPLC-MS/MS), 16S rDNA analysis, polymyxin B pretreatment, and amplicon sequencing are required to resolve ligand identity. Until these controls are applied, the mtDNA hypothesis and a manufacturing contamination hypothesis remain equally supported by the available data — a distinction with profoundly different regulatory and clinical implications for LNP-based therapeutics.