Lactobacillus rhamnosus modulates gut microbiota and tyrosine metabolism to potentiate PD-1-mediated anti-tumor immunity in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype with limited benefit from immune checkpoint blockade (ICB) and frequent immune-related adverse events (irAEs). We achieved stable overexpression of PD-1 in tumor cells through intratumoral injection of a PD-1-overexpressing plasmid. The results indicated that intratumoral PD-1 gene therapy exerted a dual effect. It significantly inhibited tumor cell growth via PD-1/PD-L1 interaction. Moreover, it promoted antitumor immunity, as evidenced by increased CD8+ T cells in the spleen, mesenteric lymph nodes (MLNs), and tumor tissues. Nevertheless, intratumoral PD-1 gene therapy induced intestinal side effects. Importantly, the probiotic Lactobacillus rhamnosus (L. rhamnosus) not only enhanced the antitumor activity of intratumoral PD-1 gene therapy but also alleviated intestinal injury. L. rhamnosus increased the expression of tight junction proteins (TJPs), including Claudin-1, Occludin, and ZO-1, upregulated anti-inflammatory cytokines (IL-10, TGF-β) and downregulated pro-inflammatory cytokines (IL-6, TNF-α). Notably, L. rhamnosus treatment also favorably reshaped the gut microbial composition, increasing the abundance of beneficial bacteria such as Lactobacillus, Bacteroides, and Parabacteroides, while reducing the levels of potential pathogenic bacteria including Prevotella, Staphylococcus, Adlercreutzia, and Desulfovibrio. Furthermore, metabolomic analysis revealed that L.rhamnosus significantly influenced tyrosine metabolism-related metabolites, downregulating the abundance of L-tyrosine, 3,4-dihydroxyphenylalanine (DOPA), N-acetyltyramine, methionyl-tyrosine, glutamic acid, and valine, while upregulating the level of butanoic acid. Collectively, these findings provide novel therapeutic strategies for enhancing the efficacy of TNBC treatment and alleviating intestinal side effects associated with immunotherapy.