Bifidobacterium infantis-mediated HSV-TK/GCV therapy modulates the tumor microenvironment through site-specific...

Bifidobacterium (BF)-mediated herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) therapy has shown antitumor activity, but its effects on protein phosphorylation remain unclear.

Quantitative phosphoproteomic analysis was performed on HCT-116 tumor xenografts to identify phosphorylation changes induced by BF-TK/GCV treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to explore the affected biological processes and signaling pathways. Immunohistochemistry (IHC) was used to validate the phosphorylation levels of key proteins. Additionally, RNA sequencing and IHC were applied to immunocompetent MC38 tumor-bearing mice to assess transcriptional changes and immune-related pathways.

Compared with the BF-TK group, BF-TK/GCV treatment resulted in 337 differentially abundant phosphopeptides (DAPs), and 108 DAPswere significantly altered relative to the BF/GCV group. GO analysis revealed enrichment in protein kinase activity, cell cycle regulation, and DNA replication. KEGG pathway analysis and IHC validation demonstrated that BF-TK/GCV differentially regulated the phosphorylation status of HIF-1α, mTOR, PKM2, and PD-L1 in a site-specific manner. Notably, IHC showed elevated immunoreactivity for several of these phosphoproteins in xenograft tumors, while phosphoproteomic comparisons also revealed decreased phosphopeptide abundance at other sites, indicating context-dependent modulation rather than uniform inhibition or activation. Notably, BF-TK/GCV suppressed tumor growth and significantly prolonged survival in tumor-bearing mice.

Collectively, BF-TK/GCV does not uniformly inhibit or activate phosphorylation but rather reprograms the phosphoproteome in a site- and pathway-specific manner. These findings provide new insights into its therapeutic potential and suggest a promising strategy for cancer treatment.