Background: Local immunomodulation with nanoparticles (NPs) and focused ultrasound (FUS) is recognized for triggering anti-tumor immunity. However, the impact of these tumor immunomodulations on sex-specific microbiome diversity at distant sites and their correlation with therapeutic effectiveness remains unknown. Here, we conducted local intratumoral therapy using immunogenic cell death-enhancing Calreticulin-Nanoparticles (CRT-NPs) and FUS in male and female mice. We identified immune-related microbiome populations, aiming to translate our findings into clinical applications. Methods: CRT-NPs were synthesized by loading CRT-delivering plasmids into cationic liposomes. Local tumor therapy was performed using CRT-NP and FUS-based histotripsy (HT) on poorly immunogenic Mouse Oral Squamous Cell Carcinoma (MOC2) in the flank regions of male and female mice. Fecal samples were collected and analyzed before and three weeks post-treatment. The microbiome features were then correlated with immune cell dynamics within tumors and systemic cytokine responses to identify prognostic biomarkers in both male and female subjects. Results: Intratumorally administered CRT-NP induced tumor remission and immune cell activation in both male and female mice, whereas HT was ineffective in males and showed efficacy only in females. Turicibacter and Peptococcus inversely correlated with tumor growth, while Enterorhabdus, Subdologranulum, Desulfovibrio, and Aldercreutzia-Asaccharobacter showed direct correlations with tumor growth. HT induced higher levels of Turicibacter in MOC2-bearing females, while males displayed increased Enterorhabdus and Streptococcus populations. Independent of sex, treatments promoting CD4+ T helper cells, functional CD8+ T cells, and total macrophage infiltration correlated with higher levels of Gastrophilales, Romboutsia, Turicibacter, and Peptococcus. Alternatively, Enterorhabdus, Desulfovibrio, Streptococcus, and Staphylococcus corresponded to poor treatment outcomes in both sexes. Conclusion: An enhanced abundance of Enterorhabdus, Desulfovibrio, Streptococcus, and Staphylococcus in response to immunomodulatory therapies could serve as predictive biomarkers in a sex-independent manner. These findings could also be potentially extended to the realm of personalized interventions through fecal transplantations to reverse immunosuppressive phenotypes in males and improve patient outcomes.
Keywords: Focused Ultrasound; Gut Microbiome; Immunomodulation; Nanoparticle; Sexual dimorphism.
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