Abstract 1281: Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota
Authors: Bernardo Giancarla , Le Noci Valentino , Montanari Elena , Ottaviano Emerenziana , Gargari Giorgio , Borghi Elisa , Guglielmetti Simone , Ambrogi Federico , Triulzi Tiziana , Tagliabue Elda , Pupa Serenella M. , Sommariva Michele , Sfondrini Lucia 
Published: March 2024

Abstract
The lung has been traditionally regarded as a sterile environment. Emerging evidence has overturned this belief unveiling the existence of a specific lung microbiota that plays a pivotal role in modulating lung immune tolerance. Recent research also sheds light on how the tissue microbiota is significantly altered by the presence of a growing tumor. Here, we investigated the compositional changes in lung-colonizing bacterial populations associated with the growth of LLC1 lung carcinoma cells in C57BL/6 mice, by analyzing the lung 16S rRNA profiling. While overall microbial diversity and structure were similar in tumor-bearing and healthy lungs, abundance analysis of amplicon sequence variants (ASVs) revealed that 48 ASVs were differentially modulated at significant level (FDR ≤ 0.05). Notably, local treatment with aerosolized vancomycin/neomycin antibiotics starting 4 days after tumor injection caused a different representation of 47 ASVs (FDR ≤ 0.05), mostly reducing those ASVs specifically emerging in the lung by the tumor growth, belonging to Bacterioidales, Clostridiales, Lactobacillales and Enterobacteriales. A different microbial composition was also suggested by the results of Partial Least Squares Discriminant Analysis (PLS-DA) that clustered lung tumor samples as saline or antibiotic-aerosolized. Based on the reported ability of some bacteria belonging to these orders to induce immunosuppressive cells in the gut, we examined the impact of antibiotic aerosol on tumor-associated Tregs in the lung. The presence of the tumor promoted the appearance of Tregs expressing CD69 and PD-1 activation markers, but vancomycin/neomycin aerosol treatment strongly dropped Treg frequency in tumor-bearing lungs, compared to saline. In a therapeutic setting, aerosolization of antibiotics starting 4 days after LLC1 tumor intravenous injection significantly reduced the number of lung metastases (p=0.0116). This effect was associated with an increased cytotoxic activity of immune infiltrating effector cells suggesting that the modulation of the lung microbiota impairs tumor growth and enhances antitumor immune response. Some cultivable bacterial strains, that were in agreement with metataxonomic profile results, were isolated from tumor-bearing lungs, but not from healthy lungs. Ongoing experiments suggest an effect of their in vivo transfer in the lung by aerosol on tumor growth and the ability to induce Treg expansion in vitro. Overall, this study unveils that the growth of the murine LLC1 tumor shapes the lung microbial composition by favoring the expansion of microbes with potential immunosuppressive activity eventually affecting the tumor progression. Moreover, our findings unravel the therapeutic potential of localized antibiotics to revert the immunosuppressive microenvironment by targeting tumor-associated bacteria.
Citation Format: Giancarla Bernardo, Valentino Le Noci, Elena Montanari, Emerenziana Ottaviano, Giorgio Gargari, Elisa Borghi, Simone Guglielmetti, Federico Ambrogi, Tiziana Triulzi, Elda Tagliabue, Serenella M. Pupa, Michele Sommariva, Lucia Sfondrini. Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1281.