Abstract 2537: <i>In vivo</i> advanced molecular imaging of the probiotic <i>L. lactis</i> expressing fluorescent proteins to monitor its transit through the gastrointestinal tract

Abstract Introduction: The real-time imaging of probiotic bacteria expressing a reporter protein represents an important area for gastrointestinal (GI) research. Fluorescent probiotic bacteria could be useful to evaluate the interaction of probiotics with the gut microbiome, either under normal conditions or dysbiosis. Also, they could be employed as diagnostic tools or in treatments preferentially directed to the harsh hypoxic, and/or acidic microenvironment of digestive diseases such as inflammatory bowel diseases (IBD), adenomatous polyps of the colon or colorectal cancer (CRC). Methods: Recombinant strains of probiotic bacterium Lactococcus lactis expressing different fluorescent proteins were orally administered to C57BL/6 and Balb/c mice, their transit through the GI tract was monitored by in vivo advanced molecular imaging and multispectral optoacoustic tomography at different time points after oral gavage inoculation. The L. lactis-iRFP intensity signal in live animals was quantified using the software Aura 4.0.7. Results: We found that the green channel to detect L. lactis-GFP results in a strong tissue autofluorescence in both C57BL/6 and Balb/c mice and the signal from L. lactis-mCherry could not be detected in neither black nor withe mice because the red wavelength lacks enough deep tissue penetrance to pass through the intestines and abdominal wall. In contrast, L. lactis-iRFP bacterium produce a strong, specific and well-localized signal, that can be detected in real-time for up to 24 h after oral gavage administration in Balb/c (white) mice, whereas in C57BL6 (black) mice it could be detected as an unspecific signal which suggests that melanin excess may interfere with the detection of L. lactis-iRFP. On the other hand, the pilot optoacoustic tomography imaging revealed that biliverdin, the iRFP substrate generated a strong signal which made it difficult to distinguish between the negative controls and L. lactis-iRFP. This result suggests that additional optimization of optoacoustic tomography imaging for iRFP is needed. Conclusions: These results suggest that L. lactis-iRFP in Balb/c mice has the desired features to serve as a fluorescent reporter for in vivo imaging that can be used to monitoring the bacterial presence, permanence and interactions with the anatomical structures while transiting through the GI tract. Additionally, optoacoustic tomography imaging requires further optimization and improvement. Citation Format: Nestor Rubio-Infante, Martin Ramos-Gonzalez, James Amos-Landgraf, Satyanarayana Rachagani, Lacey R. McNally, Jorge Gomez-Gutierrez. In vivo advanced molecular imaging of the probiotic L. lactis expressing fluorescent proteins to monitor its transit through the gastrointestinal tract [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2537.