Abstract 3987: Implementation of advanced molecular imaging to evaluate probiotic L. lactis bacterium capability to preferentially target orthotopic colorectal tumor

Abstract Colorectal cancer (CRC) is the third most diagnosed cancer and has the second highest mortality of all cancers. CRC treatments include surgery, chemotherapy, and radiation. However, CRC patients suffer from significant toxic side effects, develop resistance to the therapies, and experience recurrence and metastasis. A new strategy for CRC treatment is immunotherapy, which uses the patients’ immune system to attack cancer cells. Unfortunately, most CRC patients do not respond to immune checkpoint inhibitors (ICIs). To better treat CRC patients with immunotherapy, it is essential to improve tumor-targeting and tumor-specific uptake. Therefore, this study used advanced molecular imaging for real-time evaluation of the capability of probiotic facultative anaerobic bacterium Lactococcus lactis (L. lactis) to preferentially target orthotopic colorectal tumors. We combined bioluminescent (BL) and fluorescent imaging to simultaneously detect CRC tumors and L. lactis expressing infrared fluorescent protein (L. lactis-iRFP), respectively. To simultaneously detect BL and iRFP signals, we subcutaneously injected a CRC cell line expressing luciferase (CT26/luc) in the right flank of 6-week-old BALB/c mice. After the tumor reached a volume of 500 mm3, L. lactis-iRFP was injected intratumorally. We found that BL and iRFP signals colocalized at the tumor site without directly overlapping without interfering one with another and L. lactis-iRFP signal was detected at the tumor site up to 5 days post-injection. Next, we proceeded to generate an orthotopic CRC mouse tumor model in 6-weeks-old BALB/c mice using the colonoscopy guided inoculation technique. We included two control mice without tumors, one administered with non-fluorescent L. lactis wild type, and the other with L. lactis-iRFP to compare the bacterium retention between tumor bearing mice. At day 11, when the orthotopic CRC tumor was well-established, the animals were administered with 1x1012 cfu/200 µL of L. lactis-iRFP or wild type via oral gavage, and BL and fluorescence signals were measured. The advanced molecular imaging revealed a strong iRFP signal in the tumor-bearing animals, up to 5 days post L. lactis-iRFP inoculation. The colons were scanned ex vivo and showed BL signal in the distal location of the CRC tumor. Interestingly, only the colons with tumors showed iRFP signal, suggesting that L. lactis-iRFP preferentially targets the CRC tumor microenvironment. However, additional experiments are required to reproduce this data and support a conclusion. We demonstrated that simultaneous detection of BL and iRFP signals is feasible and that this imaging technology could be potentially implemented for preclinical real-time evaluation of the capability of other bacteria to preferentially target solid tumors. Citation Format: Jorge G. Gomez-Gutierrez, Martin Ramos-Gomzalez, Nestor Rubio-Infante, James Amos-Landgraf, Satyanarayana Rachagani, Lacey McNally, Haval Shirwan. Implementation of advanced molecular imaging to evaluate probiotic L. lactis bacterium capability to preferentially target orthotopic colorectal tumor [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 3987.