S.J. Hickman1, V. Vudatha2, J.M. Lever3, K.M. Hardiman3, J.G. Trevino2, A.M. Carter3 1Mercer University School of Medicine, Macon, GA, USA 2Virginia Commonwealth University School of Medicine, Division Of Surgical Oncology, Richmond, VA, USA 3University Of Alabama at Birmingham, Department Of Surgery, Heersink School Of Medicine, Birmingham, AL, USA
Introduction: Fusobacterium nucleatum (Fn) is an anaerobic commensal pathogen that has been identified in the tumor environment of numerous cancers. A high abundance of Fn in tumors is associated with poorer outcomes in colorectal cancer (CRC) patient populations. Of interest, methamphetamine (MA) increases the abundance of Fusobacteria within the gastrointestinal microbiome in both rodent and human models. Given the similarity in pharmacodynamics between MA and amphetamine (AMPH)-class medications, as well as the rise in prescriptions for AMPHs, further investigation into a potential relationship between AMPH-class drugs, dysbiosis, and cancer outcomes is warranted. The purpose of this study is to evaluate the level of Fn in pre-clinical models of colorectal and pancreatic cancers to facilitate their future use for testing of the effects of AMPHs on Fn abundance, tumor onset, tumor growth, and survival.
Methods: dsDNA was extracted from mice CRC and human pancreatic ductal adenocarcinoma (PDAC) tumors via Zymo Research Quick-DNA Fecal/Soil Microbe Miniprep Kit. Quantitative real-time PCR (qPCR) was performed to assess the level of Fn DNA (primers targeting Fuso 16S ribosome), total bacterial load (non-specific bacterial primers), and relative host DNA presence (primers for GAPDH) in syngeneic mouse tumors (CT26 CRC cell lines in Balb/c mice) and PDAC patient and patient-derived xenograft (PDX) tumors. A standard curve was created using known amounts of Fn DNA, in the background of tumor DNA, to establish a dynamic range, limit of detection, and limit of specificity for Fn in both mouse and PDX tumors. PCR products that produced signals near or above our limit of detection were confirmed with genomic sequencing and gel electrophoresis.
Results: A total of 8 colorectal cancer mouse tumors were analyzed. Of the 8 syngeneic mouse tumors, no tumors demonstrated detectable levels of Fn. A total of 25 PDAC and PDX tumors were screened. Of the 25 samples, one sample showed high abundance of Fn, and 7 other samples had low abundance of Fn.
Conclusion: The absence of Fn within mouse CRC tumor environments provides ideal conditions for colonization with Fn. This system allows the effect of AMPH on Fn abundance, tumorigenesis rates, and overall tumor outcomes to be studied under a defined and controlled experimental paradigm where observations can be directly linked to Fusobacteria. The identification of PDX lines with Fn provides conditions where the effect of AMPHs can be tested in the background of a more complex tumor microbiome that translates more directly to actual patient tumors. Retrospective health data from the patients will be correlated to Fn level to look for associations between Fn abundance and patients’ outcomes, as has been observed for CRC. These studies into a relationship between AMPH and Fn will provide a more complete picture on AMPH driven dysbiosis within the microbiome and its implications in disease processes such as cancer.