Background: Colorectal cancer (CRC) is the second leading cause of cancer-associated deaths in the US. Evidence from several studies suggests that the colonic immune environment can influence the risk of CRC. Bacterial minicells (MC) are spherical, non-viable nano-sized particles derived from genetically-engineered E. coli (Vaxiion Therapeutics, Inc). MC contain all of the components of parental bacteria they are derived from with the exception of the chromosome, making them non-infectious. MC used here have been engineered to display antitumor activity via integrin-specific cytotoxic effects but evidence suggests they also require a working immune system to be optimally effective in mouse models of cancer. For our colon cancer studies, we are using an immune-competent mouse model, FABP-CreXApcfl/+, which has a conditional deletion of exons 11-12 in one Adenomatous Polyposis Coli (Apc) allele in the epithelium of colon and distal ileum, due to the targeted expression of CRE, leading to colonic polyposis.
Hypothesis: Since MC have been shown to have anti-cancer activity in multiple mouse models, but even more importantly because evidence suggests they are more effective in animals with a functional immune system, we hypothesize that MC can change the immune environment of the colon and delay/decrease the onset of colon cancer in our mouse model.
Methods: We treated 14 week old FABP-CreXApcfl/+ mice with PBS or 1.5 x 109 MC in PBS intra-rectally once a week until 19 weeks of age (6 treatments total). Mice were then left untreated until 26 weeks of age, euthanized, and colons were collected to measure tumor number and size. Colonic tumor load was also compared to 24 week old untreated FABP-CreXApcfl/+ controls. RNA was isolated from tumor adjacent (TA), small tumor (≤4mm) and large tumor (>4 mm) tissue, converted to cDNA, and qPCR studies were performed. Hprt was used as a housekeeping gene control and fold-change target gene expression was reported in comparison to Hprt expression.
Results: The mean number of colonic polyps in 26 week old mice that received MC-treatment during the 14th-19th week of age was significantly lower (2.4±1.5) in comparison with mice that received PBS only (9.3±1.4, p=0.03) or 24 week-old untreated controls (8.0±1.9, p=0.02). Moreover, MC-treatment significantly restricted the development of colonic polyps >4mm in size (0.6±0.2/mouse, i.e. 60% of mice had one large tumor each) in comparison to PBS-treated (3.7±0.8/mouse, p=0.03) and 24 week-old untreated (2.0±0.2/mouse, p=0.01) controls. Furthermore, as per qPCR studies, preliminary work suggests that MC promote the expression of TH1 and cytotoxic T cell-associated immune responses (CD8a, Tbet, Ifng, perforin) as well as the TH17 (Il17a) immune gene marker in the TA areas of the colon. We also found a decline in the expression of pro-inflammatory (il23, Il6), immunosuppressive (Il10, Foxp3) and immune cell recruitment (Cx3cl1) immune markers in the large tumors of MC-treated mice. Furthermore, our preliminary data suggest the presence of ‘low’ and ‘high’ responders in MC-treated mice. ‘Low responders’ have at least one large tumor at 26 weeks of age and lower correlation of TH1 and cytotoxic T cell-associated immune responses in comparison to ‘high responders’ which have an absence of large tumors and display multi-fold higher levels of TH1 and cytotoxic T cell-associated immune responses in comparison to PBS-treated controls.
Conclusion: MC significantly delay the development of colonic polyps and our preliminary data suggest that the delay/decrease in tumorigenesis could be because of attenuation of pro-tumor inflammation as well as promotion of anti-tumor immunity in the colon. These studies also suggest our mouse model will be appropriate for the study of low and high responder anti-tumor immune phenotypes in mice, shedding light on what may be occurring in human CRC patients.
Citation Format: Mohammad W. Khan, Shingo Tsuji, MengXi Tian, Nairika Meshgin, Shea Grenier, Matthew J. Giacalone, Kathleen L. McGuire. Immunity, the colonic environment, and colon cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A05.