Title: To determine whether IFNγ-primed macrophages promote EGF-dependent epithelial Reg4+ cell hyperplasia to initiate carcinogenesis in IBD-associated cancer patients.
Problem and Rationale:Reg4, a member of the human regenerating (Reg) gene family, promotes intestinal epithelial cell survival pathways to maintain mucosal homeostasis1. Epithelial Reg4 expression is highly upregulated in chronic inflammatory bowel disease (IBD) patients in remission (on anti-TNFs) 2. Although a role for Reg4 in IBD has not been characterized yet. Reg4 overexpression is associated with an aggressive phenotype of colon cancer in dysplastic or inflamed hyperplastic tissues3, 4. However, whether it serves as risk factor for the development of IBD-associated cancer is yet unexplored.
IFNγ, an immunomodulatory cytokine, persists at high levels in chronic IBD patients in remission5. Consistently, the IFNγ-primed macrophage population is enhanced in remissive chronic IBD patients6 and patients with IBD-associated cancer7, 8. These cancer-associated macrophages are known to secrete pro-tumorigenic and angiogenic factors that promotes carcinogenesis8, 9. However, whether they contribute specifically to Reg4+ cell hyperplasia in IBD-associated cancer is still unknown. Based on these observations, we hypothesize that in IBD-associated cancer, IFNγ-primed macrophages drive the hyperplastic expansion of Reg4+ cells via activation of EGF signaling. Reg4+ cells recruit the pro-tumorigenic macrophages to the site of injured epithelia via a chemokine-dependent mechanism supporting a positive feedback loop.
Objective 1 Determine the correlation between the levels of Reg4, IFNγ-enriched immune cells, and tumor-associated macrophages (TAM), in remissive IBD patients, and/or IBD-associated colon cancer patients.The localization and quantification of Reg4 and -associated chemokines, Cxcl6, 9, 10, 1110, in the colon of remissive IBD patients, IBD-associated colon cancer patients and colon cancer patients (aggressive phenotype control for the study) will be determined via in situ hybridization, and qPCR (Fig. 1; Objective 1). Concurrently, flow cytometric analysis will determine IFNγ-enriched intestinal immune cell subsets and TAM subsets from the same patients. TAM subsets will be flow sorted for qPCR analysis of a pro-tumorigenic growth factor profile associated with crypt hyperplasia (Fig. 1; Objective 1).
Fig.1 Illustration of the research study’s’ objectives and outcomes.
Objective 2 Determine if IFNγ-primed macrophages drive the positive feedback loop promoting Reg4+ cell hyperplasia and chemokine-dependent recruitment of pro-tumorigenic macrophages to the site of epithelial injury in IBD-associated colon cancer patients.Differentiated macrophages from CD14+ monocytes, isolated from PBMCs of remissive chronic IBD patients, colon cancer patients and IBD-associated colon cancer patients, will be primed by IFNγ into activated M2 macrophages. For the epithelial experiments, we will utilize patient-specific adult colonic epithelial stem cell derived 3D mini-guts or organoids, and a pre-established AOM-DSS-injury model that induces a hyperplastic epithelial response at low concentrations in vitro (Fig. 1; Objective 2). A co-culture system employing anti-TNF treated control (non-IBD) and IBD organoids with IFNγ-primed macrophages or flow sorted TAMs, will be established either through direct interaction or via conditioned medium supplementation. Their effect on Reg4 expansion, crypt survival and chemokines expression will be determined at baseline and in the AOM-DSS-injury model, via in situ hybridization, and qPCR.
A chemotaxis assay using the above co-culture setup will quantify the chemokine-dependent TAM migration and invasion into the injured epithelium and the expansion of Reg4+ cells in response to EGF secreted by TAMs (Fig. 1; Objective 2). Spectrophotometry and immunofluorescence imaging will quantify the differences in this assay. Organoid co-cultures in the chemotaxis chambers will be supplemented with Reg4 or chemokine blocking antibodies to determine their specificity in driving TAM migration and invasion into injured epithelium. Conversely, the effect of pro-tumorigenic factors, like EGF on Reg4 expansion will be determined by a dose-dependent exposure of EGF to anti-TNF treated control and IBD organoids at baseline and in the presence of AOM-DSS-induced injury.
Significance: IBD currently affects 1 in 227 people in New Zealand, the incidence being one of the highest in the world. 60-75% of these IBD patients go on to develop colon cancer. Overall, our study seeks to identify clinically relevant markers to predict the onset of IBD-associated carcinogenesis and define the fundamental basis for a dysregulated inflammatory loop driven by epithelial Reg4 and IFNγ-primed macrophages, in immunotherapy resistant IBD patients. If our hypothesis is correct, an expanding Reg4 and IFNγ+ population in remissive chronic IBD patients will predict the initiating carcinogenic event in IBD-associated colon cancer patients. Potential drug candidates to these targets will help discern IFN-specific drugs to combine to the patient’s anti-TNF regimen to increase the efficacy of the treatment and hence reduce further hospitalizations, keeping the disease in remission.
1. Sasaki N, et al. Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon. Proc Natl Acad Sci U S A113, E5399-5407 (2016).
2. Planell N, et al. Transcriptional analysis of the intestinal mucosa of patients with ulcerative colitis in remission reveals lasting epithelial cell alterations. Gut62, 967-976 (2013).
3. Violette S, et al. Reg IV, a new member of the regenerating gene family, is overexpressed in colorectal carcinomas. Int J Cancer103, 185-193 (2003).
4. Numata M, et al. Erratum: Relationship between RegIV gene expression to outcomes in colorectal cancer. Journal of Surgical Oncology105, 625-626 (2012).
5. Rismo R, Olsen T, Cui G, Christiansen I, Florholmen J, Goll R. Mucosal cytokine gene expression profiles as biomarkers of response to infliximab in ulcerative colitis. Scand J Gastroenterol47, 538-547 (2012).
6. Nakanishi Y, Sato T, Takahashi K, Ohteki T. IFN-γ-dependent epigenetic regulation instructs colitogenic monocyte/macrophage lineage differentiation in vivo. Mucosal Immunol11, 871-880 (2018).
7. Sica A, Schioppa T, Mantovani A, Allavena P. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer42, 717-727 (2006).
8. Zhao G, et al. Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth. J Biol Chem291, 20462-20472 (2016).
9. Jetten N, Verbruggen S, Gijbels MJ, Post MJ, De Winther MP, Donners MM. Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis17, 109-118 (2014).
10. Lyons J, et al. The colonic epithelium plays an active role in promoting colitis by shaping the tissue cytokine profile. PLoS Biol16, e2002417 (2018).