Colitis is a major risk factor for the development of colorectal cancer, leading to colitis-associated colorectal cancer (CAC). The most commonly used animal model to study CAC is the azoxymethane-dextran sulphate-sodium (AOM/DSS) model. The ideal experimental conditions of this model depend on several factors, including the used mouse strain. No data on feasibility and conditions for older mice, e.g., for aging studies, have yet been reported. Thus, we conducted a descriptive, observational pilot study where CAC was induced in 14-month-old female Balb/C and C57/Bl6 mice using 12.5 mg/kg AOM i.p. and three different concentrations of DSS (1, 2, and 3%) in drinking water (ad. lib.). The mice were monitored regularly during the three-month experimental phase. After euthanasia, the colons of the mice were evaluated macroscopically and microscopically. Both the mouse strains showed a DSS-concentration-dependent induction of CAC. Carcinomas were only observed at 3% DSS. The DSS dose was found to be significantly correlated with the histology score and % Ki67 positive cells only in C57/Bl6 mice but not in Balb/C mice, which showed a variable response to the CAC induction. No differences in colon length, weight, or mucin content were observed. Optimal conditions for CAC induction in these aged animals are thus considered to be 3% DSS, as carcinomas did not develop when 2% DSS was used. On the other hand, Balb/C mice reacted severely to 3% DSS, indicating that 2.5% DSS may be the “sweet spot” for future experiments comparing CAC in aged Balb/C and C57/Bl6 mice. This model will allow investigation of the effect of aging on CAC development and therapy.

Inhibition of the epidermal growth factor receptor (EGFR) shows clinical benefit in metastatic colorectal cancer (CRC) patients, but KRAS-mutations are known to confer resistance. However, recent reports highlight EGFR as a crucial target to be co-inhibited with RAS inhibitors for effective treatment of KRAS mutant CRC. Here, we investigated the tumor cell-intrinsic contribution of EGFR in KRAS ^G12D tumors by establishing murine CRC organoids with key CRC mutations (KRAS, APC, TP53) and inducible EGFR deletion. Metabolomic, transcriptomic, and scRNA-analyses revealed that EGFR deletion in KRAS-mutant organoids reduced their phenotypic heterogeneity and activated a distinct cancer-stem-cell/WNT signature associated with reduced cell size and downregulation of major signaling cascades like MAPK, PI3K, and ErbB. This was accompanied by metabolic rewiring with a decrease in glycolytic routing and increased anaplerotic glutaminolysis. Mechanistically, following EGFR loss, Smoc2 was identified as a key upregulated target mediating these phenotypes that could be rescued upon additional Smoc2 deletion. Validation in patient-datasets revealed that the identified signature is associated with better overall survival of RAS mutant CRC patients possibly allowing to predict therapy responses in patients.

Introduction: The prostaglandin E₂ (PGE₂) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE₂ pathway regulation in colon cancer cells and the colon in vitro and in vivo.Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE₂ levels. In CaSR-transfected HT29^CaSR-GFP and Caco-2^CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE₂ pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E₂ synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29^CaSR-GFP but not in Caco-2^CaSR-GFP cells. Other genes involved in the PGE₂ pathway (COX-1, or the PGE₂ receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29^GFP and Caco-2^GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR.In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE₂-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE₂ pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

Background/Objectives: The extracellular calcium-sensing receptor (CaSR) is a multifunctional receptor proposed as a possible drug target for inflammatory bowel disease. We showed previously that CaSR inhibition with NPS 2143, a negative allosteric modulator of the CaSR, somewhat ameliorated the symptoms of chemically induced severe colitis in mice. However, it was unclear whether the potential of CaSR inhibition to reduce colitis may have been overshadowed by the severity of the induced inflammation in our previous study. Therefore, we tested if CaSR inhibition could prevent medium-grade colitis. Methods: Female BALB/c mice were treated with NPS 2143 or a vehicle prior to the induction of colitis with 2.5% DSS. On the day of sacrifice, colons and plasma were collected. The histology score was determined based on hematoxylin-eosin-stained sections. Mucin content, proliferation (Ki67), and immune cell infiltration (CD3 and CD20) were quantified based on immunostainings. Gene expression was measured by RT-qPCR. Results: Treatment with NPS 2143 had no effect on the clinical symptom score of the mice. However, the colons of the mice in the treated group were significantly longer (p < 0.05), and NPS 2143 significantly reduced colon ulceration (p < 0.05). The treatment also significantly reduced the expression of COX2 in the proximal colon and IL-22 in the distal colon. The proliferation of cells in the lymph nodes was significantly lower after the treatment, but no difference was observed in the epithelial cells. Conclusions: In summary, while NPS 2143 had an anti-inflammatory effect on medium-grade colitis, this effect appeared to be milder than in severe colitis, as observed previously, indicating that the effectiveness of CaSR inhibition as an anti-inflammatory measure in the colon is proportional to disease severity.