Abstract 657: The comparison of calcium-induced differentiation in colonoid cultures derived from the normal human colon tissue and colonic adenomas
Authors: Aslam Muhammad N. , McClintock Shannon D. , Rizvi Areeba H. , Attili Durga , Pandya Shailja , Rehman Humza , Nadeem Daniyal M. , Turgeon D. Kim , Varani James 
Published: August 2019

Abstract
Introduction: Previous ex vivo studies with colon cancer cell lines and human colon adenoma derived colonoid cultures have demonstrated that calcium and Aquamin (a calcium-rich, multi-mineral natural product) suppressed proliferation and induced differentiation. Aquamin was effective at a lower dose as compared to calcium.
Methods: Colonoid cultures were established from histologically-normal human colon tissue and maintained in a low-calcium (0.25 mM) medium (control) or supplemented with an amount of calcium or Aquamin (1.5 mM), that was shown previously to induce differentiation in human adenoma colonoids. Quantitative immunohistochemistry and differential proteomic expression were assessed after 4 weeks of in vitro intervention with calcium or Aquamin, as performed in the past with adenoma-derived cultures.
Results: The normal colonoids underwent differentiation as indicated by morphological features, a low proliferative index and high-level expression of cytokeratin 20 (CK20) under control conditions. Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). This was an altered response as compared to adenoma-derived colonoids which differentiated in response to calcium or Aquamin at 1.5mM calcium. In spite of this, proteomic analysis and immunohistochemistry of normal colonoids revealed that both interventions induced strong up-regulation of several proteins involved in cell-cell and cell-matrix interaction and in barrier formation. Among these were cadherins, keratins, claudins, CAECAMS, laminin subunits, and desmosomal proteins. Transmission electron microscopy showed an increase in desmosomes in response to intervention.
Conclusion: These findings suggest that normal colonoids were inherently more calcium-sensitive than adenoma colonoids and were readily differentiated at a lower calcium concentration as present in the control.
Table:Quantitative expression of immunohistochemical markers in colonoid culturesHuman Colonic Adenoma (n=3)Normal Colon (n=3)IHC MarkersControlCalcium 1.5mMAquamin 1.5mMControlCalcium 1.5mMAquamin 1.5mMKi6784.8±9.250.1±14.8*41.8±23.6*33.2±26.127.5±26.827.1±24.3CK200.23±0.130.54±0.13*0.53±0.10*0.78±0.190.73±0.220.76±0.20E-Cadherin0.44±0.060.70±0.09*0.70±0.07*0.49±0.100.61±0.09*0.58±0.08**reflects significance as compared to the corresponding control (p&lt;0.05).Each condition has 20-30 crypts in colonoid culture per adenoma or normal colon.
Citation Format: Muhammad N. Aslam, Shannon D. McClintock, Areeba H. Rizvi, Durga Attili, Shailja Pandya, Humza Rehman, Daniyal M. Nadeem, D. Kim Turgeon, James Varani. The comparison of calcium-induced differentiation in colonoid cultures derived from the normal human colon tissue and colonic adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 657.