PO-121 ALK dependant phosphoproteome and gene expression profiling of neuroblastoma cells

Abstract

IntroductionThe calcium-sensing receptor (CaSR) is a heterotrimeric G protein-coupled receptor that was firstly identified as a key regulator of Ca++ homeostasis in the blood. Ubiquitously expressed in the body, the CaSR controls also cell proliferation, differentiation and apoptosis. In colorectal cancer the CaSR slows cell proliferation and induces apoptosis, however, it is down-regulated during tumorigenesis due to unclear mechanisms. Moreover, a common view on CaSR localization within normal intestinal crypts is still missing.We hypothesise that restoring CaSR expression inhibits tumour progression. Therefore, it is of paramount importance to understand the mechanisms that regulate CaSR expression in normal colonic epithelia and in tumours.Our aim is to find ways to restore CaSR expression by inducing differentiation in colonic organoids.Material and methodsWe used a 3D cell culture methodology, culturing colon organoids in matrigel. The organoids were generated extracting colonic crypts from the intestine of the mice and were kept in standard stem cell media to guarantee stem cell poll.We induced lineage-specific differentiation of colonic organoids either into enterocytes or goblet cells by altering the composition of the culturing media. We assessed CaSR expression by RT-qPCR and immunofluorescence, testing also specific differentiation markers such as FABP2 for enterocytes and MUC2 for goblet cells.Results and discussionsOur preliminary data showed that the CaSR was not expressed in the organoids cultured in stem cell media where even the differentiation markers were barely detectable. On the other hand, organoids cultured for 7 days in enterocyte-specific differentiating condition expressed both CaSR and FABP2, while in goblet cells CaSR expression remained undetectable. Our results not only show that inducing differentiation leads to higher CaSR levels, but also suggest that the CaSR is expressed in enterocytes rather than MUC2-expressing goblet cells.ConclusionWe conclude that, like poorly differentiated colorectal tumour cells, the colonic stem cells do not express the CaSR, which is expressed in mature intestinal cells. Further experiments will allow us to undoubtedly determine in which subtypes of differentiated cells the CaSR is preferentially localised.