Abstract
Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour–stroma interaction.
Highlights
The risk to develop colorectal cancer (CRC) is high and the danger to die from metastases is evident
It was shown that cancer cells repel endothelial cells [15], and here we investigated whether fibroblasts may as well retract from CRC spheroids
Blood endothelial cells (BECs) were included to this model and b Fig. 1 Colon cancer spheroid-induced circular chemorepellent induced defects’’ (CCIDs) formation in cancer-associated fibroblasts (CAFs) SW620 spheroids was transferred on a cell-tracker stained CT5.3 or b more densely seeded CT5.3 (50% of CT5.3 were stained with green cell-tracker the other 50% were stained with red celltracker) or c on cell-tracker stained primary CAF3 layers
Summary
The risk to develop colorectal cancer (CRC) is high and the danger to die from metastases is evident. The CRC cell lines Caco, SW480 and SW620 express steadily increasing levels of ALOX12 and secrete increasing amounts of 12(S)-HETE, respectively, and this is directly proportional to their metastasising potential i.e. increased growth in soft agar and enhanced migration [13]. In line with this observation, CYP1A1 mRNA is over-expressed in CRC liver metastases as compared to normal colonic epithelium, as detected by RNASeq, whereas CYP1A1 mRNA and protein expression does not vary, between normal colonic tissue and CRC. Taken together there is ample evidence that 12(S)-HETE and the ZEB pathway contribute to malignancy and studying the interface between cancer cells and their immediate stromal environment is relevant to understand tumour spreading
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