Colorectal cancer (CRC) is the second leading cause of cancer death in Canada, with the major risk factor being chronic inflammation. How inflammation leads to cancer is not well understood. Our recent work has focused on a colonic epithelial cell known as the tuft cell that uniquely expresses the protein doublecortin-like kinase 1 (Dclk1). Using Cre-dependent lineage tracing of Dclk1-expressing cells, we showed that Dclk1 labels long-lived quiescent cells in the colon that serve as a cellular origin of CRC upon inflammatory injury. The aim of the study was to determine the generalizability of inflammation-induced tumor promotion from genetically susceptible Dclk1+ cells and explore the mechanism by which inflammation contributes to tuft cell cancer initiation. We hypothesized that various colonic inflammatory insults lead to dedifferentiation of Dclk1+ tuft cells to a stem cell state susceptible to tumor initiation. To investigate the various forms of injury or infection that can activate quiescent tuft cells, we crossed our transgenic Dclk1-CreERT2 mice to both ROSA26-tdTomato and APCf/f mice (Dclk1/APCf/f). Following tamoxifen induction, mice were treated with the colitis-inducing agents dextran sodium sulfate (DSS), trinitrobenzene sulfonic acid (TNBS), oxazolone or Citrobacter rodentium. To examine the role of dedifferentiation in colonic tumor initiation, we crossed Lgr5-DTR-eGFP mice to our Dclk1/APCf/f mice. The mice were then given tamoxifen and DSS to induce tumorigenesis and diphtheria toxin (DT) post DSS injury to ablate Lgr5+ intestinal stem cells. Treatment with DSS, TNBS, oxazolone, or C. rodentium induced colonic inflammation as detected by significantly increased myeloperoxidase (MPO) activity and histologic analysis. DSS administration led to Dclk1+ cell-derived colonic tumors as previously reported. Surprisingly, administration of TNBS, oxazolone, or C. rodentium in Dclk1/APCf/f mice did not lead to colonic tumorigenesis up to 52 weeks following induction of colitis. Interestingly, ablation of Lgr5+ intestinal stem cells post colitis significantly reduced colonic tumors in DSS-treated Lgr5-DTR-eGFP/Dclk1/APCf/f mice. Our data suggests that a specific inflammatory response unique to DSS-induced colitis, and not TNBS, oxazolone or C. rodentium infection, results in colonic tumor formation. Interestingly, the colonic transformation of Dclk1+ tuft cells in DSS colitis appears to be mediated through Lgr5-expressing cells. These findings provide insight into the molecular pathways by which Dclk1-derived colonic tumors arise. CAG, CIHRCFI
Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents.In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition.These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative 'instant histology' of fresh tissue biopsies.
of villus formation at embryonic day (E)14.5. This analysis revealed crack-like extensions of the lumen from the apical surface into the epithelium, which lengthen over time. The tips of these cracks are associated with dividing cells. Because of this association and a precedent for lumen-forming cell divisions in the zebrafish neural keel, we speculated that these cells might generate cracks by trafficking apical proteins to the cytokinetic plane. Using intestinal explant cultures in concert with nocodazole-mediated synchronization of the cell cycle, we examined the trafficking of the apical protein Ezrin during cell division. We found that Ezrin is deposited along the cytokinetic plane of divisions at crack tips, defining new villus domains and segregating daughter cells onto adjacent villi. We call these specialized cell divisions e-divisions (lumen extending). We also noted a second type of cell division, which functions in intestinal growth, but is not associated with apical cracks and does not define new villus domains. We have named these divisions g-divisions (growth building). Eand g-divisions differ cell biologically in several ways: localization of apical proteins, segregation of daughter cells, midbody morphology, and angle of the mitotic spindle relative to the nearest overlying apical surface. Ezrin null mice have fused villi with underlying isolated secondary lumens. Such a phenotype has been thought to support the secondary lumen model of normal villus morphogenesis, because it was believed that the loss of Ezrin perturbs their fusion with the main lumen. However, we have now determined that dividing cells in Ezrin null mice have mis-oriented mitotic spindles; we speculate that mis-orientation of e-divisions may in fact cause ectopic lumens and fused villi in this model. Indeed, the characteristic phenotype of Ezrin null mice can be recapitulated in wild-type intestines after treatment with blebbistatin, a myosin II inhibitor that results in mis-oriented spindles both in vitro and in intestinal explant cultures. This indicates that correct spindle orientation is indispensible for proper villus morphogenesis. Further understanding of this process will open up new avenues of in vitro bioengineering of intestinal surface, potentially providing life-saving therapies for those with intestinal failure.
Protease-activated receptor-4 (PAR(4)), the most recently discovered member of the PARs family, is activated by thrombin, trypsin and cathepsin G, but can also be selectively activated by small synthetic peptides (PAR(4)-activating peptide, PAR(4)-AP). PAR(4) is considered a potent mediator of platelet activation and inflammation. As both PAR(1) and PAR(2) have been implicated in the modulation of nociceptive mechanisms, we investigated the expression of PAR(4) in sensory neurons and the effects of its selective activation on nociception.We demonstrated the expression of PAR(4) in sensory neurons isolated from rat dorsal root ganglia by reverse transcription-polymerase chain reaction and immunofluorescence. We found that PAR(4) colocalized with calcitonin gene-related peptide and substance P. We also showed that a selective PAR(4)-AP was able to inhibit calcium mobilization evoked by KCl and capsaicin in rat sensory neurons. Moreover, the intraplantar injection of a PAR(4)-AP significantly increased nociceptive threshold in response to thermal and mechanical noxious stimuli, while a PAR(4) inactive control peptide had no effect. The anti-nociceptive effects of the PAR(4)-AP were dose-dependent and occurred at doses below the threshold needed to cause inflammation. Finally, co-injection of the PAR(4)-AP with carrageenan significantly reduced the carrageenan-induced inflammatory hyperalgesia and allodynia, but had no effect on inflammatory parameters such as oedema and granulocyte infiltration.Taken together, these results identified PAR(4) as a novel potential endogenous analgesic factor, which can modulate nociceptive responses in normal and inflammatory conditions.
