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Lrig1 Marks a Distinct Population of Proliferative and Quiescent IntestinalStem Cells and Acts as a Tumor SuppressorAnne Powell, Yina Li, Yang Wang, Jeff Franklin, Kevin Haigis, Robert J. Coffey

Whether proliferative and quiescent intestinal stem cells coexist in normal and canceroustissue is controversial. Due to heightened transcript expression in the intestinal epithelialstem cell compartment and detection in quiescent hair follicle stem cells, we hypothesizedthat Leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1) would marka novel population of intestinal stem cells and may be important for intestinal homeostasisand in cancer. To this end, we generated Lrig1-CreERT2/+; R26R-LacZ mice for Lrig1intestinal epithelial cell lineage analysis. Examining adult mice at baseline and after irradiationdamage, we show that Lrig1 labels both quiescent and proliferative stem cells and thesecells are distinct from the well-defined Lgr5-expressing proliferative stem cell population.In addition, in a regenerative response to gut injury, these singly-labeled Lrig1-expressingcells proliferated and gave rise to clusters of labeled daughter cells, demonstrating the abilityof these quiescent stem cells to become activated. Interestingly, loss of Lrig1 resulted inheightened expression of ErbB1-3 in the normal intestine, duodenal adenomas and carcin-oma, supporting a role for Lrig1 in the maintenance of intestinal epithelial homeostasis andits ability to act as a tumor suppressor. To directly compare the role of the Lrig1- and Lgr5-expressing cell populations in tumorigenesis, we used both Lrig1-CreERT2/+;Apcfl/fl andLgr5-EGFP-IRES-CreERT2;Apcfl/fl mice to delete Apc. Five days after deletion, Lrig1-CreERT2/+;Apcfl/fl mice have increased duodenal size and marked histological changes,which are absent in the Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice, further supporting a distinctand important role for the Lrig1 population in maintaining intestinal homeostasis. Finally,stochastic loss of Apc in Lrig1-expressing cells in Lrig1-CreERT2/+;Apcfl/+ mice results inmultiple, large distal colonic tumors and less frequent, smaller intestinal tumors- commonfeatures of human familial polyposis- 4.5 months after Cre activation. In summary, we showthat Lrig1 is a distinct marker of both proliferative and quiescent intestinal stem cells, isimportant for intestinal homeostasis and acts as a tumor suppressor.

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CDC42 is Required for Intestinal Stem Cell Self-Renewal but NotDifferentiationLei Chen, Gene LeSage

Adult intestinal stem cells (ISCs) possess long term self-renewal and multilineage differenti-ation properties to maintain intestinal epithelium. CDC42, a member of the Rho GTPasefamily, determines cell polarity by regulating actin, microtubule and protein distributionthrough Par6/aPKC signaling. Cdc42 is involved in the regulation of cell polarity, nicheinteraction, differentiation and proliferation of hematopoietic, neural and epidermis stemcells. Aims: To determine the effect of CDC42 loss of function on ISC self-renewal anddifferentiation and cell proliferation and migration along the crypt-villus axis. Methods:We employed an tamoxifen-inducible conditional gene targeting approach (Cdc42loxp/loxp;CK19CreERT;Rosa-Stop-YFP mice) where loss of CDC42 and expression of YPF onlyoccurs after the administration of tamoxifen and in CK19 expressing cells. For controlstudies, we used CK19CreERT;Rosa-stop-YFP mice. Cell polarity was determined by immun-ofluorescence microscopy for Zo-1, actin and beta-Catenin, proliferation by BrdU labelingand Ki76 staining, apoptosis by Caspase3 staining, and differentiation by FABP, MUC2,chromogranin and MMP7 staining. Mispositioning of cells was determined from EphrinB(villus) and EphB2 (crypt) distribution. Results: After the administration of tamoxifen, wedemonstrated complete loss of CDC42 only in YFP+ cells. We found that Cdc42 deletiondisrupted cell polarity and proliferation in the crypt (progenitor) cells but not villus (differenti-ated) cells without an increase in apoposis. Cell migration (determined by pulse BrdUlabeling) and the expression of specific differentiation markers was the same in YPF positiveand negative cells, indicating that CDC42 deficiency does not alter differentiation or migra-tion. Administration of a single dose of tamoxifen to CK19CreERT;Rosa-stop-YFP miceresulted in long term retention (> 6 months) of YPF positive cells extending throughout thecrypt-villus length, indicating permanent labeling of ISCs. Consistent with CDC42 beingrequired for ISC self-renewal, we observed complete loss of YFP+, CDC42 deficient cells inCdc42loxp/loxp;CK19CreERT;Rosa-Stop-YFP mice, first disappearing in the crypt by day 4and then from the villus by day 10 following tamoxifen administration. Cdc42 deficientcells in the intervillus regions are EphrinB negative and EphB2 positive, which suggestsCDC42 deficient cells mislocate to the intervillus region. There was a loss of the expressionof Lrig1, a stem cell marker, and a decrease in pGSK3beta, pPKC, c-Myc and nuclear beta-Catenin levels in the Cdc42 deficient crypt cells. Summary and conclusions: Our findingsdemonstrate an essential role of Cdc42 in self-renewal of intestinal stem/progenitor cellsand retaining their localization in the crypts.

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Insulin-Like Growth Factor-I Promotes Intestinal Stem Cell Expansion DuringCrypt Regeneration and Mucosal Healing Following RadiationLaurianne Van Landeghem, Maria A. Santoro, Adrienne E. Krebs, Amanda T. Mah, JeffreyJ. Dehmer, Adam D. Gracz, Scott T. Magness, Pauline K. Lund

High dose irradiation induces massive loss of intestinal epithelial crypts. After radiation,surviving intestinal stem cells (ISC) are thought to expand and regenerate the crypts. Insulin-like growth factor-I (IGF-I) has potent trophic effects on intestinal epithelium and mediatesthe actions of hormones used clinically to promote intestinal growth such as glucagon-likepeptide-2 and growth hormone. Several studies indicate that IGF-I may exert selective orpreferential anti-apoptotic effects on ISC. However, direct visualization of ISC regenerationafter irradiation, or trophic effects of IGF-I on ISC, have not been demonstrated due to lackof ISC biomarkers. This study used a transgenic reporter mouse with EGFP expressiondriven by genomic regulatory sequences of the Sox9 gene which has been shown to markmultipotent ISC and permit their quantification or isolation (Gracz et al, Am J Physiol GI;298(5):G590-600, 2010). We hypothesized that Sox9-EGFP ISC expand and regenerate crypts

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after irradiation and that IGF-I enhances these effects. Methods: Sox9-EGFP mice were givenIGF-I (5μg/g body weight/day) or vehicle immediately after 14Gy abdominal irradiation andcompared with non-irradiated controls. Histology and flow cytometry assessed Sox9-EGFPISC between days 3 and 9 after irradiation. Expression of the type I IGF receptor (IGF-IR)was studied by immunohistochemistry and real-time qPCR on FACS-isolated cells. Results:Irradiation induced complete crypt loss by day 3. By day 4, there was massive expansionof Sox9-EGFP ISC, with a maximum 4.5 ±0.4 fold increase in ISC at day 5 and a returntoward baseline by day 9. Co-localization of Sox9-EGFP and EdU, a proliferation marker,revealed that at day 5 after radiation, the proportion of proliferating Sox9-EGFP ISC was2.8 ±0.2 fold higher than in non-irradiated controls. IGF-I treatment significantly increasedSox9-EGFP ISC by 1.7 ±0.2 fold in non-irradiated mice and further enhanced the maximumpost-radiation induced increase in regenerating Sox9-EGFP ISC (+6.6 ±0.6 vs. non-irradiatedcontrols). IGF-IR immunoreactivity was highly localized to crypt based columnar cells/ISC.IGF-IR mRNA expression was 9.4 ±1.0 fold higher in Sox9-EGFP-sorted ISC than in unsortedcells. Conclusions: We conclude that after radiation, Sox9-EGFP ISC expand and have directroles in crypt regeneration. IGF-I potently and preferentially increases ISC numbers innormal or irradiated intestine, likely by signaling through IGF-IR whose expression is highlyup-regulated in ISC. Ongoing microarray analyses are defining the molecular phenotype ofregenerating or IGF-I stimulated ISC. IGF-I or hormones that induce IGF-I may promoteISC expansion in patients at risk of intestinal failure.

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Fusion of MSC With Gastric Epithelial Cells Increases Invasion andMetastasis of Gastric CancerHanchen Li, Calin Stoicov, Jian Hua Liu, Jean Marie Houghton

Background The C57BL/6 mouse is a model of Hf induced gastric cancer. Infection causesgastritis, atrophy, metaplasia, dysplasia and gastric intraepithelial neoplasia (GIN). Priorstudies from our group have shown that bone marrow (BM) derived mesenchymal stemcells (MSC) home into areas of inflammation, engraft into the mucosa and are the cell oforigin of gastric cancer. In this study, we address what role fusion has in this process, anddefine the fusion partner within the gastric mucosa, and address the implication of fusionbetween epithelium and MSC carrying p53 mutations. Third, we determine if recruitmentoccurs at one point during infection or if it is an ongoing process. Methods P53MSC andwt MSC have been isolated and characterized by our laboratory. MSC populations werestably transfected with a modified ZEG construct. MSC ZEG are RFP/beta galactosideasepositive in the absence of fusion, and after fusion with a cre-recombinase expressing cell,both RFP and GFP positive. Mice used were wt (no cre expression), K19-cre (expressionof cre is limited to GI epithelial cells) and CMV-cre (all cell types express cre) transgenicmice. We infected mice with H.felis according to our standard protocol. At 9 months micereceived a single iv injection of 1 x 106 MSC. 8 weeks later (11 months of infection) allmice were euthanized, stomachs removed, weighed, and processed for histology, immuno-fluorescence and immunohistochemisty. Full necropsy was performed. Results At 11 monthsof infection, wt, CMV-cre and K-19 cre mice +/- wt MSC had similar histological findings.Findings included chronic inflammation, mucus cell metaplasia and parietal cell loss. Nomouse had dysplasia evident and there was no extra-gastric disease noted. Infected wt,CMV-cre and K19-Cre mice which had received p53 MSC containing the ZEG constructhad widespread atrophy, dysplasia, invasion of glands into the submucosal area and apparentinvasion of cells into the gastric vasculature. 50% of the mice (10/20) had multiple pulmonarynodules consistent with metastatic disease. The majority of dysplastic glands within thestomach were RFP+ thus defining them as originating from MSC cells. All of the RFP+ cellsexpressed GFP in both the K19 and CMV-cre model, (but not in the wt model) suggestingfusion between the MSC and the epithelial cell occurred. An epithelial phenotype was furtherconfirmed by cytokeratin IHC. Lung nodules from K19 cre mice receiving p53MSC-ZEGwere epithelial tumors with abundant stroma which stained positive for GFP, RFP andcytokeratin by specific IHC. Conclusions MSC are recruited to the Hf infected gastric mucosaon an on-going basis where they fuse with existing neoplastic and preneoplastic epithelialcells. Fusion between p53MSC and metaplastic epithelial cells increases the progression oflocal disease and lung metastasis.

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Deletion of Bak Leads to Expansion of the Stem Cell Niche in Murine ColonicCryptsCarrie A. Duckworth, Tanya Stezhka, David M. Pritchard

Background: Intestinal epithelia are in a constant state of regeneration, sustained by stemcells which are thought to be located at or near the base of crypts. The intestinal stem cellniche is maintained by bidirectional signalling between epithelial stem cells and adjacentstromal pericryptal myofibroblasts. Bak is a pro-apoptotic bcl-2 family member that we havepreviously shown to be an important regulator of spontaneous and induced apoptosis,proliferation and differentiation in the colon. Reduced bak expression also increased suscept-ibility to colorectal carcinogenesis. We have currently investigated whether bak exerts theseeffects by altering the composition of the colonic stem cell niche. Methods: Tissue sectionswere prepared from formalin fixed and paraffin embedded or frozen samples of small intestineand colon from 10-12 week wild-type (C57BL/6) and bak-null mice. Haematoxylin andeosin staining was carried out to assess crypt morphology. Immunohistochemistry wasperformed for Ki67, DCAMKL-1 and CD24 (the latter two being potential stem cell markersin intestinal epithelia) and scored on a cell positional basis along the crypt axis. α-smoothmuscle actin (α-SMA) immunohistochemistry was used to assess pericryptal myofibroblastnumbers. Results: The colonic crypts of bak-null were significantly longer than those fromC57BL/6 mice (bak-null, 33.5 ± 0.9; C57BL/6, 24.8 ± 0.3 cells per hemi-crypt; p<0.0001)and also showed a significant increase in Ki67 labelling index between cell positions 2-34.The percentage of Ki67 positive colonic epithelial cells was significantly increased in bak-null mice compared with C57BL/6 mice, thus discounting the possibility that the observeddifferences were simply due to crypt hyperplasia (C57BL/6, 18.6 ± 2.3%; bak-null, 31.9 ±1.9%, p<0.01). DCAMKL-1 and CD24 were expressed at the base and mid-crypt regionsof colonic crypts in both bak-null and C57BL/6 mice, but bak-null mice showed significantly

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