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Bone marrow-derived epithelial cells (BMDECs) are recruited to sites of chronic inflammation; however, their clinical significance has not been determined. Here, we used gender-mismatched allogeneic bone marrow transplantation (BMT) in the context of the classical multistage model for murine cutaneous carcinogenesis to probe the recruitment of BMDECs in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with the phorbol ester, 12-O-tetradecanolyphorbol-13-acetate (TPA). We detected clusters of bone marrow-derived cells (BMDCs) in over 40% of papillomas, where they occupied 25% or more of the epithelial lesional area. In the dysplastic ulcers which arose in some irradiated DMBA/TPA treated mice, the magnitude of the recruitment was greater. The BMDCs clustered in the cutaneous epithelium where they became immunoreactive to epidermal keratins, and proliferated and stratified, thereby contributing to the lesions comparably with the progeny of hair follicle stem cells in engrafted Krt1-15Cre;R26R mice. Moreover, cytokeratins were detected by immunostaining and Q-PCR in plastic adherent bone marrow cells (BMCs) cultured in the presence of filter-separated epidermal keratinocytes (KCs). Cytokeratin production was enhanced by bone morphogenetic protein 5 (BMP5). Moreover, BMCs migrated towards the alarmin protein, High Mobility Group Box 1 (HMGB1), as well as towards epidermal KCs in ex vivo invasion assays. Furthermore, when naïve female mice received BMTs from donors previously treated with DMBA, several papillomas and a squamous cell carcinoma were observed after TPA promotion alone. We conclude that surprisingly large numbers of BMDECs are recruited to a subset of cutaneous papillomas and dysplastic ulcers and reflect a previously unrecognized systemic contribution to these lesions. We also conclude that carcinogen-exposed cells originating in the BMCs are sufficient to initiate a subset of benign and malignant lesions upon tumor promotion. Ultimately, these findings may provide novel targets for treatment of non-melanoma skin cancers as well as other cancers. Citation Format: Heuijoon Park, Sonali Lad, Kelsey Boland, Kelly Johnson, Nyssa Readio, Guangchun Jin, Samuel A sfaha, Kelly S. Patterson, Ashok Singh, Xiangdong Yang, Douglas Londono, Anupama Singh, Carol Trempus, Derek Gordon, Timothy C. Wang, Rebecca J. Morris. Chronic inflammation-mediated contribution of bone marrow-derived epithelial cells and hair follicle stem cells to development of cutaneous neoplasms [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 LB-039.

// Guangchun Jin 1, 2 , Kosuke Sakitani 1 , Hongshan Wang 1, 3 , Ying Jin 1 , Alexander Dubeykovskiy 1 , Daniel L. Worthley 4 , Yagnesh Tailor 1 and Timothy C. Wang 1 1 Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA 2 The Research Institute, Yanbian University Hospital, Jilin, China 3 Department of General surgery, Zhongshan Hospital, Fudan University, Shanghai, China 4 South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, South Australia, Australia Correspondence to: Timothy C. Wang, email: tcw21@columbia.edu Keywords: GPR56, progastrin, proliferation, stem cell, colorectal cancer Abbreviations: Human progastrin (hGAS), G-protein coupled receptor 56 (GPR56), 5-bromo-2’-deoxyuridine (BrdU), Bone morphogenetic protein (BMP). Received: November 02, 2016 Accepted: February 22, 2017 Published: March 23, 2017 ABSTRACT Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro , colonic organoids cultured from GPR56 −/− mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro , and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo , deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b +Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b + Ly6G + cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+IMCs in early cancer development. © 2011 Nature America, Inc. All rights reserved.

Bone marrow mesenchymal stem cells (MSCs) have been shown to have immune modulatory effects. Despite efforts to identify these cells in vivo, to date, MSCs have been defined mainly by their in vitro cell characteristics. Here, we show that Lin−CD44hiSca1−cKit+CD34− cells make up ∼0.5%–1% of murine whole bone marrow cells and yield nearly an equal amount of fibroblastic colony-forming units (CFU-F) as whole bone marrow. After transplantation into lethally irradiated recipients, Lin−CD44hiSca1−cKit+CD34− cells engrafted in the bone marrow long-term and demonstrated characteristics of MSCs, including capacity to differentiate into osteoblasts and adipocytes. To examine whether Lin−CD44hiSca1−cKit+CD34− cells have immune modulatory effects, in vitro coculture with activated CD4+ T-cells resulted in decreased Th17 cell differentiation by Lin−CD44hiSca1−cKit+CD34− cells. Furthermore, serial infusions with Lin−CD44hiSca1−cKit+CD34− cells reduced the progression to low-grade gastric dysplasia in mice infected with chronic Helicobacter felis (p = .038). This correlated with reduced gastric interleukin (IL)-17F, IL-22, and ROR-γt gene expression in responding mice (p < .05). These data suggest that bone marrow derived Lin−CD44hiSca1−cKit+CD34− cells have characteristics of MSCs and reduce progression of early gastric tumorigenesis induced by chronic H. felis infection. The prevention of dysplastic changes may occur through inhibition of Th17-dependent pathways.Disclosure of potential conflicts of interest is found at the end of this article.

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor ( Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5 neg or low cell population but was distinct from typical antral Lgr5 high stem cells. Treatment with progastrin interconverts Lgr5 neg or low CCK2R+ cells into Lgr5 high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers., Bone marrow-derived epithelial cells can be recruited to sites of chronic inflammation. Here, the authors using allogenic bone marrow transplantation in a multistage murine cutaneous carcinogenesis model show that bone marrow-derived epithelial cells and hair follicle stem cells are recruited to cutaneous neoplasms during tumor promotion of carcinogen-exposed skin and bone marrow.

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.

Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human progastrin. Murine deletion of Cck2r abrogated progastrin-dependent increases in colonic proliferation, mucosal thickness, and beta-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase-like-1 (DCAMKL1), stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human progastrin-overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer.

Background: The tumor microenvironment plays a pivotal role in carcinogenesis. Myofibroblasts, a major component of tumor stroma, have been demonstrated to be derived in a large part from bone marrow. However, the specific contributions of bone marrow-derived myofibroblasts to cancer growth and progression are not completely understood. In this study, we established bone marrow-derived (BMD) inflammation-related gastric myofibroblasts (MF) and investigated the role of BMD-MF in tumor growth. Methods: Gastric myofibroblast were isolated from EGFP bone marrow-transplanted IL-1β transgenic mice and wild type mice. Gene expression was assessed by real-time RT-PCR, immunocytochemistry staining and Western blot. The levels of cytokines and chemokines were measured by ELISA. Cell migration and invasion were determined by transwell migration assay and 3-D organic co-culture system, respectively. Tumor growth in vivo was determined in a SCID xenograft model. Results: We isolated and cultured successfully gastric myofibroblasts from BMT-IL-1β mice and WT mice. The pure BMD-MF (EGFP+) expressed myofibroblast markers (a-SMA and vimentin), but did not expressed markers of epithelial cells (E-cadherin). The mRNA expression of IL-1β, TNF-α, IL-6, SDF-1, MMP-1, TGF-β and COX-2 was significantly higher in the BMD-IL-1β-MF than WT-MF. Furthermore, BMD-IL-1β-MF secreted much higher levels IL-6 and SDF1 peptides in the culture medium than did WF-MF. The BMD-IL-1β MF significantly increased gastric cancer cell migration and invasion compared to WT-MF, and CXCR4 inhibitor (AMD3100) reduced cell migration induced by both myofibroblasts, indicating that the SDF1/CXCR4 axis is responsible for cell migration and invasion. Furthermore, in xenograft studies SCID mice co-injected gastric cancer cells (105 MKN45) and BMD-IL-1β MF formed larger size tumor compared those co-injected with WT-MF and those injected alone with gastric cancer cells. Mice injected with MKN45 alone on the left side and BMD-IL-1β-MF alone on the right side, surprisingly formed larger size tumors on the left side compared to those non-injected with BMD-IL-1β-MF on the right side. Furthermore, the EGFP+ MF could be detected in the xenograft tumor on the left side, suggesting that MF can migrate to the contralateral tumor and promote tumor growth. Moreover, treatment of CXCR4 inhibition (AMD3100) reduced tumor growth and the migration of MF to the contralateral tumor, indicating that SDF1/CXCR4 axis plays an important in the migration of myofibroblast and tumor growth. Conclusion: Our results for the first time show that tumor-associated myofibroblasts in the IL-1β gastric cancer model are bone marrow derived and have a stronger capacity to promote growth and invasion. The project was support by NIH grant 5U54CA126513, R01CA093405 and R01CA120979 (Wang TC) and NIH grant R01A120915 (Yang CS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1412.

Background & Aims: We have previously reported that overexpression of amidated gastrin in transgenic (INS-GAS) mice with Helicobacter felis (H. felis) infection accelerated gastric corpus carcinogenesis, while overexpression of non-amidated glycine-extended gastrin in transgenic (MTI-G-gly) mice inhibited parietal cell loss and atrophy in stomach. We have also reported that overexpression of the full length progastrin in transgenic (hGAS) mice stimulated colon carcinogenesis in azoxymethane-treated mice. In this study, we investigated the role of progastrin for Helicobacter-associated gastric carcinogenesis in mice. Methods: hGAS, INS-GAS and non-transgenic wild type (B6 wt) mice on a C57BL/6 background were infected with H. felis for 12 or 18 months (m). We also analyzed male hGAS, INS-GAS and wild type (FVB wt) mice on a FVB/N background with H. felis infection for 7 m. Infection status was assessed by realtime PCR, microscopic evaluation and serum titer of H. felis-specific IgG antibodies. Proliferating cells and parietal cells were investigated by immunohistochemistry (IHC) with Ki-67 and HK-ATP-beta antibodies, respectively. Expression of stem cell-related genes such as CD44 & DCAMKL-1 was also analyzed by IHC. Results: At 12 m post infection (p.i.), INS-GAS mice had mild corpus dysplasia and B6 wt mice had severe gastritis or metaplasia, while hGAS mice had only mild to moderate gastritis, respectively. At 18 m p.i., both INS-GAS and B6 wt mice had severe atrophic gastritis and corpus dysplasia, while hGAS mice showed only moderate gastritis with mild gastric atrophy but no corpus dysplasia. In contrast, hGAS mice had moderate to severe antral or pyloric dysplasia, while INS-GAS and B6 wt mice did not. H. felis colonization remained stable over time among 3 groups of mice. The serum titer of H. felis-specific IgG antibody as well as Th1-Th2 polarization calculated by IgG1/IgG2c subclass ratio showed no significant differences among 3 groups. At 18 m p.i., the number of Ki-67(+) cells per gastric corpus gland was the highest in INS-GAS and the lowest in hGAS mice. Parietal cell loss was remarkable in INS-GAS and B6 wt, but minimal in hGAS mice. CD44(+) and DCAMKL-1(+) cell numbers were significantly increased in INS-GAS and B6 wt, while essentially unchanged in hGAS mice. On a FVB/N background, INS-GAS mice with H. felis infection for 7 m showed gastric corpus adenocarcinoma, and infected FVB wt mice showed severe gastric atrophy with intestinal metaplasia, while infected hGAS mice showed only moderate gastritis without metaplasia or dysplasia. Conclusions: These results point to a protective effect of progastrin on Helicobacter-associated gastric corpus carcinogenesis perhaps by inhibiting the loss of parietal cells. In contrast antral pathology is increased in hGAS mice. We conclude that the different forms of gastrin have contrasting effects on Helicobacter-associated gastric corpus carcinogenesis in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4159.

G A A b st ra ct s by ELISA. The colon cancer cell line MC38 was used for wound healing and cell proliferation assays using MSC conditioned (50% MSC / 50% with fresh) or control (50% MC38/ 50% fresh) media, examined at intervals in triplicate. For In Vivo studies, 8 week old APC min mice were injected intravenously with 5 million RFP-MSC. At four weeks, small intestine and colons were removed, adenomas enumerated, examined by direct fluorescent microscopy and standard H&E. RESULTS: WT mice receiving sham injections or RFP-MSC did not develop tumors. APC min mice receiving sham injection did not develop colon tumors. APC mice receiving MSCs developed 3 colonic tumors (n=4 each group). One mouse had one tumor, one had 2 tumors and 2 mice did not have any tumors. There was no statistical difference between small bowel adenomas in the APC min mice +/-MSC. Tumors were adenomas, and adenocarcinoma. Colonic epithelium was host derived, with CD44+, alpha smooth muscle actin + RFP-MSC incorporated into the stroma as myofibroblasts. MSC conditioned media contains TNF-alpha (216+/-48 pg/ml), MCP-1 (153+/-75pg/ml), CCL-5 (12804+/-9750pg/mml, IL6(3971+/-98 pg/ml) and undetectable levels of IFN, IL2, IL4 or IL10. Conditioned media increased proliferation of MC38 monolayers at all time points and increased monolayer wound healing compared to control media at 18 and 21 hours. Addition of anti-TNF antibody decreased proliferation and wound healing to control levels stressing the importance of this cytokine. Conclusion: Culture derived MSC migrate to at-risk tissue and initiate colon neoplasia by incorporating into stroma. MSCs produce factors (such as TNF-alpha) that provide colon adenocarcinoma cells (MC38) with migration and growth advantage. A model in which we can manipulate stromal-tumor interactions will be useful for identifying new targets for colon cancer prevention and therapy.