This study characterizes the contribution of bone marrow-derived cells (BMDCs) to Barrett's adenocarcinoma of the esophagus using a mouse surgical model of disease and human specimens. Transplantation of bone marrow expressing beta galactosidase into a wild-type mouse, followed by surgical esophagojejunostomy, allowed tracking of BMDCs into the surgical anastomosis and resulting Barrett's metaplasia. Human tissue from a male patient who had been transplanted with female bone marrow and later developed esophageal adenocarcinoma allowed us to tract donor-derived cells into the tumor. Using a combination of antibodies directed against beta-galactosidase (animal studies) and X/Y fluorescent in situ hybridization (FISH) (human studies), combined with specific lineage staining directed against epithelial, fibroblast, endothelial, and leukocyte markers, we show that bone marrow cells contribute to both the epithelial and stromal component of esophageal adenocarcinoma. These findings demonstrate that BMDCs can generate cancer-associated fibroblasts as well as contribute directly to epithelial cells in cancer of the esophagus.

Tumor recurrence after chemotherapy is a major cause of patient morbidity and mortality. Recurrences are thought to be secondary to small subsets of cancer cells that are better able to survive traditional forms of chemotherapy and thus drive tumor regrowth. The ability to isolate and better characterize these therapy-resistant cells is critical for the future development of targeted therapies aimed at achieving more robust and long-lasting responses. Using a novel application for the proliferation marker carboxyfluorescein diacetate, succinimidyl ester (CFSE), we have identified a population of slow-cycling, label-retaining tumor cells in both in vitro sphere cultures and in vivo xenograft models. Strikingly, label-retaining cells exhibit a multifold increase in ability to survive traditional forms of chemotherapy and reenter the cell cycle. Further, we demonstrate the innovative application of CFSE to live sort slow-cycling tumor cells and validate their chemoresistance and tumorigenic potential.

Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to malignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk tissue. To address this question, ApcMin/+ and ApcMin/+ Rag2−/− mice, which have a predilection to mammary carcinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC) transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the marrow cavity where they could be recovered up to a year later without apparent effect on the health of the mouse. No mammary tumors were found. However, in mice carrying the ApcMin/+ mutation, p53MSC homed to mammary tissue and significantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-α-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent disease. The increased cancer phenotype was completely preventable with neutralization of TNF-α or by transfer of CD4+ regulatory T cells from immune competent donors, demonstrating that immune competency to regulate inflammation was sufficient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and stromal mutations. The significant synergy between host immunity and mesenchymal cells identified here may restructure treatments to restore an anticancer microenvironment.

Background and aim

Analysis of clinical colon cancer specimens show alterations in the CD95 (Fas Ag/Fas L) pathway as tumors progress from local to metastatic disease, suggesting this pathway may play a role in invasive behavior of colon cancer. However, direct causality between these alterations and clinical disease progression has not been shown.

Methods

Surgically resected metastatic colon cancer samples were evaluated for Fas Ag/L and apoptosis. Alterations in the Fas signaling pathway found in human samples was recreated through a series of staged transfection experiments in the MC38 mouse colon cancer cell line and the effects on growth tested in vitro, and in vivo.

Results

Expression of FLICE like inhibitory protein (FLIP) confers apoptosis resistance, increasing the incidence of primary tumors through a survival advantage by avoiding apoptosis and inducing Fas mediated proliferation. Co-expression of Fas L enables colon cancer cells to metastasize to the liver from local tumors as well as from intravenous injection of cells. MC38-FasL/FLIP colon cancer cells induce apoptosis in hepatocytes via activation of type 2 Fas Ag signaling, thus creating a niche conducive to tumor growth and fueling their own growth via Fas proliferative signaling.

Conclusion

Alterations in the Fas Ag pathway which inhibit apoptosis and increase Fas mediate proliferation directly increases local colon cancer growth and enhances metastasis to the liver. Delineating points in the pathway responsible for growth and metastasis will offer targets which may be exploited for therapy.

Helicobacter infection, one of the most common bacterial infections in man worldwide, is a type 1 carcinogen and the most important risk factor for gastric cancer. Helicobacter pylori bacterial factors, components of the host genetics and immune response, dietary cofactors and decreased acid secretion resulting in bacterial overgrowth are all considered important factors for induction of gastric cancer. Components found in green tea have been shown to inhibit bacterial growth, including the growth of Helicobacter spp. In this study, we assessed the bactericidal and/or bacteriostatic effect of green tea against Helicobacter felis and H. pylori in vitro and evaluated the effects of green tea on the development of Helicobacter-induced gastritis in an animal model. Our data clearly demonstrate profound growth effects of green tea against Helicobacter and, importantly, demonstrate that green tea consumption can prevent gastric mucosal inflammation if ingested prior to exposure to Helicobacter infection. Research in the area of natural food compounds and their effects on various disease states has gained increased acceptance in the past several years. Components within natural remedies such as green tea could be further used for prevention and treatment of Helicobacter-induced gastritis in humans.

Trefoil family factor 2 (TFF2), also known as spasmolytic peptide, is a low-molecular-weight protein that is upregulated in gastric tissues infected with Helicobacter or having other inflammatory conditions, but a precise function is yet to be elucidated. The role of TFF2 in the development of gastritis, colitis, and inflammatory cytokine responses was examined both in vivo and in vitro using wild-type and TFF2 knockout mice. TFF2 knockout and wild-type mice were infected with Helicobacter felis (H. felis) to induce gastritis. Colitis was induced in TFF2 knockout and wild-type mice by administering dextran sodium sulfate (DSS) in drinking water. Histopathology, clinical disease (colitis), and antibody levels (H. felis) were examined. TFF2 expression in tissues was determined by reverse transcriptase PCR, and the inflammatory and proliferative responses of TFF2-expressing macrophages and spleen cells were examined by cytokine enzyme-linked immunosorbent assay, thymidine incorporation, and gene array studies. TFF2 knockout mice have increased susceptibility to H. felis-induced gastritis, with enhanced gastric inflammation. They were also more susceptible to DSS-induced colitis, with prolonged colonic hemorrhage and persistent weight loss. Remarkably, TFF2 expression was not limited to the gastrointestinal tract, as suggested in previous studies, but was also present in macrophages and lymphocytes. The inflammatory and proliferative responses of these immune cell types were dysregulated in TFF2 knockout mice. TFF2−/− cells were hyperresponsive to interleukin 1 beta stimulation but showed normal responses to lipopolysaccharide, suggesting a specific role for TFF2 in interleukin 1 receptor but not Toll-like receptor 4 signaling via their Toll-interleukin 1 resistance domains. TFF2−/− lymphocytes also produced higher levels of interleukin 2 than wild-type cells. Thus, TFF2 was expressed in the gastrointestinal cells and in immune cells and was a negative regulator of gastrointestinal inflammation and immune cell cytokine responses. Our studies suggest that TFF2 not only controls gastrointestinal repair but also regulates mononuclear cell inflammatory responses.

Escape from normal apoptotic controls is thought to be essential for the development of cancer. During Helicobacter pylori infection, the leading cause of gastric cancer, activation of the Fas antigen (Fas Ag) apoptotic pathway is responsible for early atrophy and tissue loss. As disease progresses, metaplastic and dysplastic glands arise which express Fas Ag but are resistant to apoptosis and are believed to be the precursor cells for adenocarcinoma. In this report, we show that one mechanism of acquired Fas resistance is inhibition of receptor aggregation via a major histocompatibility complex class II (MHCII)-mediated, actin-dependent mechanism. For these studies we used the well-described C57BL/6 mouse model of Helicobacter pylori and Helicobacter felis infection. Under normal conditions, Fas Ag is expressed at low levels, and MHCII expression on gastric mucosal cells is negligible. With infection and inflammation, both receptors are upregulated, and 6.1% of gastric mucosal cells express MHCII in combination with Fas Ag. Using the rat gastric mucosal cell line RGM-1 transfected with murine Fas Ag and MHCIIαβ chains, we demonstrate that MHCII prevents Fas receptor aggregation and inhibits Fas-mediated signaling through its effects on the actin cytoskeleton. Depolymerization of actin with cytochalasin D allows receptors to aggregate and restores Fas sensitivity. These findings offer one mechanism by which gastric mucosal cells acquire Fas resistance.

Molecular and genetic studies have demonstrated that members of the Toll-like receptor (TLR) family are critical innate immune receptors. TLRs are recognition receptors for a diverse group of microbial ligands including bacteria, fungi, and viruses. This study demonstrates that distinct TLRs are responsible for the recognition of Helicobacter lipopolysaccharide (LPS) versus intact Helicobacter bacteria. We show that the cytokine-inducing activity of Helicobacter LPS was mediated by TLR4; i.e., TLR4-deficient macrophages were unresponsive to Helicobacter pylori LPS. Surprisingly, the cytokine response to whole Helicobacter bacteria (H. pylori, H. hepaticus, and H. felis) was mediated not by TLR4 but rather by TLR2. Studies of HEK293 transfectants revealed that expression of human TLR2 was sufficient to confer responsiveness to intact Helicobacter bacteria, but TLR4 transfection was not sufficient. Our studies further suggest that cag pathogenicity island genes may modulate the TLR2 agonist activity of H. pylori as cagA+ bacteria were more active on a per-cell basis compared to cagA mutant bacteria for interleukin-8 (IL-8) cytokine secretion. Consistent with the transfection studies, analysis of knockout mice demonstrated that TLR2 was required for the cytokine response to intact Helicobacter bacteria. Macrophages from both wild-type and TLR4-deficient mice produced a robust cytokine secretion response (IL-6 and MCP-1) when stimulated with intact Helicobacter bacteria. In contrast, macrophages from TLR2-deficient mice were profoundly unresponsive to intact Helicobacter stimulation, failing to secrete cytokines even at high (100:1) bacterium-to-macrophage ratios. Our studies suggest that TLR2 may be the dominant innate immune receptor for recognition of gastrointestinal Helicobacter species.

Fas-mediated gastric mucosal apoptosis is gaining attention as a cause of tissue damage due to Helicobacter pylori infection. We explored the effects of H. pylori directly, and the effects of the inflammatory environment established subsequent to H. pylori infection, on Fas-mediated apoptosis in a nontransformed gastric mucosal cell line (RGM-1). Exposure to H. pylori-activated peripheral blood mononuclear cells (PBMCs), but not H. pylori itself, induced Fas antigen (Fas Ag) expression, indicating a Fas-regulatory role for inflammatory cytokines in this system. Of various inflammatory cytokines tested, only interleukin 1β and tumor necrosis factor alpha induced Fas Ag expression, and removal of either of these from the conditioned medium abrogated the response. When exposed to Fas ligand, RGM-1 cells treated with PBMC-conditioned medium underwent massive and rapid cell death, interestingly, with a minimal effect on total cell numbers early on. Cell cycle analysis revealed a substantial increase in S phase cells among cells exposed to Fas ligand, suggesting an increase in their proliferative response. Taken together, these data indicate that the immune environment secondary to H. pylori infection plays a critical role in priming gastric mucosal cells to undergo apoptosis or to proliferate based upon their Fas Ag status.