Carlos H.F. Chan

Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohns disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-ΔfimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.

IL-17 Promotes p38 MAPK-Dependent Endothelial Activation Enhancing Neutrophil Recruitment to Sites of Inflammation

Neutrophilic inflammation plays an important role in lung tissue destruction occurring in many chronic pulmonary diseases. Neutrophils can be recruited to sites of inflammation via the action of the cytokine IL-17. In this study, we report that IL-17RA and IL-17RC mRNA expression is significantly increased in asthmatic bronchoscopic biopsies and that these receptors are not only expressed on epithelial and inflammatory cells but also on endothelial cells. IL-17 potently stimulates lung microvascular endothelial cells to produce chemoattractants (CXCL8 and derivatives of the 5-lipoxygenase pathway) that selectively drive neutrophil but not lymphocyte chemotaxis. Moreover, IL-17 promotes endothelial activation by inducing the expression of endothelial adhesion markers (E-selectin, VCAM-1, and ICAM-1) in a p38 MAPK-dependent manner. This increased expression of adhesion molecules stimulates the trans-endothelial migration of neutrophils, as well as the transmigration of HT-29 colon carcinoma cells, suggesting a further role in promoting lung metastasis. Finally, IL-17 increased neutrophil adhesion to the endothelium in vivo as determined by intravital microscopy of mice cremaster muscle. Overall, our results demonstrate that IL-17 is a potent activator of the endothelium in vivo leading to neutrophil infiltration. Therefore, preventing neutrophil recruitment by blocking the action of IL-17 on endothelial cells may prove to be highly beneficial in diseases in which neutrophilic inflammation plays a key role.

LPS-Induced TLR4 Signaling in Human Colorectal Cancer Cells Increases β1 Integrin-Mediated Cell Adhesion and Liver Metastasis

Infectious complications resulting from resection of colorectal cancer (CRC) elevates the risk of cancer recurrence and metastasis, but the reason for this risk relationship is unknown. Defining the mechanisms responsible may offer opportunities to improve outcomes in a majority of patients whose tumors are resected as part of their therapy. The complex formed between Toll receptor TLR4 and myeloid differentiation factor MD2 defines a major cell surface receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen that has been implicated in infectious complications after CRC resection. As the TLR4/MD2 complex is expressed on CRC cells, we hypothesized that LPS may promote liver metastasis in CRC by stimulating TLR4 signaling. In support of this hypothesis, we report here that LPS enhances liver metastasis of human CRC cells that express TLR4/MD2 after intrasplenic graft of immunocompromised nude mice. Compared with TLR4 nonexpressing, nonmetastatic CRC cells, we observed increased in vitro adherence to different extracellular matrices and human umbilical vein endothelial cells (HUVEC). Furthermore, we observed an increased likelihood of in vivo capture within hepatic sinusoids after LPS treatment. No differences were apparent in phosphorylation of p38 and MAPK isoforms, but in metastatic CRC cells expressing surface TLR4 treatment with LPS increased Ser473 phosphorylation of AKT kinase. We showed that enhanced adherence elicited by LPS in these cells could be blocked at three different levels, using Eritoran (TLR4 small molecule antagonist), PI-103 (PI3K inhibitor), or anti-β1 integrin blocking antibodies. Taken together, the results indicate that stimulation of the TLR4/MD2 complex by LPS activates PI3K/AKT signaling and promotes downstream β1 integrin function, thereby increasing the adhesiveness and metastatic capacity of CRC cells. Our findings suggest that inhibiting LPS-induced TLR4 signaling could improve therapeutic outcomes by preventing cancer metastasis during the perioperative period of CRC resection.

Global analysis of neutrophil responses to Neisseria gonorrhoeae reveals a self-propagating inflammatory program.

An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with Neisseria gonorrhoeae. Neutrophils undergo an aggressive opsonin-independent response to N. gonorrhoeae, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa-expressing bacteria. In this study, we sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an in vivo model of N. gonorrhoeae infection, we show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa-dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of N. gonorrhoeae infection.

Colorectal Hyperplasia and Dysplasia Due to Human Carcinoembryonic Antigen (CEA) Family Member Expression in Transgenic Mice

CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin α5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer.

The impact of incidental gastrointestinal stromal tumours on patients undergoing resection of upper gastrointestinal neoplasms

BACKGROUND Emerging data suggest asymptomatic gastrointestinal stromal tumours (GISTs) of the upper gastrointestinal (UGI) tract are not uncommon. We sought to determine their incidence in patients undergoing resection for UGI neoplasms and their impact on surgical and adjuvant treatment. METHODS We accessed a database prospectively listing all patients undergoing resection of non-GIST neoplasms of the stomach and esophagus at a single university centre over a 4.5-year period and reviewed pathology reports for the presence of synchronous GISTs in the UGI tract. We compared patient demographic and tumour characteristics, operative procedures and postoperative outcomes. RESULTS In all, 207 patients undergoing gastrectomy or esophagectomy for non- GIST neoplasms were included. We identified 15 synchronous GISTs in the UGI tract of 11 (5.3%) patients (1 preoperatively, 4 intraoperatively and 10 on final pathology), with an average age of 67 years. Most patients were men. Additional resections were required for GISTs identified pre- or intraoperatively. Final pathology revealed completely resected c-kit positive tumours of an average size of 0.5 (range 0.1-4.0) cm with low or very low risk of malignant potential. No patients received adjuvant therapy for the GISTs. After a median follow-up of 11 (range 2-36) months, 5 patients died from their primary cancer, 3 were alive with primary cancer recurrence, and 3 were alive without disease. No patients experienced GIST recurrence. CONCLUSION Incidentally finding a synchronous GIST during resection of UGI neoplasms is not uncommon; it may alter surgical treatment but is unlikely to impact longterm survival.

Massive upper gastrointestinal bleeding secondary to duodenal metastasis of transitional cell carcinoma of the urinary bladder

Acute upper gastrointestinal (UGI) bleeding is a common problem in our clinical practice and is often due to peptic ulcer diseases. Occasionally, malignancy may be implicated in these situations. Here we report a rare case of UGI bleeding secondary to metastatic transitional cell carcinoma (TCC) of the urinary bladder. A 62-year-old man with a history of stage IIIb TCC of the urinary bladder presented with hematemesis. Endoscopy showed a large tumor in the second stage of the duodenum that occupied 40% of the duodenal circumference, over 7 cm in length. Biopsies revealed a poorly differentiated malignant neoplasm consistent with metastasis from urothelial carcinoma that was identical to the previous surgical specimen of the urinary bladder. He was treated with supportive therapy and intravenous proton pump inhibitor and was discharged home 2 weeks later. Two weeks after discharge, the patient returned to the hospital with a painful swelling of the floor of his mouth. Biopsy again showed the same cancer type. He had unremitting bleeding from his mouth requiring multiple transfusions and a course of palliative radiation therapy. He progressively deteriorated in his cardiopulmonary and neurological functions and expired with cardiopulmonary arrest one month later.

Abstract 1394: Acute bacterial infection increases lung cancer metastasis via toll-like receptors 2 and 4

INTRODUCTION: Surgery is required for curative treatment of lung cancer but is associated with a high rate of bacterial infections such as pneumonia. There is emerging clinical evidence to suggest that infectious complications may decrease survival after cancer surgery, but the mechanism is unclear. Interactions between bacteria and host cells are mediated by specialized pathogen recognition receptors, of which the Toll-like receptors (TLRs) are best characterized. We and others have shown that cancer cells express TLRs, but their function is largely unknown. The aim of this study is to investigate the mechanisms of bacterial infection-facilitated lung cancer metastasis with a particular focus on the role of TLRs. METHODS: Two lung cancer cell lines (murine H-59 and human A549) were incubated a) directly with heat-inactivated bacteria (S.pneumonia or E.coli) or purified bacterial antigens (lipopolysaccharide or lipoteichoic acid), or b) indirectly with an in vitro pneumonia model using conditioned media from pulmonary bronchial epithelial cells incubated with heat-inactivated bacteria or bacterial antigens. Migratory and metastatic ability of thus treated cells were determined in vitro (adhesion, migration) and in vivo (hepatic intravital microscopy and intra-splenic liver metastasis). A clinically relevant in vivo model of severe infection, cecal ligation and puncture (CLP) or sham control, was employed in some animals. To assess the role of TLRs, we used transgenic TLR4 knockout mice, and small molecule inhibiting molecules or antibodies of TLRs. RESULTS: Direct and indirect (in vitro pneumonia) infection of H-59 and A549 increased adhesion to extracellular matrix components approximately 2-8 fold and 2-4 fold over negative control, respectively. In vitro pneumonia increased in vitro migration of H-59 approximately 2-5 fold. Direct and indirect incubation of H-59 with S.pneumonia and E.coli increased the in vivo adhesion to liver sinusoids 2-4 fold. CLP (in vivo infection model) enhanced the in vivo adhesion of H-59 3-fold and increased number of liver metastases at 2 weeks post-injection approximately 12 fold. These effects were attenuated in TLR4 -/- mice and with TLR2 and TLR4 inhibition. CONCLUSION: Activation of TLR2 or TLR4 on lung cancer cells, either directly by S.pneumonia and E.coli, indirectly though activated bronchial epithelial cells, or via an in vivo infection, increases cancer cell adhesion, migration and metastasis. TLRs thus represent a potential therapeutic target in bacterial infection facilitated cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1394. doi:1538-7445.AM2012-1394

Abstract 5279: LPS-TLR4 binding increases metastatic potential of colorectal cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objective: Infectious complications in colorectal cancer patients undergoing curative resection are associated with cancer recurrence, but the exact mechanism is not well understood. Toll-like receptor 4 (TLR4) is the sole receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen involved in such infectious complication. TLR4 expression has been found on several cancer types. We hypothesize that LPS promotes cancer cell metastatic potential via TLR4 signaling in colorectal cancer cells. Methods: We compared the effects of LPS stimulation on two well-characterized colorectal cancer cell lines: Caco-2 (non-metastatic) and HT-29 (metastatic). Flow cytometry was performed to detect cell surface TLR4 and various integrin expression. Cancer cells were incubated with or without 0.1 µg/mL LPS for 4 hours. In vitro static adhesion assay was used to study the ability of cell adhesion to extracellular matrix proteins (fibronectin, collagen I and collagen IV). Intravital microscopy, a technique used to study microcirculation in vivo, was carried out to observe dynamic attachment of intra-splenic injected cancer cells to C57/BL6 mouse liver sinusoids. Results: Caco-2 cells lack TLR4 receptors and are unresponsive to LPS treatment. In contrast, HT-29 cells were shown to express TLR4 receptors and HT-29 responds to LPS by a 2-fold increase of β1, but not α5, integrin expression in LPS-treated HT-29 cells (Mean Fluorescence Intensity:1041 vs. 458(untreated)). LPS-treated HT-29 cells showed enhanced attachment to fibronectin (1.6-fold; p < 0.01), collagen I (3-fold; p < 0.01) and collagen IV (2-fold; P < 0.01) relative to controls in vitro. Similarly, adherence of HT-29 cells to hepatic sinusoids in vivo was increased by LPS treatment (2 fold; p < 0.05). Conclusion: These data suggest that LPS-TLR4 signaling in colorectal cancer cells increases the metastatic potential. LPS-TLR4 mediated signaling cascade may be a valuable therapeutic target for the prevention of cancer metastasis. 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 5279.

Abstract 5172: Role of CEACAM1 on cancer cell endothelial adhesion and liver metastasis in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: There are emerging data to suggest that tumor over-expression of the carcinoembryonic antigen-related cell adhesion molecules (CEACAM1, 5 and 6) may influence cancer metastasis; however, the mechanisms for this are unclear. CEACAM1 was shown to bind homotypically to itself and heterotypically to CEACAM5 and CEACAM6. Since CEACAM1 is the only known CEACAM family member expressed on the hepatic endothelium, we hypothesized that CEACAM1 promotes cancer metastasis by directly mediating binding between circulating tumor cells and the sinusoidal endothelium. Methods: Using intra-vital microscopy (IVM), a physiologically relevant model system to assess the early stages of liver metastasis in vivo, we have compared the hepatic endothelial adhesion of intra-splenically injected MC38 cells (mouse colon cancer cell line with minimal CEACAM1 expression) between Ceacam1+/+ and Ceacam1−/− mice. A CEACAM1-negative population of MC38 cells (MC38-null) was sorted out by FACS using specific anti-CEACAM1 antibodies. MC38-null cells were then infected with retroviruses produced from ψ2 packaging cells carrying pLXSN-CEACAM1 expression construct and infected cells were selected using neomycin-containing media. A CEACAM1-positive population of MC38 cells (MC38-CC1) was sorted out by FACS using specific anti-CEACAM1 antibodies. The migratory ability of MC38-null and MC38-CC1 cells were compared by IVM in Ceacam1+/+ mice as mentioned above. Liver metastasis assays were performed by means of intra-splenic injection of MC38 cells into the Ceacam1+/+ and Ceacam1−/− mice. Data expressed as mean ± SEM, student t-test determined significance (*p<0.05). Results: By using IVM, we observed a 3-fold decrease in hepatic sinusoidal endothelial adhesion of intra-splenically injected MC38-null cells in Ceacam1−/− mice compared to Ceacam1 +/+ mice (0.8±0.2 vs 2.5±0.3 adhered cells)*. Forced expression of CEACAM1 in MC38 cells however did not significantly increase hepatic endothelial adhesion in the Ceacam1+/+ mice (2.7±0.9 adhered MC38-CC1 cells vs 2.5±0.2 adhered MC38-null cells). Development of liver metastasis after intra-splenic injection of MC38-null cells was significantly reduced in the Ceacam1−/− mice (81% reduction in comparison to Ceacam1+/+ mice)*. Conclusion: Sinusoidal endothelial cell, but not cancer cell, CEACAM1 expression supports liver metastasis by increasing the adherence of circulating tumor cells to hepatic endothelium. Further investigation will be required to dissect the exact mechanism of how endothelial CEACAM1 influence hepatic endothelial adhesion of cancer cells. 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 5172.