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Intravenously-applied bacteria tend to accumulate in tumors and can sporadically lead to tumor regression. Systemic administration of attenuated Salmonella typhimurium is safe and has shown no significant adverse effects in humans. The purpose of this study was to test the hypothesis that engineering S. typhimurium to express a chemokine, CCL21, would increase anti-tumor activity. We engineered an attenuated strain of S. typhimurium to produce the chemokine CCL21. Attenuated S. typhimurium expressing CCL21 significantly inhibited the growth of primary tumors and pulmonary metastases in preclinical models of multi-drug-resistant murine carcinomas, while control bacteria did not. Histological analysis of tumors showed marked inflammatory cell infiltrates in mice treated with CCL21-expressing but not control bacteria. Levels of cytokines and chemokines known to be induced by CCL21 [e.g., interferon-gamma (INFgamma), CXCL9, and CXCL10] were significantly elevated in tumors of mice treated with CCL21-expressing but not control S. typhimurium. The anti-tumor activity was found to be dependent on CD4- and CD8-expressing cells, based on antibody-mediated in vivo immuno-depletion experiments. Anti-tumor activity was achieved without evidence of toxicity. In summary, chemokine-expressing, attenuated bacteria may provide a novel approach to cancer immunotherapy for effective and well-tolerated in vivo delivery of immunomodulatory proteins.

Intravenous administration of bacteria leads to their accumulation in tumors and to sporadic tumor regression. We therefore explored the hypothesis that Salmonella typhimurium engineered to express the proapoptotic cytokine Fas ligand (FasL) would exhibit enhanced antitumor activity. Immunocompetent mice carrying tumors derived from syngeneic murine D2F2 breast carcinoma or CT-26 colon carcinoma cells were treated intravenously with FasL-expressing S. typhimurium or with phosphate-buffered saline (PBS; control). Treatment with FasL-expressing S. typhimurium inhibited growth of primary tumors by an average of 59% for D2F2 tumors and 82% for CT-26 tumors (eg, at 25 days after initial treatment, mean volume of PBS-treated CT-26 colon carcinomas = 1385 mm(3) and of S. typhimurium FasL-treated CT-26 tumors = 243 mm(3), difference = 1142 mm(3), 95% confidence interval = 800 mm(3) to 1484 mm(3), P < .001). Pulmonary D2F2 metastases (as measured by lung weight) were reduced by 34% in S. typhimurium FasL-treated mice compared with PBS-treated mice. FasL-expressing S. typhimurium had similar effects on growth of murine B16 melanoma tumors in wild-type mice but not in lpr/lpr mice, which lack Fas, or in mice with disrupted host inflammatory responses. Antitumor activity was achieved without overt toxicity. These preclinical results raise the possibility that using attenuated S. typhimurium to deliver FasL to tumors may be an effective and well-tolerated therapeutic strategy for some cancers.

Intravenously administered bacteria reportedly accumulate in tumors. Furthermore, systemic administration of attenuated Salmonella typhimurium has little or no significant side-effects in humans. Consequently, we engineered such bacteria to improve their oncolytic activity by stably inserting a gene encoding LIGHT, a cytokine known to promote tumor rejection. Unlike control bacteria, attenuated S. typhimurium expressing LIGHT inhibited growth of primary tumors, as well as the dissemination of pulmonary metastases, in various mouse tumor models employing murine carcinoma cell lines in immunocompetent mice. Antitumor activity was achieved without significant toxicity and was associated with infiltration of inflammatory cells and dependent on the LIGHT receptors, herpes virus entry mediator (HVEM), and lymphotoxin-beta receptor (LTbetaR). These findings provide evidence that nonvirulent bacteria can be exploited as targeting vehicles for local generation of therapeutic proteins in tumors.

Cytotoxic necrotizing factor type 1 (CNF1) from Escherichia coli activates the small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42) by catalyzing their deamidation at a specific glutamine residue. Since RhoA, Rac, and Cdc42 play a pivotal role in cell migration during the early phase of wound repair, we investigated whether CNF1 was able to interfere with wound healing in intestinal epithelial monolayers (T84 cells). After mechanical injury, we found that CNF1 blocks epithelial wound repair within 48 h. This effect was characterized by cell elongation and filopodium formation on the leading edge, in association with permanent phosphorylation of the focal adhesion kinase (FAK) via Rho activation. Moreover, inhibition of Rho kinase with Y-27632 decreased CNF1-mediated permanent FAK phosphorylation, leading to complete restitution of wound repair within 24 h. In addition, we found that CNF1 induced upregulation of mitogen-activated protein kinases (MAPK) activation. Moreover, activation of Rac and MAPK by CNF1 increased matrix metalloproteinase 9 expression in wounded T84 monolayers. Taken together, these results provide evidence that CNF1 strongly impairs intestinal epithelial wound healing.

Helicobacter pylori has been shown to induce chronic active gastritis and peptic ulcer and may contribute to the development of duodenal ulcer. Previous studies have shown that H. pylori mediates apoptosis of gastric epithelial cells via a Fas-dependent pathway. However, evidence for the induction of such a mechanism in intestinal epithelial cells (IEC) by H. pylori infection has not been demonstrated yet. This study was performed (i) to ascertain that H. pylori can induce IEC apoptosis; (ii) to delineate the role of the cag pathogenicity island (PAI), cagE, and vacA gene products in this process; and (iii) to verify whether the Fas-dependent pathway is involved in this phenomenon. When T84 cells were exposed to VacA(+)/cag PAI(+) H. pylori strains (CCUG 17874 and 60190), they exhibited apoptosis hallmarks as assessed by morphological studies, as well as annexin V and 3,3'-dihexyloxacarbocyanine iodide staining. In contrast, few or no apoptotic features could be detected after incubation with an isogenic mutant of strain 60190 in which the cagE gene was disrupted (60190:C(-) strain) or with a VacA(-)/cag PAI(-) H. pylori strain (G21). In addition, activation of caspase-3 during infection with VacA(+)/cag PAI(+) H. pylori strains was inhibited by pretreatment of IEC with an antagonistic anti-Fas antibody (ZB4). Taken together, these findings indicate that H. pylori triggers apoptosis in IEC via a Fas-dependent pathway following a process that depends on the expression of the cag PAI.

Acute colitis is characterized by a large number of polymorphonuclear leukocytes (PMNLs) migrating across the columnar epithelium in response to inflammatory stimuli. Several of these inflammatory factors have been characterized as proapoptotic inducers for intestinal epithelial cells. Our aim was to elucidate the role of PMNL transmigration in the onset of intestinal epithelial cell apoptosis. We found that PMNL migration, in response to N-formyl-methionyl-leucyl-phenylalanine across monolayers of intestinal epithelial cells (T84), was associated with activation of caspase-2, -3, and -9 and poly(ADP-ribose) polymerase cleavage within epithelial cells. Moreover, dihydrocytochalasin B treatment of T84 cells induced apoptosis with similar characteristics. Although Fas and Fas ligand were expressed on T84 cells and PMNLs, treatment of epithelial cells with an antagonistic anti-Fas antibody failed to prevent apoptosis induced by migrating PMNLs. Owing to the F-actin reorganization accompanying PMNL transmigration, these findings indicate a direct relationship between PMNL migration and induction of apoptosis in epithelial cells. This apoptotic process appears to involve remodeling of the actin cytoskeleton of enterocytes independent of the Fas/Fas ligand pathway.