Gastrointestinal Tract Dysbiosis Enhances Distal Tumor Progression through Suppression of Leukocyte Trafficking.

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNFα levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNFα supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer control. SIGNIFICANCE: Antibiotic-induced dysbiosis enhances distal tumor progression by altering host cytokine levels, resulting in suppression of tumor endothelial adhesion molecules and activated and effector CD8+ T cells in the tumor.

Cigarette smoke-induced disruption of pulmonary barrier and bacterial translocation drive tumor-associated inflammation and growth.

Microorganisms have an important role in tumorgenesis by the induction of inflammation and by a direct impact on tumor cells. Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for lung cancer and microbial colonization. We asked whether bacterial pathogens act as tumor promoters during CS-induced pulmonary inflammation. In a metastatic lung cancer (LC) model, Lewis lung carcinoma (LLC) cells were injected in mice to initiate the growth of tumors in the lung. Exposure to the combination of cigarette smoke (CS) and nontypeable Haemophilus influenzae (NTHi) synergistically increased metastatic growth. Lung levels of albumin and LDH, translocation of bacterial factors into tumor tissue, tumor inflammation, and tumor proliferation were significantly increased in mice exposed to CS in combination with NTHi. Bacterial pathogens increased the proliferation of cultured LLC cells and human cancer cell lines. Metastatic growth induced by the exposure to CS in combination with NTHi was reduced in mice deficient for IL-17. Our data provide evidence that CS-induced loss of pulmonary barrier integrity allows bacterial factors to translocate into tumor tissue and to regulate tumor-associated inflammation and tumor proliferation. Translocation of bacterial factors in tumor tissue links CS-induced inflammation with tumor proliferation.

Microbiota modulate tumoral immune surveillance in lung through a γδT17 immune cell-dependent mechanism.

Commensal bacteria are crucial to maintain immune homeostasis in mucosal tissues and disturbances in their ecology can affect disease susceptibility. Here, we report evidence that commensal bacteria shape the efficiency of immune surveillance in mucosal tissues. Antibiotic-treated (Abt) mice were more susceptible to development of engrafted B16/F10 melanoma and Lewis lung carcinoma, exhibiting a shortened mean survival time with more numerous and larger tumor foci in the lungs. The defective antitumor response of Abt mice was independent of dehydration caused by antibiotics. Host defenses relied upon intact commensal bacteria with no class specificity. Mechanistic investigations revealed a defective induction of the γδT17 cell response in lungs of Abt mice; here, more aggressive tumor development was observed, possibly related to a reduction in IL6 and IL23 expression there. Adding normal γδT cells or supplementing IL17 restored the impaired immune surveillance phenotype in Abt mice. Overall, our results demonstrated the importance of commensal bacteria in supporting the host immune response against cancer, defined an important role for γδT17 responses in the mechanism, and suggested deleterious effects of antibiotic treatment on cancer susceptibility and progression.

Transmigration and phagocytosis of macrophages in an airway infection model using four-dimensional techniques.

During infection, recruited phagocytes transmigrate across the epithelium to remove the pathogens deposited on the airway surface. However, it is difficult to directly observe cellular behaviors (e.g., transmigration) in single-cell layer cultures or in live animals. Combining a three-dimensional (3D) cell coculture model mimicking airway infection with time-lapse confocal imaging as a four-dimensional technique allowed us to image the behaviors of macrophages in 3D over time. The airway infection model was moved to a glass-bottomed dish for live-cell imaging by confocal laser scanning microscopy. Using time-lapse confocal imaging, we recorded macrophages transmigrating across the polyethylene terephthalate (PET) membrane of the inserts through the 5-µm pores in the PET membrane. Macrophages on the apical side of the insert exhibited essentially three types of movements, one of which was transmigrating across the epithelial cell monolayer and arriving at the surface of monolayer. We found that adding Staphylococcus aureus to the model increased the transmigration index but not the transmigration time of the macrophages. Only in the presence of S. aureus were the macrophages able to transmigrate across the epithelial cell monolayer. Apical-to-basal transmigration of macrophages was visualized dynamically. We also imaged the macrophages phagocytizing S. aureus deposited on the surface of the monolayer in the airway infection model. This work provides a useful tool to study the cellular behaviors of immune cells spatially and temporally during infection.

Bacterial-mediated knockdown of tumor resistance to an oncolytic virus enhances therapy.

Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.

Salmonella typhimurium A1-R tumor targeting in immunocompetent mice is enhanced by a traditional Chinese medicine herbal mixture.

We have developed a bacterial cancer therapy strategy using the genetically-engineered strain Salmonella typhimurium A1-R (A1-R). A1-R is auxotrophic for leu and arg which attenuates bacterial growth in normal tissue but allows high tumor virulence. A1-R is effective against metastatic human and murine cancer cell lines in clinically-relevant nude-mouse models. However, A1-R treatment of tumors in immunocompetent mouse models with high doses is limited by toxicity. The current study evaluated a traditional Chinese medicine (TCM) herbal mixture in combination with A1-R therapy in a syngeneic metastatic immunocompetent mouse model of highly aggressive lung cancer. In a model of Lewis lung carcinoma, the combination of a TCM herbal mixture and S. typhimurium A1-R enabled bacteria to be safely administered at the large dose of 2 × 10(7) colony forming units once a week i.v. with increased treatment efficacy and reduced toxicity compared to monotherapy with A1-R. The herbal mixture prevented body weight loss, spleen weight gain and liver infection by A1-R, as well as hemorrhagic lesions on the skin, liver, and spleen, all observed with A1-R monotherapy. The results of the present study suggest that the combination of A1-R and TCM has important potential for therapy of highly aggressive types of cancer, including those which are resistant to standard therapy.

Primer dosing of S. typhimurium A1-R potentiates tumor-targeting and efficacy in immunocompetent mice.

We developed the tumor-targeting strain Salmonella typhimuium A1-R (A1-R) and have shown it to be active against a number of tumor types in nude mice. However, in immunocompetent mice, dosing of A1-R has to be adjusted to avoid toxicity. In the present study, we developed a strategy to maximize efficacy and minimize toxicity for A1-R tumor-targeting in immunocompetent mice implanted with the Lewis lung carcinoma. A small primer dose of A1-R was first administered (1×10(6) colony forming unit [cfu] i.v.) followed by a high dose (1×10(7) cfu i.v.) four hours later. The primer-dose strategy resulted in smaller tumors and no observable side-effects compared to treatment with high-dose-alone. The serum level of tumor necrosis factor (TNF-α) was elevated in the mice treated with primer dose compared to mice only given the high dose. Tumor vessel destruction was enhanced by primer dosing of A1-R in immuno-competent transgenic mice expressing the nestin-driven green fluorescent protein, which is selectively expressed in nascent blood vessels. The primer-dose may activate TNF-α and other cytokines in the mouse, necessary for invasion of the tumor by the bacteria, as well as enhance tumor vessel destruction, thereby allowing a subsequent therapeutic dose to be effective and safe. The results of the present study suggest effective future clinical strategies of bacterial treatment of cancer.

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice.

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host, in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well tolerated by the immunocompetent host. Weekly intravenous injection with 2 × 10(7) bacteria and twice a week intravenous injection with 10(7) bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intra-thoracic administration was carried out with 10(8) bacteria A1-R injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration, without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.

Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors.

A fundamental obstacle in gene therapy for cancer is the specific delivery of an anticancer gene product to a solid tumor, and yet no systemic delivery system that specifically targets solid tumors currently exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized and proliferated in several types of mouse solid tumors after systemic application. In this report, we further describe a novel approach to cancer gene therapy in which genetically engineered Bifidobacterium is used as a tumor-specific vector. Similarly to wild-type B. longum, genetically engineered B. longum could be detected in tumor tissue only and was not found in a large survey of normal mouse tissues after intravenous injection. This finding strongly suggests that obligate anaerobic bacteria such as Bifidobacterium can be used as highly specific gene delivery vectors for cancer gene therapy.

Effect of Escherichia coli L-form cytoplasmic membranes on the interaction between macrophages and Lewis lung carcinoma cells: scanning electron microscopy.

Scanning electron microscopy (SEM) investigations on the interactions between peritoneal macrophages from Lewis lung carcinoma (LLC)-bearing mice and LLC tumour cells during 21 days after tumour implantation were carried out. The action of lipopolysaccharide (LPS)-containing cytoplasmic membranes (CM), from the stable protoplast type L-form of Escherichia coli, on the activity of in vitro phagocytosis was studied; CM induced a continuous increase in macrophage numbers. Activation of macrophage surfaces in healthy and tumour-bearing mice was established. Lamelipods, pseudopods and migration fringes 14 days after CM application were seen. Crater-like cavities deeply in the macrophage cells as well as adherent or prominent engulfed tumour cells within macrophages were observed during in vitro interaction with LLC cells. Macrophages from tumour-bearing mice without CM treatment showed less activation evaluated by SEM during earlier stages of tumour growth. The SEM investigation proved the temporary stimulating effect of E. coli L-form CM on the cell surface activation of peritoneal macrophages in healthy and LLC-bearing mice.