Oral microbiota affects the efficacy and prognosis of radiotherapy for colorectal cancer in mouse models.

Radiotherapy is inevitably intertwined with various side effects impairing the quality of life of cancer patients. Here, we report the possibility that alterations of the oral microbiota influence the therapeutic efficacy and prognosis of radiotherapy for primary rectal cancer and colorectal cancer (CRC) liver metastases that pathologically disrupt gastrointestinal integrity and function. 16S rRNA sequencing shows that oral microbiota alterations change the gut bacterial composition within tumors but not in adjacent peritumor tissues in CRC mouse models. Specifically, buccal Fusobacterium nucleatum migrates to the CRC locus and impairs the therapeutic efficacy and prognosis of radiotherapy. Administration of a specific antibiotic, metronidazole, abrogates the adverse effects of oral microbiome fluctuation on radiotherapy for CRC. The oral microbiota were also associated with radiation-induced intestinal injury via intestinal microbes. Our findings demonstrate that the oral microbiome in synergy with its intestinal counterparts impinges on the efficacy and prognosis of radiotherapy for CRC.

Metabolic modulation of tumours with engineered bacteria for immunotherapy.

The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1-4. Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.

Smectite promotes probiotic biofilm formation in the gut for cancer immunotherapy.

Administration of probiotics to regulate the immune system is a potential anti-tumor strategy. However, oral administration of probiotics is ineffective because of the poor inhabitation of exogenous bacteria in host intestines. Here we report that smectite, a type of mineral clay and established anti-diarrhea drug, promotes expansion of probiotics (especially Lactobacillus) in the murine gut and subsequently elicits anti-tumor immune responses. The ion-exchangeable microstructure of smectite preferentially promotes lactic acid bacteria (LABs) to form biofilms on smectite in vitro and in vivo. In mouse models, smectite laden with LAB biofilms (Lactobacillus and Bifidobacterium) inhibits tumor growth (when used alone) and enhances the efficacy of chemotherapy or immunotherapy (when used in combination with either of them) by activating dendritic cells (DCs) via Toll-like receptor 2 (TLR2) signaling. Our findings suggest oral administration of smectite as a promising strategy to enrich probiotics in vivo for cancer immunotherapy.

Streptococcus thermophilus Inhibits Colorectal Tumorigenesis Through Secreting ß-Galactosidase.

BACKGROUND & AIMS: Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multicohort fecal samples. Here, we aim to investigate whether this bacterium could act as a prophylactic for CRC prevention. METHODS: The antitumor effects of S thermophilus were assessed in cultured colonic epithelial cells and in 2 murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S thermophilus was identified by mass spectrometry and followed by ß-galactosidase activity assay. The mutant strain of S thermophilus was constructed by homologous recombination. The effect of S thermophilus on the gut microbiota composition was assessed by shotgun metagenomic sequencing. RESULTS: Oral gavage of S thermophilus significantly reduced tumor formation in both Apcmin/+ and azoxymethane-injected mice. Coincubation with S thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. ß-Galactosidase was identified as the critical protein produced by S thermophilus by mass spectrometry screening and ß-galactosidase activity assay. ß-Galactosidase secreted by S thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S thermophilus without functional ß-galactosidase lost its tumor-suppressive effect. Also, S thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via ß-galactosidase. ß-Galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathway kinases, which partially mediated the anticancer effects of S thermophilus. CONCLUSION: S thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S thermophilus is mediated at least by the secretion of ß-galactosidase.

PD-1 Signaling Promotes Tumor-Infiltrating Myeloid-Derived Suppressor Cells and Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.

Development of Oxytolerant Salmonella typhimurium Using Radiation Mutation Technology (RMT) for Cancer Therapy.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Colon Cancer Prevention with Walnuts: A Longitudinal Study in Mice from the Perspective of a Gut Enterotype-like Cluster.

There is limited understanding of how walnut consumption inhibits the development of colorectal cancer. A possible mechanism may involve alterations to the gut microbiota. In this study, the effects of walnut on gut microbiota were tested in a mouse tumor bioassay using the colonotropic carcinogen, azoxymethane (AOM) added to the total Western diet (TWD). 16S rRNA pyrosequencing identified three enterotype-like clusters (E1, E2, and E3) in this murine model. E1, E2, and E3 are associated with AOM exposure, walnut consumption, and TWD diet, respectively. E2 and E3 showed distinct taxonomic and functional characteristics, while E1 represented an intermediate state. At the family level, E1 and E3 were both enriched with Bacteroidaceae, but driven by two different operational taxonomic units (OTU; OTU-2 for E1, OTU-4 for E3). E2 was overrepresented with Porphyromonadaceae and Lachnospiraceae, with OTU-3 (family Porphyromonadaceae) as the "driver" OTU for this cluster. Functionally, E3 is overrepresented with genes of glycan biosynthesis and metabolism, xenobiotic metabolism, and lipid metabolism. E2 is enriched with genes associated with cell motility, replication and repair, and amino acid metabolism. Longitudinally, E2 represents the gut microbial status of early life in these mice. In comparison with E1 and E3, E2 is associated with a moderate lower tumor burden (P = 0.12). Our results suggest that walnuts may reduce the risk of colorectal cancer within a Western diet by altering the gut microbiota. Our findings provide further evidence that colorectal cancer risk is potentially modifiable by diet via alterations to the microbiota.

Toll-like Receptor-6 Signaling Prevents Inflammation and Impacts Composition of the Microbiota During Inflammation-Induced Colorectal Cancer.

Tightly regulated immune responses must occur in the intestine to avoid unwanted inflammation, which may cause chronic sequela leading to diseases such as colorectal cancer. Toll-like receptors play an important role in preventing aberrant immune responses in the intestine by sensing endogenous commensal microbiota and delivering important regulatory signals to the tissue. However, the role that specific innate receptors may play in the development of chronic inflammation and their impact on the composition of the colonic microbiota is not well understood. Using a model of inflammation-induced colorectal cancer, we found that Lactobacillus species are lost more quickly in wild-type (WT) mice than TLR6-deficient mice resulting in overall differences in bacterial composition. Despite the longer retention of Lactobacillus, the TLR6-deficient mice presented with more tumors and a worse overall outcome. Restoration of the lost Lactobacillus species suppressed inflammation, reduced tumor number, and prevented change in the abundance of Proteobacteria only when given to WT mice, indicating the effect of these Lactobacillus are TLR6 dependent. We found that the TLR6-dependent effects of Lactobacillus could be dissociated from one another via the involvement of IL10, which was necessary to dampen the inflammatory microenvironment, but had no effect on bacterial composition. Altogether, these data suggest that innate immune signals can shape the composition of the microbiota under chronic inflammatory conditions, bias the cytokine milieu of the tissue microenvironment, and influence the response to microbiota-associated therapies.

Traditional Processed Meat Products Re-designed Towards Inulin-rich Functional Foods Reduce Polyps in Two Colorectal Cancer Animal Models.

Inulin-rich foods exert a prebiotic effect, as this polysaccharide is able to enhance beneficial colon microbiota populations, giving rise to the in situ production of short-chain fatty acids (SCFAs) such as propionic and butyric acids. These SCFAs are potent preventive agents against colorectal cancer due to their histone deacetylases inhibitory properties, which induce apoptosis in tumor colonocytes. As colorectal cancer is the fourth most common neoplasia in Europe with 28.2 new cases per 100,000 inhabitants, a cost-effective preventive strategy has been tested in this work by redesigning common porcine meat products (chorizo sausages and cooked ham) consumed by a substantial proportion of the population towards potential colorectal cancer preventive functional foods. In order to test the preventive effect of these inulin-rich meat products against colorectal cancer, an animal model (Rattus norvegicus F344) was used, involving two doses of azoxymethane (10 mg/kg) and two treatments with dextran sodium sulfate (DSS) during a 20-week assay period. Control feed, control sausages, functional sausages (15.7% inulin), control cooked ham and functional cooked ham (10% inulin) were used to feed the corresponding animal cohorts. Then, the animals were sacrificed and their digestive tract tissues were analyzed. The results showed a statistically significant 49% reduction in the number of colon polyps in the functional meat products cohorts with respect to the control meat products animals, as well as an increase in the cecum weight (an indicator of a diet rich in prebiotic fiber), a 51.8% increase in colon propionate production, a 39.1% increase in colon butyrate concentrations, and a reduction in the number of hyperplastic Peyer's patches. Metagenomics studies also demonstrated colon microbiota differences, revealing a significant increase in Bacteroidetes populations in the functional meat products (mainly due to an increase in Bacteroidaceae and Prevotellaceae families, which include prominent propionate producers), together with a reduction in Firmicutes (especially due to lower Lachnospiraceae populations). However, functional meat products showed a remarkable increase in the anti-inflammatory and fiber-fermentative Blautia genus, which belongs to this Lachnospiraceae family. The functional meat products cohorts also presented a reduction in important pro-inflammatory bacterial populations, such as those of the genus Desulfovibrio and Bilophila. These results were corroborated in a genetic animal model of CRC (F344/NSlc-Apc1588/kyo) that produced similar results. Therefore, processed meat products can be redesigned towards functional prebiotic foods of interest as a cost-effective dietary strategy for preventing colorectal cancer in human populations.

Editing of the gut microbiota reduces carcinogenesis in mouse models of colitis-associated colorectal cancer.

Chronic inflammation and gut microbiota dysbiosis, in particular the bloom of genotoxin-producing E. coli strains, are risk factors for the development of colorectal cancer. Here, we sought to determine whether precision editing of gut microbiota metabolism and composition could decrease the risk for tumor development in mouse models of colitis-associated colorectal cancer (CAC). Expansion of experimentally introduced E. coli strains in the azoxymethane/dextran sulfate sodium colitis model was driven by molybdoenzyme-dependent metabolic pathways. Oral administration of sodium tungstate inhibited E. coli molybdoenzymes and selectively decreased gut colonization with genotoxin-producing E. coli and other Enterobacteriaceae. Restricting the bloom of Enterobacteriaceae decreased intestinal inflammation and reduced the incidence of colonic tumors in two models of CAC, the azoxymethane/dextran sulfate sodium colitis model and azoxymethane-treated, Il10-deficient mice. We conclude that metabolic targeting of protumoral Enterobacteriaceae during chronic inflammation is a suitable strategy to prevent the development of malignancies arising from gut microbiota dysbiosis.

Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic.

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.

Comparison of the common bacteria in human and mouse tumours using high-throughput sequencing.

Evidence has indicated that gut bacteria may serve an important role in cancer development and therapy, while little work has been done to explore the microbial diversity inside tumours. In the present study, high­throughput sequencing was first used to identify and compare the microbial diversity in human and mouse tumours. Principal component analysis (PCA) and ß-diversity indicated a low microbial similarity among mouse artificial tumours (M.AT group), mouse spontaneous tumours (M.T group) and human tumours (H.T group), and Serratia (35.85 vs. 32.64 vs. 73.32%), Pseudomonas (24.10 vs. 16.62 vs. 1.72%) and Ochrobactrum (6.28 vs. 11.08 vs. 11.90%) were identified as dominant bacteria at the genus level. In addition, Venn results indicated 103 common operational taxonomic units (OTUs) in the M.AT, M.T and H.T groups, and only 2 and 1 OTUs belonged to Lactobacillus and Escherichia, respectively, while no OTUs belonging to Salmonella, Bifidobacteria or Clostridium were identified. In the present study, the common bacteria between human and mouse tumours were identified, which may serve as potential strains for bacteriotherapy of cancers.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Procedure for Horizontal Transfer of Patient-Derived Xenograft Tumors to Eliminate Corynebacterium bovis.

Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.

EGFR-targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells.

Tumor-targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)-targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC-10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co-expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile. Biotechnol. Bioeng. 2016;113: 2698-2711. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

Salmonella overcomes tumor immune tolerance by inhibition of tumor indoleamine 2, 3-dioxygenase 1 expression.

Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments.

Engineering nanoparticle-coated bacteria as oral DNA vaccines for cancer immunotherapy.

Live attenuated bacteria are of increasing importance in biotechnology and medicine in the emerging field of cancer immunotherapy. Oral DNA vaccination mediated by live attenuated bacteria often suffers from low infection efficiency due to various biological barriers during the infection process. To this end, we herein report, for the first time, a new strategy to engineer cationic nanoparticle-coated bacterial vectors that can efficiently deliver oral DNA vaccine for efficacious cancer immunotherapy. By coating live attenuated bacteria with synthetic nanoparticles self-assembled from cationic polymers and plasmid DNA, the protective nanoparticle coating layer is able to facilitate bacteria to effectively escape phagosomes, significantly enhance the acid tolerance of bacteria in stomach and intestines, and greatly promote dissemination of bacteria into blood circulation after oral administration. Most importantly, oral delivery of DNA vaccines encoding autologous vascular endothelial growth factor receptor 2 (VEGFR2) by this hybrid vector showed remarkable T cell activation and cytokine production. Successful inhibition of tumor growth was also achieved by efficient oral delivery of VEGFR2 with nanoparticle-coated bacterial vectors due to angiogenesis suppression in the tumor vasculature and tumor necrosis. This proof-of-concept work demonstrates that coating live bacterial cells with synthetic nanoparticles represents a promising strategy to engineer efficient and versatile DNA vaccines for the era of immunotherapy.

Genetically modified bacteria as a tool to detect microscopic solid tumor masses with triggered release of a recombinant biomarker.

Current tomographic methods of cancer detection have limited sensitivity and are unable to detect malignant masses smaller than half a centimeter in diameter. Mortality from tumor recurrence and metastatic disease would be reduced if small lesions could be detected earlier. To overcome this limitation, we created a detection system that combines the specificity of tumor-targeting bacteria with the sensitivity of a synthetic biomarker. Bacteria, specifically Salmonella, preferentially accumulate in tumors and microscopic metastases as small as five cell layers thick. To create tumor detecting bacteria, an attenuated strain of Salmonella was engineered to express and release the fluorescent protein ZsGreen. A single-layer antibody method was developed to measure low concentrations of ZsGreen. Engineered bacteria were administered to a microfluidic tumor-on-a-chip device to measure protein production. In culture, half of produced ZsGreen was released by viable bacteria at a rate of 87.6 fg bacterium(-1) h(-1). Single-layer antibody dots were able to detect bacterially produced ZsGreen at concentrations down to 4.5 ng ml(-1). Bacteria colonized in 0.12 mm(3) of tumor tissue in the microfluidic device released ZsGreen at a rate of 23.9 µg h(-1). This release demonstrates that ZsGreen readily diffuses through tissue and accumulates at detectable concentrations. Based on a mathematical pharmacokinetic model, the measured rate of release would enable detection of 0.043 mm(3) tumor masses, which is 2600 times smaller than the current limit of tomographic techniques. Tumor-detecting bacteria would provide a sensitive, minimally invasive method to detect tumor recurrence, monitor treatment efficacy, and identify the onset of metastatic disease.

Context-dependent role of IL-18 in cancer biology and counter-regulation by IL-18BP.

IL-18 is a proinflammatory and immune regulatory cytokine, member of the IL-1 family. IL-18 was initially identified as an IFN-γ-inducing factor in T and NK cells, involved in Th1 responses. IL-18 is produced as an inactive precursor (pro-IL-18) that is enzymatically processed into a mature form by Casp1. Different cells, such as macrophages, DCs, microglial cells, synovial fibroblasts, and epithelial cells, express pro-IL-18, and the production of bioactive IL-18 is mainly regulated at the processing level. PAMP or DAMP molecules activate inflammasomes, which trigger Casp1 activation and IL-18 conversion. The natural inhibitor IL-18BP , whose production is enhanced by IFN-γ and IL-27, further regulates IL-18 activity in the extracellular environment. Inflammasomes and IL-18 represent double-edged swords in cancer, as their activation may promote tumor development and progression or oppositely, enhance anti-tumor immunity and limit tumor growth. IL-18 has shown anti-tumor activity in different preclinical models of cancer immunotherapy through the activation of NK and/or T cell responses and has been tested in clinical studies in cancer patients. However, the dual role of IL-18 in different experimental tumor models and human cancers raises critical issues on its therapeutic use in cancer. This review will summarize the biology of the IL-18/IL-18R/IL-18BP system and will address the role of IL-18 and its inhibitor, IL-18BP, in cancer biology and immunotherapy.

Murine solid tumours as a novel model to study bacterial biofilm formation in vivo.

Bacteria of many species are able to invade and colonize solid tumours in mice. We have focused on Salmonella enterica serovar Typhimurium. Detailed analysis revealed that such tumour-invading Salmonella form biofilms, thus providing a versatile in vivo test system for studying bacterial phenotypes and host-pathogen interactions. It appears that biofilm formation by S. typhimurium is induced as a defence against the immune system of the host, and in particular against neutrophils. Further, we extended our work to the clinically more relevant biofilm infection by Pseudomonas aeruginosa. The induction of P. aeruginosa biofilms in neoplastic tissue appears to be elicited as a reaction against the immune system. Reconstitution experiments reveal that T cells are responsible for biofilm induction. Isogenic mutants that are no longer able to form biofilms can be used for comparison studies to determine antimicrobial resistance, especially therapeutic efficacy against P. aeruginosa located in biofilms.