Rapid dietary changes, such as switching from high-forage to high-grain diets, can modify the rumen microbiome and initiate gastrointestinal distress, such as bloating. In such cases, feed additives, including prebiotics and live microbials, can be used to mitigate these negative consequences. Bio-Mos® is a carbohydrate-based prebiotic derived from yeast cells that is reported to increase livestock performance. Here, the responses of rumen bacterial cells to Bio-Mos® were quantified, sorted by flow cytometry using fluorescently-labeled yeast mannan, and taxonomically characterized using fluorescence in situ hybridization and 16S rRNA sequencing. Further, to evaluate the effects of bovine-adapted Bacteroides thetaiotaomicron administration as a live microbial with and without Bio-Mos® supplementation, we analyzed microbial fermentation products, changes to carbohydrate profiles, and shifts in microbial composition of an in vitro rumen community. Bio-Mos® was shown to be an effective prebiotic that significantly altered microbial diversity, composition, and fermentation; while addition of B. thetaiotaomicron had no effect on community composition and resulted in fewer significant changes to microbial fermentation. When combined with Bio-Mos®, there were notable, although not significant, changes to major bacterial taxa, along with increased significant changes in fermentation end products. These data suggest a synergistic effect is elicited by combining Bio-Mos® and B. thetaiotaomicron . This protocol provides a new in vitro methodology that could be extended to evaluate prebiotics and probiotics in more complex artificial rumen systems and live animals.
<div>Abstract<p>Several approaches to manipulate the gut microbiome for improving the activity of cancer immune-checkpoint inhibitors (ICI) are currently under evaluation. Here, we show that oral supplementation with the polyphenol-rich berry camu-camu (CC; <i>Myrciaria dubia</i>) in mice shifted gut microbial composition, which translated into antitumor activity and a stronger anti–PD-1 response. We identified castalagin, an ellagitannin, as the active compound in CC. Oral administration of castalagin enriched for bacteria associated with efficient immunotherapeutic responses (<i>Ruminococcaceae</i> and <i>Alistipes</i>) and improved the CD8<sup>+</sup>/FOXP3<sup>+</sup>CD4<sup>+</sup> ratio within the tumor microenvironment. Moreover, castalagin induced metabolic changes, resulting in an increase in taurine-conjugated bile acids. Oral supplementation of castalagin following fecal microbiota transplantation from ICI-refractory patients into mice supported anti–PD-1 activity. Finally, we found that castalagin binds to <i>Ruminococcus bromii</i> and promoted an anticancer response. Altogether, our results identify castalagin as a polyphenol that acts as a prebiotic to circumvent anti–PD-1 resistance.</p>Significance:<p>The polyphenol castalagin isolated from a berry has an antitumor effect through direct interactions with commensal bacteria, thus reprogramming the tumor microenvironment. In addition, in preclinical ICI-resistant models, castalagin reestablishes the efficacy of anti–PD-1. Together, these results provide a strong biological rationale to test castalagin as part of a clinical trial.</p><p><i>This article is highlighted in the In This Issue feature, p. 873</i></p></div>
Abstract Introduction The gut microbiota influences immune checkpoint inhibitors (ICI) efficacy, and immunogenic commensals bacteria such as Akkermansia muciniphila (AM) and Ruminoccocus are enriched in ICI responder patients. Therefore, strategies to beneficially shift microbiota composition represent a novel therapeutic avenue. Myrciaria dubia (MD) is a polyphenol-rich berry prebiotic that has been recently shown to improve the gut microbiota composition and dampen inflammation in metabolic diseases. Methods Daily oral administration of MD or water was performed in MCA-205 (PD-1 sensitive) and E0071 (PD-1 resistant) murine tumor models treated with iso-control or anti-PD-1. 16s rRNA gene sequencing and qPCR were used to profile the murine fecal microbiota. Tumor immune infiltration was analyzed by flow cytometry and RNA sequencing. Bile acid levels were measured using liquid chromatography coupled to tandem mass spectrometry in mouse feces and plasma at sacrifice. MD supplementation was also tested in antibiotic-treated mice that had undergone a fecal microbiota transplantation (FMT) using feces from non-responder (NR) and responder (R) non-small cell lung cancer (NSCLC) patients. Finally, to determine the bioactive compound, reverse-phase high performance liquid chromatography was used to isolate fractions that were tested in the MCA-205 tumor model. Results In MCA-205, MD gavage alone significantly reduced tumor size and in both tumor models MD increased the efficacy of anti-PD-1 compared to water + anti-PD-1. The activity of MD was inhibited upon antibiotics administration. FMT using mouse feces previously treated with MD recreated the effect in MD-naïve mice. Increases in alpha diversity, and AM and Ruminococcaceae were observed in the feces of MD supplemented mice. Serum and fecal CA, αMCA primary and HDCA and DCA secondary bile acids were upregulated in mice treated with MD. The combination of MD + anti-PD-1 increased the frequency of CD8+/CD4+ Treg and ICOS+ expression on CD8+ T cells, while the depletion of the CD8+ cells inhibited the anti-cancer activity. Next, we validated that FMT from NSCLC ICI patients recreated the patients' phenotypes in the MCA-205 tumor model (R vs NR). Treatment with MD resorted anti-PD-1 anti-tumor efficacy post FMT from NR patients. Lastly, we isolated the bioactive polyphenol molecule from MD and confirmed its anti-cancer effect in vivo. Supplementation of AM and ruminococcus further increased anti-PD1 activity. Conclusion We isolated the bioactive polyphenol molecule from the MD berry and demonstrated that its potent adjuvant effect on anti-PD-1 was associated with a beneficial shift in microbiome and bile acid composition in a CD8+ T cell dependent manner. The modification of the gut microbiota with polyphenol supplementation provides a new therapeutic approach in immuno-oncology. Citation Format: Meriem Messaoudene, Florent Cauchois, Reilly Piedgeon, Khoudia Diop, Corentin Richard, Thibault Varin, Jocelyn Trottier, Olivier Barbier, Geneviève Pilon, Simon Turcotte, André Marette, Bastien Castagner, Bertrand Routy. A new polyphenol prebiotic isolated from Myrciaria dubia improves gut microbiota composition and increases anti-PD-1 efficacy in murine cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5730.
Abstract For automated oligosaccharide synthesis to impact glycobiology, synthetic access to most carbohydrates has to become efficient and routine. Methods to install “difficult” glycosidic linkages have to be established and incorporated into the overall synthetic concept. Described here is the first automated solid‐phase synthesis of oligosaccharides containing the challenging β‐mannosidic linkage. Carboxybenzyl mannoside building blocks proved effective β‐mannosylation agents and resulted in excellent conversion and good to moderate selectivities. [(Triisopropylsilyl)oxy]‐methyl ether (Tom), served as an orthogonal, minimally intrusive, and readily cleavable protecting group for the elongation of the C3 position of mannose. The desired oligosaccharide products were readily separated from by‐products containing unwanted stereoisomers using reverse‐phase HPLC. The methods described here expand the scope of carbohydrates currently accessible by automation as many oligosaccharides of biological interest contain β‐mannosidic linkages.
Abstract Despite many years of research and a few success stories with gene therapeutics, efficient and safe DNA delivery remains a major bottleneck for the clinical translation of gene-based therapies. Gene transfection with calcium phosphate (CaP) nanoparticles brings the advantages of low toxicity, high DNA entrapment efficiency and good endosomal escape properties. The macroscale aggregation of CaP nanoparticles can be easily prevented through surface coating with bisphosphonate conjugates. Bisphosphonates, such as alendronate, recently showed promising anticancer effects. However, their poor cellular permeability and preferential bone accumulation hamper their full application in chemotherapy. Here, we investigated the dual delivery of plasmid DNA and alendronate using CaP nanoparticles, with the goal to facilitate cellular internalization of both compounds and potentially achieve a combined pharmacological effect on the same or different cell lines. A pH-sensitive poly(ethylene glycol)-alendronate conjugate was synthetized and used to formulate stable plasmid DNA-loaded CaP nanoparticles. These particles displayed good transfection efficiency in cancer cells and a strong cytotoxic effect on macrophages. The in vivo transfection efficiency, however, remained low, calling for an improvement of the system, possibly with respect to the extent of particle uptake and their physical stability. Graphical abstract
Inositol phosphates are essential for mammalian cell signalling with critical roles in cellular processes. The fully phosphorylated inositol phosphate, myo-inositol hexakisphosphate (IP6), modulates numerous eukaryotic proteins and virulence factors. It has been suggested that the high charge density of IP6 causes restructuring of virulence factors in mammalian cells, activating their site-specific enzymatic activity. IP6 is challenging to study due to its phytase instability, hydrophilicity, and propensity to precipitate. Here we suggest the thiophosphate bioisostere, myo-inositol hexakisthiophosphate (IT6) will mitigate these issues, as thiophosphate substitution has been found to be phytase resistant, improve lipophilicity, and solubility. Assessment of the chemical properties of IT6 has indeed validated these characteristics. In addition, we performed biophysical characterization of the IT6 binding event with the virulence factors Salmonella enterica serovar Typhimurium AvrA, Vibrio parahaemolyticus VopA, and Clostridioides difficile TcdB. Our data shows that the higher charge density of IT6 increased its binding affinity and residence time to the proteins, which improved stabilization of the bound-state. IT6 is a valuable tool for structural biology research and the described biophysical characteristics of thiophosphate substitution is of value in medicinal chemistry.
Clostridioides difficile is a bacterium that causes life-threatening intestinal infections. Infection symptoms are mediated by a toxin secreted by the bacterium. Toxin pathogenesis is modulated by the intracellular molecule, inositol-hexakisphosphate (IP6). IP6 binds to a cysteine protease domain (CPD) on the toxin, inducing auto-proteolysis, which liberates a virulence factor in the cell cytosol. We developed second-generation IP6 analogs designed to induce auto-proteolysis in the gut lumen, prior to toxin uptake, circumventing pathogenesis. We synthesized a panel of thiophosphate-/sulfate-containing IP6 analogs, and characterized their toxin binding affinity, auto-proteolysis induction and cation interactions. Our top candidate was soluble in extracellular cation concentrations, unlike IP6. The IP6 analogs were more negatively charged than IP6, which improved affinity and stabilization of the CPD, enhancing toxin auto-proteolysis. Our data illustrate the optimization of IP6 with thiophosphate biomimetics which are more capable of inducing toxin auto-proteolysis than the native ligand, warranting further studies in vivo.
