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Resurrecting the intestinal microbiota to combat
antibiotic-resistant pathogens / Cancer immunotherapy
  April 2016
"there is little solid evidence in humans of their effectiveness in enhancing health, promoting longevity, or reducing infections."
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Download the PDF here
Download the PDF here
Download the PDF here
Download the PDF here
For now, clinical studies need to be performed to establish effectiveness and safety, and such studies require funding.
Unlike chemical drugs, whose composition and purity can be determined with precision, live bacteria must be cultured, often in complex media, and, even if maintained in pure form, can mutate and either gain or lose functions upon extended culturing. Thus, guaranteeing uniformity, purity, and effectiveness becomes a substantial challenge that can be overcome with a high level of vigilance. Thus, guaranteeing uniformity, purity, and effectiveness becomes a substantial challenge that can be overcome with a high level of vigilance. Development and safe delivery of a medicine consisting of live bacteria poses special but not unprecedented challenges.
The limited regulation of probiotics has occasionally resulted in exaggerated claims that have left at least some health professionals skeptical of the entire field. The recently characterized bacterial species that constitute a small fraction of the commensal microbiota but have been shown in the laboratory to provide colonization resistance will be new members of the probiotic club; presumably, specific claims about their benefits will be substantiated by careful clinical studies to demonstrate safety and effectiveness.
The idea that administration of live bacteria to humans can lead to health benefits likely originated with elie Metchnikov, a Nobel laureate in 1908. Toward the end of his career, he became fascinated with the longevity of yogurt-eating people in Bulgaria (29). He postulated that lactate-producing bacteria were health-promoting and that other bacteria inhabiting the host were responsible for age-related decompensation and tissue destruction. A century later, this general notion remains popular and has led to the concept that certain bacteria-in particular, lactate-producing genera such as Lactobacillus-promote health and thus are referred to as probiotics (30). Other probiotic bacteria, such as Bifidobacteria, have similarly been proposed to enhance health and have recently been associated with enhanced responses to cancer immunotherapy (31) and resistance to infection by enteropathogenic Escherichia coli (32) in animal models. Despite the popularity of these probiotics, there is little solid evidence in humans of their effectiveness in enhancing health, promoting longevity, or reducing infections.
The intestinal microbiota, which is composed of diverse populations of commensal bacterial species, provides resistance against colonization and invasion by pathogens. Antibiotic treatment can damage the intestinal microbiota and, paradoxically, increase susceptibility to infections. Reestablishing microbiota-mediated colonization resistance after antibiotic treatment could markedly reduce infections, particularly those caused by antibiotic-resistant bacteria. Ongoing studies are identifying commensal bacterial species that can be developed into next-generation probiotics to reestablish or enhance colonization resistance. These live medicines are at various stages of discovery, testing, and production and are being subjected to existing regulatory gauntlets for eventual introduction into clinical practice. The development of next-generation probiotics to reestablish colonization resistance and eliminate potential pathogens from the gut is warranted and will reduce health care-associated infections caused by highly antibiotic-resistant bacteria.
This review summarizes recent studies identifying protective commensal bacterial species and discusses the need for a development path for these potential next-generation probiotics that demonstrates their effectiveness, ensures their safety, and promotes their eventual production, distribution, and affordability.
Because specific health claims are not made, most probiotics are not regulated as drugs by the U.S. Food and Drug Administration.
Movement of laboratory discoveries to the clinic requires clinical trials, safe manufacturing, distribution, and, ultimately, delivery to the patient.
Unlike chemical drugs, whose composition and purity can be determined with precision, live bacteria must be cultured, often in complex media, and, even if maintained in pure form, can mutate and either gain or lose functions upon extended culturing.
Could microbial therapy boost cancer immunotherapy?

Immunotherapies known as checkpoint blockades are rapidly changing standard treatment and outcomes for patients with advanced malignancies, as they lead to long-term disease control in a subset of patients (1). On pages 1084 and 1079 of this issue, Sivan et al. (2) and Vetizou et al. (3), respectively, illustrate an important role for the gut microbiome in modulating the efficacy of this treatment.
Administration of specific bacterial species or combinations of bacteria that would enhance responses to therapy would be preferable but will require extensive development and testing. For now, additional studies on patient populations are warranted. Stool samples can be collected and the microbiota analyzed, and thus prospective collection from all members of phase 2 or 3 clinical study is feasible. The findings of Sivan et al. and Vetizou et al. show that collection of fecal samples should be considered going forward in immunotherapy studies to characterize and ultimately manipulate this factor to favor response in immunotherapy-treated cancer patients.
Sivan et al. and Vetizou et al. provide strong evidence for the role of stool microbiota (i.e., intestinal microbes) in response and resistance to immunotherapy. Sivan et al. illustrate the importance of Bifidobacterium to antitumor immunity and anti-PD-L1 antibody against (PD-1 ligand) efficacy in a mouse model of melanoma. The authors demonstrate that mice raised in two different facilities [Jackson Laboratory (JAX) and Taconic Farms (TAC)] that are known to harbor distinct microbiota exhibit differential tumor growth that disappears upon cohousing of the animals. Furthermore, when fecal material from JAX mice, whose tumors grow more slowly, was transferred into the intestine of TAC mice, the latter exhibited delayed tumor growth and enhanced CD8+ T cell infiltration of the tumor. Anti-PD-L1 therapy was more effective in JAX mice, and the combination of JAX fecal transfer to TAC mice undergoing anti-PD-L1 therapy was more effective than either intervention alone. Sivan et al. also show that Bifidobacterium confers nearly the same effect as JAX stool transfer and that bacteria must be alive for the treatment to be effective. Investigation of the underlying mechanisms of this effect reveals that Bifidobacterium alters dendritic cell activity, which in turn leads to improved tumor-specific CD8+ T cell function.
Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy
T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the mechanisms underlying variable immune responses between individuals are not well understood. One possible modulator could be the intestinal microbiota. We compared melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or after fecal transfer. Sequencing of the 16S ribosomal RNA identified Bifidobacterium as associated with the antitumor effects. Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein 1 ligand 1 (PD-L1)-specific antibody therapy (checkpoint blockade), and combination treatment nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced CD8+ T cell priming and accumulation in the tumor microenvironment mediated the effect. Our data suggest that manipulating the microbiota may modulate cancer immunotherapy.
Our studies demonstrate an unexpected role for commensal Bifidobacterium in enhancing antitumor immunity in vivo......At the sequence level, Bifidobacterium operational taxonomic unit OTU_681370 showed the largest increase in relative abundance in JAX-fed TAC mice (table S1) and the strongest association with antitumor T cell responses across all permutations (Fig. 3D and table S3). .....Our studies demonstrate an unexpected role for commensal Bifidobacterium in enhancing antitumor immunity in vivo. Given that beneficial effects are observed in multiple tumor settings and that alteration of innate immune function is observed, this improved antitumor immunity could be occurring in an antigen-independent fashion. The necessity for live bacteria may imply that Bifidobacterium colonizes a specific compartment within the gut that enables it to interact with host cells that are critical for modulating DC function or to release soluble factors that disseminate systemically and lead to improved DC function.
Gut microbes affect immunotherapy
The unleashing of antitumor T cell responses has ushered in a new era of cancer treatment. Although these therapies can cause dramatic tumor regressions in some patients, many patients inexplicably see no benefit. Mice have been used in two studies to investigate what might be happening. Specific members of the gut microbiota influence the efficacy of this type of immunotherapy (see the Perspective by Snyder et al.). Vetizou et al. found that optimal responses to anticytotoxic T lymphocyte antigen blockade required specific Bacteroides spp. Similarly, Sivan et al. discovered that Bifidobacterium spp. enhanced the efficacy of antiprogrammed cell death ligand 1 therapy.
Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota
Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy. We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species. In mice and patients, T cell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Tumors in antibiotic-treated or germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage with B. fragilis, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis-specific T cells. Fecal microbial transplantation from humans to mice confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties. This study reveals a key role for Bacteroidales in the immunostimulatory effects of CTLA-4 blockade.
Probiotic Gut Bacteria Enhance Cancer Immunotherapy in a Mouse Model of Melanoma Sivan A, Corrales L, Hubert N, et al. Commensal Bifidobacterium Promotes Antitumor
Immunity and Facilitates Anti-PD-L1 Efficacy. Science 2015;350:1084-1089.
The exciting fields of cancer immunotherapy and the gut microbiome converge in a recent study from Sivan et al (Science 2015;350:1084-1089), in which the investigators elegantly illustrate the beneficial effects of gut Bifidobacteria in a mouse model of melanoma. The investigators suggest that the gut microbiota may be as critical to tumor suppression as therapy with the immune checkpoint inhibitor anti-PD-L1. Moreover, when administration of Bifidobacteria was combined the immunotherapy in mice implanted with melanoma, tumor growth was essentially arrested.
The described discovery sequence mirrors what others have previously reported in colitis models. The investigators first noted that mice of the same strain (C57BL/6) exhibited differential melanoma growth rates and tumor-specific immune cell infiltrate based on the mouse vendor. Tumors grew more aggressively in mice from Taconic Biosciences (TAC) than in mice from Jackson Labs (JAX). These differences normalized when TAC mice were either transplanted with fecal microbiota from JAX mice or cohoused with JAX mice (leveraging coprophagic behavior of mice with consequent transfer of gut microbes), thus illustrating that specific gut microbiota could prevent tumor growth. Next, the investigators tested the possibility that manipulation of gut microbiota could treat tumors by first implanting tumor and then manipulating the gut microbiota. To do so, they examined the effects of JAX fecal microbiota transplantation alone or in combination with anti-PD-L1 therapy in TAC mice. Both strategies were effective alone, and synergistic when combined.
To identify the bacterial mediators of this protective/therapeutic effect, the investigators availed 16S ribosomal RNA sequencing and thereby identified Bifidobacteria as the genus-level taxon associated with antitumor T-cell responses. Further analyses narrowed the important bifidobacterial species to B breve, B longum, and B adolescentis. Noting that the first 2 of these bacterial species are common components of over-the-counter probiotic supplements, the investigators then demonstrated that a commercial cocktail of this supplement recapitulated the effects they observed with the fecal transplant. Finally, the investigators demonstrated that live Bifidobacteria are required and act indirectly through stimulation of host antitumor T-cell responses. Supporting this, neither heat-killed Bifidobacteria nor live Lactobacillus supplementation could recapitulate the observed effects, and Bifidobacteria were ineffective in CD8 T-cell-depleted mice. The investigators found no evidence for translocation of Bifidobacteria into mesenteric lymph nodes, spleen, or tumor.
Although melanoma research may not typically pique the interest of gastroenterologists or GI researchers, we should take note of this exciting study. Cancer, a multifaceted condition that spans all organ systems and persists as a global epidemic, represents a nascent application for microbiota-related research. As a community, we have already established important links between the gut microbiota, physiology, and a list of diseases that now includes skin cancer.
Metastatic melanoma is the model example for cancer immunotherapy success. Beginning with the introduction of ipilimumab (an anti-CTLA-4 antibody) and now with the addition of PD-L1 inhibitors, significant gains have been seen for the first time in controlling disease progression. Even these therapies offer room for improvement with progression-free survival remaining at <1 year (N Engl J Med 2015;373:23-34). The gut microbiota is a potentially modifiable factor influencing tumor immunity that may enable even greater efficacy, or even perhaps a better chance of preventing cancer altogether.
Bifidobacteria are identified by these investigators as commensal microbes that enhance antitumor immunity. As discussed, Bifidobacteria are presently sold as over-the-counter, first-generation probiotic supplements (Clin Gastroenterol Hepatol 2012;10:960-968). The molecular mechanism(s) by which these bacteria impact dendritic cells is not delineated in this study, but elucidating this may unlock further opportunities for targeted therapeutics. Is the key feature a metabolic bacterial trait? If so, such knowledge may permit the design of next-generation probiotics. To clarify the pertinent host-microbe interactions, an investigation of additional gut microbiota is warranted: given the diversity of gut microbes, one might expect other bacterial players to bear similar effects. Finally, if gut bacteria can enhance antitumor immunity, it stands to reason that certain commensal microbes may specifically antagonize antitumor immunity as well. The ability to identify such bacterial taxa may improve our understanding of variability in the natural history of disease, permit more accurate prognostication, and potentially enable a microbiota-based therapeutic intervention.
As is reported in the same issue of Science, PD-L1 is not the only checkpoint inhibitor therapy that can be influenced by gut microbiota. Vetizou and colleagues concurrently report that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species (Science 2015;350:1079-1084). These 2 important studies add additional complexity to understanding how gut microbes may influence a variety of cancer therapies.
Additionally, it was reported previously that gut microbiota could modulate the anticancer immune effects of cyclophosphamide (Science 2013;342:971-976). This study differed from the more recent studies in that the postulated mechanism of action involved translocation of bacteria into secondary lymphoid organs with consequent priming of the antitumor immune response: a breach of small intestinal microbial barrier function induced by cyclophosphamide set up this cascade of events. In that study, Lactobacillus species were implicated as key bacterial mediators.
Several major challenges must be overcome before applying these findings to clinical practice. First, the human gut microbiota is tremendously diverse, much more so than the 2 microbiota studied here. Variability and dynamics of human Bifidobacterial representation, as well as of microbiota community structure and function, must be studied and understood in this context. Many standard concomitant therapies including antibiotics have poorly understood effects in altering antitumor immunity. The effect of diet, a major factor shaping the gut microbiota (Science 2011;333:101-104), is not described in this study. However, as we have recently shown, even a single diet ingredient can influence host-microbe interactions with significant physiologic consequences (Cell 2015;163:95-107).
Clinical trials proposing to incorporate the addition of live probiotic bacteria to cancer therapy may have to overcome several barriers. Several cytotoxic and immune checkpoint inhibitor chemotherapies, as well as radiation therapy, affect GI barrier function. Although many currently used probiotics appear relatively safe even in the setting of cancer therapy, infectious complications could occur. In our experience, the US Food and Drug Administration has required a strong burden of proof of the safety of probiotics in the cancer population and will likely continue to do so (Curr Opin Support Palliat Care 2015;9:157-162). These potential risks and the anticipated regulatory burden provide further motivation for investigators to identify clearly the molecular mechanisms by which Bifidobacteria influence the tumor. This knowledge will help to determine whether or not specific probiotic-derived molecules might be combined with cancer therapy in a purified form.
Altogether, the data presented in this study represent an exciting step in applying microbiota-based therapies toward shaping cancer therapy. In gastroenterology, we seek efficacious therapies for metastatic cancers of the pancreas, liver, and colon. Given the observations of Sivan et al, the gut microbiota may provide the missing link.
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