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Gut microbiota modulation of and

James L. Alexander1,3, Ian D. Wilson3, Julian Teare1, Julian R. Marchesi1–3, Jeremy K. Nicholson3 and James M. Kinross1,3 Abstract | Evidence is growing that the gut microbiota modulates the host response to chemotherapeutic , with three main clinical outcomes: facilitation of efficacy; abrogation and compromise of anticancer effects; and mediation of toxicity. The implication is that gut microbiota are critical to the development of personalized cancer treatment strategies and, therefore, a greater insight into prokaryotic co‑ of chemotherapeutic drugs is now required. This thinking is based on evidence from human, animal and in vitro studies that gut are intimately linked to the pharmacological effects of (5‑fluorouracil, cyclophosphamide, irinotecan, oxaliplatin, gemcitabine, methotrexate) and novel targeted immunotherapies such as anti‑PD‑L1 and anti-CLTA‑4 therapies. The gut microbiota modulate these agents through key mechanisms, structured as the ‘TIMER’ mechanistic framework: Translocation, Immunomodulation, Metabolism, Enzymatic degradation, and Reduced diversity and ecological variation. The gut microbiota can now, therefore, be targeted to improve efficacy and reduce the toxicity of current chemotherapy agents. In this Review, we outline the implications of pharmacomicrobiomics in cancer therapeutics and define how the microbiota might be modified in clinical practice to improve efficacy and reduce the toxic burden of these compounds.

Cancer survival has dramatically increased in the past the interface of multiple anti-neoplastic and carcino- half century, driven by a deeper understanding of cancer genic metabolic, immune and inflammatory pathways12. biology, improved surgical outcomes and increasingly However, the gut microbiota also have a major role in efficacious multimodal chemotherapy and radiotherapy defining the efficacy and toxicity of a broad range of 1 13,14 1 regimens . Cytotoxic drugs continue to be the mainstay drugs . by intestinal microorganisms Centre for Digestive and Gut 15 Health, Department of of medical treatment for most patients with advanced dis- has been well recognized since the 1960s ; for example, Surgery & Cancer, 10th Floor ease, yet they have an unpredictable treatment response microbiota-driven drug metabolism is essential for the QEQM Building, St Mary’s and considerable treatment-related morbidity and activation of certain azo prodrugs such as prontosil and Hospital Campus, mortality2. The next generation of personalized ­cancer neoprontosil, and can affect drug disposition16 and tox­ Praed Street, Imperial College 3 17 London, London, W2 1NY, therapies are now emerging , which exploit advances in icity . However, advances in high-throughput sequenc- 4,5 UK. molecular and phenotypic heterogeneity , tumour evo- ing and other ‘–omics’ platforms have led to the concept 2School of Biosciences, lution, immunotherapy and vaccination. However, even of ‘pharmacomicrobiomics’, and the importance of the The Sir Martin Evans targeted therapies, which have revolutionized outcomes gut microbiota for chemotherapeutic drug modulation Building, Cardiff University, in cancers such as malignant melanoma, suffer from and is now increasingly recognized18. Museum Avenue, Cardiff, CF10 3AX, UK. novel problems such as acquired resistance, idiosyncratic Despite this progress, we are still lacking a complete 6–8 3Computational and Systems adverse effects and high costs . map of microbiota–host–drug interactions in cancer, and , Department of Systems medicine science provides multiparametric, biological complexity remains a considerable obstacle to Surgery & Cancer, time-specific insights into complex biological pathogenic the development of these ‘precision’ therapies. For exam- Sir Alexander Fleming 9 Building, Imperial College states . A major contribution of systems biology to mod- ple, the gut microbiota exert both direct and indirect London, South Kensington, ern medicine has been the rediscovery of the importance effects on chemotherapy toxicity and efficacy through London, SW7 2DD, UK. of the gut microbiota to almost all aspects of human a large suite of chemical signalling cascades (FIG. 1); 10 Correspondence to J.R.M. health . However, the major focus of ‘oncomicrobiome’ as a result, it seems to exert its influence on almost all [email protected] research to date has been on the microbiome’s role in the classes of chemotherapeutic agents and it achieves this 11 doi:10.1038/nrgastro.2017.20 aetiology of cancer and cancer risk . This focus is because on a moving playing field. Not only are the gut micro- Published online 8 Mar 2017 the gut microbiota co‑develops with the host and sits at biota niche-specific and highly personalized, but their

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Key points accumulation of inflammatory cells and discontinuity of the intestinal barrier, with accompanying translocation • Evidence is increasing that the gut microbiota modulate the actions of of commensal bacteria into secondary lymphoid organs chemotherapeutic drugs used in cancer and other diseases in a mouse model26. Although there was no reduction in • We propose the ‘TIMER’ mechanistic framework to explain how gut bacteria influence the total bacterial counts in the small intestine after 7 days chemotherapy effects on the host: Translocation, Immunomodulation, Metabolism, of treatment with cyclophosphamide, there was a reduc- Enzymatic degradation and Reduced diversity and ecological variation tion in the abundance of lactobacilli and enterococci. • A number of tools for manipulating the gut microbiota in this context, including dietary Furthermore, a specific set of Gram-positive bacteria modifications, probiotics and synthetically engineered bacteria, are in development (segmented filamentous bacteria and Lactobacillus john- • The gut microbiota will be central to the future of personalized cancer treatment sonii, Lactobacillus murinus and Enterococcus hirae) was strategies necessary to mediate cyclophosphamide-driven accumu-

lation of type 17 T helper (TH17) cell and type 1 T helper

(TH1) cell responses, and long-term treatment with ecology is also dynamic. For example, the majority of antibiotics reduced the capacity of cyclophosphamide cancers strike at extremes of age, in which the struc- to cure P815 mastocytomas26. Pre-conditioning with tural ecology of the gut might be immature or perturbed vancomycin, which is an antibiotic active against Gram- through exposure to a lifetime of environmental modifi- positive microorganisms, and to a lesser extent colistin ers19. Moreover, pathological disease states or surgical and (active against Gram-negative microorganisms), inhib- medical therapies might also create a state of dysbiosis, ited the anticancer effects of cyclophosphamide in mice further exaggerating the influence of deleterious bac­teria, inoculated with MCA205 tumours. Indeed, a follow‑up reducing efficacy and exacerbating the toxicity of chemo- study from the same group, in addition to validating therapy. A paradox also exists, as chemothera­peutics the primacy of E. hirae in restoring cyclophosphamide-­ might further exacerbate any dysbiotic state rather than induced antitumour effects to microbiota-­depleted mice, correct it, with potentially serious implications for drug also identified the colonic Gram-negative microorgan- toxicity. The gut microbiota must, therefore, have a crit- ism Barnesiella intestinihominis as an orchestrator of ical role in the development of precision treatment strat- cyclophosphamide effects27. Interestingly, some of the egies for cancer and they are increasingly seen as a target actions of Barnesiella in this setting — increasing sys- for next-generation cancer therapies. temic levels of a polyfunctional subset of cytotoxic CD8+

This Review describes the established mechanisms T cells (Tc1) and TH1 cell responses and re‑­instating through which chemotherapy treatment efficacy is facili­ intra­tumoural IFNγ‑producing γδ tumour-infiltrating tated and abrogated by the gut microbiota, through what lymphocytes — were distinct from those mechanisms we have described as the ‘TIMER’ mechanistic framework previously identified­ for other microbiota. — Translocation, Immunomodulation, Metabolism, Also using mouse models, Iida and co‑workers28 Enzymatic degradation, and Reduced diversity (TABLE 1). provided further evidence that gut bacteria regulate We review evidence that commensals are implicated in the immune response to chemotherapy. In mice treated oncological treatment-related toxicity and the role of eco- with intratumoural CpG oligodeoxynucleotides they logical engineering in modulating chemotherapy efficacy. showed that the microbiota primes tumour-associated Finally, we discuss the implications of such discoveries on innate myeloid cells for inflammatory cytokine produc- future treatment for patients with cancer and outline how tion, and that antibiotic treatment or germ-free (sterile) the gut microbiota could be modulated for developing status attenuates this response. Moreover, the ability precision strategies in the treatment of cancer. to eradicate tumours and prolong survival in control mice was markedly reduced with antibiotic treatment, Translocation and immunomodulation whereas germ-free animals failed to respond. For the Almost 1 in 10 patients with cancer will require a hos- platinum-salt oxaliplatin, the antibiotic effect manifested pital visit for an infection during chemotherapy20. within 2 days of treatment, implying that the effect in this Translocation describes the process through which com- case was due to direct suppression of early cytotoxicity, mensal or pathogenic bacteria pass across the gut barrier rather than as a result of later cell death and subsequent into the systemic milieu, where they can contribute to immune responses. the morbidity of chemotherapy21. Although it was first The importance of gut microbial facilitation of described in the 1960s22, and risk factors for its presence immune responses in cancer treatment extends to novel were established, the microbial and host mechanisms that targeted immunotherapies, such as synthetic mono­clonal drive this event are not fully defined. antibodies against cytotoxic T‑lymphocyte-associated However, studies have begun to question whether protein 4 (CTLA‑4) and anti-programmed cell death 1 translocation modifies not only the risk of sepsis, but also 1 (anti‑PD‑L1). MCA205 sarcoma progression chemotherapeutic efficacy. Cyclophosphamide exerts its is controlled by CTLA‑4‑specific antibodies in specific anti-neoplastic effects through a variety of immuno­ pathogen-free mice, but not in germ-free mice or those logical pathways23–25. Viaud and colleagues26 conducted treated with broad-spectrum antibiotics29. In the latter a series of experiments in mouse models, investigating groups, there was a decrease in activation of splenic the role of small intestinal commensal microorganisms effector CD4+ T cells and tumour-infiltrating lympho- in cyclophosphamide treatment. Both cyclophosphamide cytes. Clinical response to anti‑PD‑L1 is more frequent and doxorubicin cause shortening of intestinal villi, focal in patients who show evidence of T‑cell response in

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the tumour microenvironment30. This phenomenon with induction of tumour-specific T cells and increased was explored by studying melanoma growth in geneti- T cells in the tumour microenvironment31. This effect cally similar and the same strain of mice from different was abrogated by depletion of CD8+ T cells and by facilities (Jackson Laboratory (JAX) and Taconic Farms heat inactivation of bacteria. The absence of bacteria (TAC)), which differ in the composition of their gut bac- in secondary lymphoid organs suggests that systemic teria31. Tumours grew more aggressively in TAC mice immunological effects occurred independently of bac- than JAX mice, but differences in tumour growth and terial translocation. Instead, the authors concluded that immune responses were ablated by co‑housing. Transfer Bifidobacterium-derived signals are mediated through of faecal material from JAX to TAC mice delayed tumour dendritic cell activation, as they demonstrated using growth and enhanced immune responses in the latter; genome-wide transcriptional profiling and functional corres­ponding transfer in the opposite direction had in vitro experiments. no effect on JAX mice. Furthermore, combination of CTLA‑4-blockade-induced colitis, an unwelcome JAX microbiota and anti‑PD‑L1 monoclonal antibody phenomenon resulting from mucosal immune dysregu­ improved tumour control more than individual treatment lation, is seen in approximately one-third of patients with either in isolation. Sequencing of the 16S ribosomal receiving ipilimumab treatment against metastatic RNA gene also demonstrated that Bifidobacterium spp. mela­noma7,32. Dubin and co‑workers33 have demon- showed the largest increase in relative abundance in strated that over-representation of the JAX-microbiota-fed TAC mice, and also exhibited the phylum correlates with resistance to this particular form strongest association with antitumour T‑cell responses. of colitis in humans, and that a lack of genetic pathways Finally, mice treated with a Bifidobacterium com- involved in polyamine transport and B vitamin synthesis mercial cocktail markedly improved tumour control, is associated with increased risk of this type of colitis.

Indirect mechanisms • Reduction Direct mechanisms Inactive irinotecan • Hydrolysis Doxorubicin (SN-38G) • Dehydroxylation Methotrexate • Dealkylation Cyclophosphamide Pro-drugs Active metabolites • Demethylation Toxicity and Gemcitabine β-glucuronidase mucositis • Decarboxylation • Acetylation Shortening of villi SN-38 • Deamination Mucosal injury • Deconjugation Gut Bacteriocidal or bacterial overgrowth microbiota Secondary metabolites Biliary

Liver Intestinal PRR (e.g. TLR) MyD88 epithelial Cancer cell NF-κB

Oxaliplatin Systemic circulation Pro-inflammatory excretion via kidney gene expression APC CpG oligodeoxynucleotides Translocation Toxicity and Immunomodulation Monoclonal antibody TLR mucositis Treg cell T 1 T 17 TAMC Metabolism H H (e.g. Ipilimumab and anti-PD-L1) Enzymatic degradation Reduced diversity Sepsis Figure 1 | An overview of the TIMER microbiota–host interactions that might potentiate desirable effects, abrogate efficacy or liberate toxic Nature Reviews | Gastroenterology & Hepatology modulate chemotherapy efficacy and toxicity. Chemotherapeutics can compounds. The microbiota harbour a large suite of indirect metabolic exert their influence through multiple pathways, which are outlined here. processes (such as reduction, hydrolysis, dehydroxylation, dealkylation, etc.) Translocation: cyclophosphamide can cause shortening of the intestinal villi which could be used for drug metabolism. These secondary metabolites are and damage to the mucosal barrier, permitting commensal microorganisms secreted into the circulation and excreted by the kidney, which might in to cross the intestinal barrier and enter secondary lymphoid organs. turn cause toxicity. Gut bacteria possess β-glucuronidases that cleave the Immunomodulation: intestinal microbiota facilitate a plethora of glucuronide from the inactive metabolite of irinotecan (SN-38G), releasing chemotherapy-­induced immune and inflammatory responses. For example, active metabolite (SN‑38) in the gut causing diarrhoea. Reduced diversity: Lactobacillus and segmented filamentous bacteria mediate the chemotherapy induces changes in the diversity of the mucosal and faecal

accumulation of type 17 T helper (TH17) cell and type 1 T helper (TH1) cell microbiota through altered biliary excretion and secondary metabolism or responses26. Bifidobacteria also modify tumour-specific T‑cell induction and associated antibiotic use and dietary modifications. As a result, pathobionts increase T-cell numbers in the tumour microenvironment in patients might predominate, leading to deleterious effects such as diarrhoea and treated with anti‑PD‑L1 immunomodulator. Metabolism and enzymatic colitis. PRR, pattern-recognition ; TAMC, tumour-associated

degradation: direct and indirect bacterial modification of pharmaceuticals myeloid cell; TLR, Toll-like receptor; Treg cell, regulatory T cell.

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Table 1 | A summary of the ‘TIMER’ effects of the gut microbiota on chemotherapy efficacy and toxicity Mechanism Chemotherapy Bacteria Effect Translocation Cyclophosphamide, Gram-positive microorganisms Commensal bacteria cross the intestinal barrier to enter doxorubicin (Lactobacillus johnsonii, L. murinus secondary lymphoid organs26,27 and Enterococcus hirae)

Immunomodulation Cyclophosphamide Lactobacillus, segmented Gram-positive commensals mediate accumulation of TH17 26 filamentous bacteria and TH1‑cell response CpG Ruminococcus, Alistipes Priming of tumour-associated myeloid inflammatory oligodeoxynucleotides responses28 Oxaliplatin Not known Mediated by TLR4 and reactive oxygen species production by myeloid cells28 CTLA‑4 blockade Bacteroidales Decreased activation of splenic effector CD4+ T cells and tumour-infiltrating lymphocytes29 Anti‑PD‑L1 Bifidobacterium Tumour-specific T‑cell induction and increased T cells in tumour microenvironment31 Methotrexate Not known Gut microbes regulate against chemotherapy-induced small bowel injury via TLR2 signalling and drug transporter p-gp36 Metabolism CPT‑11 (Irinotecan) Not known Streptomycin treatment inhibits absorption of CPT‑11 and reduces activity of epithelial carboxylesterase106 Ipilimumab Bacteroidetes Bacterially mediated B vitamin production and polyamine transport deficiencies associated with increased risk of CTLA‑4 blockade-induced colitis33 Enzymatic • 5‑Fluor‑2ʹ‑deoxyuridine Mycoplasma encoded nucleoside phosphorylases restrict degradation and 5‑trifluorothymidine cytostatic activity53,54 • Gemcitabine CPT‑11 (Irinotecan) β‑glucuronidase-expressing Bacterial β‑glucuronidase cleaves glucuronide from inactive gut bacteria metabolite, releasing active metabolite (SN‑38) in the gut45 Reduced diversity Carmustine, etoposide, , , Chemotherapy associated with reduction in bacteria and ecological cytarabine and melphalan that limit inflammation and increase in bacteria associated network function combination with colitis58 Methotrexate Anaerobes, streptococci, Reduced diversity and shifts in relative abundance Bacteroides associated with chemotherapy-induced diarrhoea55

TH, T helper; TLR, Toll-like receptor.

In line with these findings, a cocktail of Bacteroidales and Microbial enzymatic degradation and metabolism Burkholderiales ameliorates CTLA‑4‑blockade-induced Gastrointestinal toxicity in the form of mucositis, caus- subclinical colitis and colon inflammatory scores in ing diarrhoea, pain and weight loss, is a common adverse antibiotic-treated mice29. effect of chemotherapy, resulting in morbidity and Evidence is also emerging that the interface of innate mortality38. Moreover, it often leads to dose-limitation,­ immunity and the microbiota mediates chemo­therapy- which reduces the efficacy of anticancer treatment. induced toxicity. Toll-like receptors (TLRs) bind bac­ In addition to the multiple host pro-inflammatory and terial molecular patterns, and evidence from in vitro apoptotic pathways activated by chemotherapy, the gut IBD models­ suggests this interaction acts to regulate microbiota are central to the pathogenesis of mucositis39 inflammation and aids healing in the healthy colon34,35. and we draw readers’ attention to an excellent system- Frank and colleagues36 showed that mice depleted in gut atic review on this topic by Touchefeu and colleagues40. microorganisms after treatment with broad-spectrum­ However, there remains a paucity of effective interven- anti­biotics were more susceptible to methotrexate-­ tions to prevent gastrointestinal toxicity or mitigate induced small intestinal injury, but this effect was rescued against its symptoms41. by administration of a TLR2 ligand. Further experiments Irinotecan causes severe diarrhoea in up to 40% of pointed to myeloid-cell-expressed transmembrane patients, requiring dose reduction and, in many cases, p‑glycoprotein (p‑gp) as critical in this pathway. The premature termination of the drug42. The effect of findings were validated in vitro using human duodenal CPT‑11 on the intestinal microbiota composition has epithelial tissue. Conversely, knockout of Tlr4 in mice been explored in rats43. CPT‑11 increased abundance is associated with improvement in symptoms of toxicity­ of caecal Clostridium cluster XI and Enterobacteriaceae, from irinotecan (CPT‑11, a topoisomerase I poison),­ both of which are potentially pathogenic. However, despite modestly elevated levels of Proteobacteria, subsequent analysis of the beneficial effects of dietary which express ­β‑glucuronidases and, therefore, might fibres in this context suggests that dysbiotic changes increase reactivation­ of SN‑38 (the active metabolite might not be a major cause of CPT‑11 toxicity, point- of irinotecan)37. ing instead to the importance of microbial production

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of the short-chain fatty acid (SCFA) butyrate, which of an active gut microbiota capable of producing BVU correlates with genetic and phenotypic markers of produces an increase in systemic concentrations of 5‑FU, host health17. The implication is that functional, rather and this combination was associated with the death of than compositional, changes of the microbiota are of 16 patients in Japan50,51. greater importance. The enzymatic functions of specific target bacteria Irinotecan’s active metabolite, SN‑38, is converted also have a key role in modifying the toxic profile of some in the liver to an inactive glucuronide (SN‑38G), which chemotherapeutics. For example, Mycoplasma hyorhinis is excreted into the gut via the bile duct44. Wallace has been associated with a range of different cancers, and colleagues­ 45 showed that bacterial β‑glucuroni- including gastric carcinoma52. This bacterium encodes dases cleave the glucuronide moiety for use as a car- a thymidine phosphorylase that markedly restricts the bon source, releasing the active SN‑38 metabolite cytostatic activity of pyrimidine nucleoside analogue into the bowel lumen, generating diarrhoea. Bacterial compounds such as 5‑fluoro‑2ʹ‑deoxyuridine and ­β‑glucuronidases (or ­possible candidate structures) are 5‑trifluorothymidine in vitro53. However, the opposite found in 43% of species in The is true for the capecitabine metabolite 5‑fluoro‑5ʹ‑­ database. Furthermore, the bacterial enzyme contains deoxyuridine, which is more effective in the presence a ‘bacterial loop’ not found in the human form of the of M. hyorhinis infection, as this pro-drug is activated enzyme, enabling­ highly selective inhibitors of the bac- by the same enzyme. For the cancer drug gemcitabine, terial enzyme to be developed, two of which blocked Mycoplasma pyrimidine nucleoside phosphorylase and the active site of the E. coli ­β‑glucuronidase, but had no cytidine deaminase enzymes have deleterious effects on effect on bovine liver glucuronidase. The quinolone anti- its therapeutic efficacy54. This example illustrates a major biotic ciprofloxacin has also been shown to inhibit this challenge for the design of more personalized chemo- enzyme, and low doses of amoxapine, a known inhib­ therapeutic strategies based on microbiota modulation, itor of bacterial β‑glucuronidases, suppressed diarrhoea as the same strain has two contrasting influences on two associated with irinotecan in a rat model46,47. An analysis different targets based on the same enzyme. of crystal structures of representative β­ ‑glucuronidases from Streptococcus agalactiae and Clostridium perfrin- Reduced diversity and ecological variation gens and the Proteobacteria Escherichia coli and the Our understanding of novel interactions determining the Bacteroidetes Bacteroides fragilis has demonstrated that effects of the microbiota on chemotherapeutics must be these enzymes have marked differences in catalytic prop- further developed to account for reciprocal modification erties and propensities for inhibition, suggesting that the of the microbiota by these agents. Data from a study in gut microbiome is able to ensure functional diversity in rats treated with methotrexate showed that animals that orthologous enzymes. Small changes in the structure of developed mucositis also demonstrated a global reduc- designed inhibitors can induce major conformational tion in microbial abundance, with an absolute and rela­ changes in the β‑glucuronidase active site48. An alterna- tive decrease of anaerobes (13‑fold) and streptococci tive strategy to reduce irinotecan-related gastrointestinal (296‑fold) with relative increase of Bacteroides, and that toxicity, which, by contrast, requires active biotransfor- this change in microbiota composition was associated mation via bacterial β‑glucuronidase to exert its restor- with diarrhoea and reduced villous length55. The struc- ative effect on the intestinal epithelium, is provided by a ture of the human gut microbiota after treatment with Chinese herbal medicine (PHY906), which achieves this chemotherapy is also characterized by reductions in step through regeneration of intestinal stem cells and diversity and richness, with similar trends in the phyla potentiation of Wnt signalling49. affected; for instance, abundance of Proteobacteria gen- An interesting microbiota-driven drug–drug inter- erally increase56,57. Importantly, attempts are now being action with potentially serious consequences, including made to understand the functional effects of chemo- death, has also been seen with the antiviral drug sorivu­ therapy on the gut microbiota. Studying 28 patients with dine (1‑β‑D‑arabinofuranosyl‑5-(E)-(2‑bromovinyl) non-Hodgkin lymphoma undergoing a 5‑day myelo­ uracil), an antiviral that was sometimes co‑administered ablative chemotherapy regimen before human haemato- with 5‑fluorouracil (5‑FU). Gut bacterial phosphorolytic poietic stem cell transplantation (HSCT), all of whom enzymes hydrolyse sorivudine to (E)-5-(2‑bromovinyl) developed gastro­intestinal-mucositis-related symptoms, uracil (BVU), with high hydrolytic activity seen with Montassier and co‑workers58 observed profound alter­ the Bacteroides species, B. vulgatus, B. thetaiotaomicron, ations in gut microbial community structure and reduc- B. fragilis, B. uniformis and B. eggerthii. The effect of the tion in diversity. Proteobacteria abundance was increased, microbiota was demonstrated by the fact that admin- with ­levels of Firmicutes and Actinobacteria decreased. istration of ampicillin, , or a cocktail of Taxa known to diminish inflammation by modulation bacitracin, neomycin and streptomycin, reduced the of the NF‑κB pathway and through production of SCFAs exposure of rats to circulating BVU whereas concen- were depleted after chemotherapy. Enterobacteriaceae trations were increased after administration of the anti- abundance increased following chemotherapy, showing biotic kanamycin (which is selective for aerobes over similarities to the changes observed in an active colitis anaerobes). BVU inactivates hepatic dihydropyrimidine model59. Metabolic pathways associated with intestinal dehydrogenase, an enzyme that is responsible for the inflammation, including cell motility, glycan metabolism detoxification of 5‑FU. The co-administration of sorivu­ and xenobiotic degradation, were negatively correlated dine and 5‑FU (or pro-drugs thereof) in the presence with abundance of Firmicutes58.

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‘Pharmacomicrobiomics’ and cancer therapy could be targeted to influence therapeutic efficacy. But, The concept that bacteria or their products have a thera- for this vision to be delivered in practice, several critical peutic part to play in cancer is not novel. In 1891, Coley unmet needs must be overcome (FIG. 2). used the toxins from Streptococcus erysipelas and Bacillus prodigiosus (now referred to as Serratia maracens) to treat Phenome trials and systems oncology. The complexity of sarcoma60, and mycobacteria are still used in the treatment the gut microbiome means that novel approaches for stud- of bladder cancer61. Bacteria exert their chemo­therapeutic ying its role in drug efficacy are required, and consider­ effect by competing for nutrients, secreting toxins and ations must be given to the study of the interaction of eliciting host immune responses. However, we are now genes and the environment (phenomics). This complexity entering a new age in which the entire ecology of the gut necessitates a detailed functional map of the vast number

Personalized longitudinal microbiota modelling for precision chemotherapy: microbiome-enhanced trial

Diet/nutrition Synthetic bacteria Diet/nutrition Synthetic bacteria FMT Drug delivery FMT Drug delivery Antibiotics Diet/nutrition Antibiotics Diet/nutrition Prebiotics/probiotics FMT Prebiotics/probiotics FMT Antibiotics Antibiotics Prebiotics/probiotics Prebiotics/probiotics Clinical interventions

Microbiome sequencing (16S rRNA genes and metagenomics) and multi‘omics’ functional analysis Faeces Tissue Biofluids (e.g. urine)

Pre-treatment Neoadjuvant Surgery Adjuvant Cancer dysbiosis Treatment dysbiosis Surgery dysbiosis Treatment dysbiosis Cure

Local recurrence

Systemic recurrence Treatment journey Treatment

Death

• Prevention of • Maintenance of chemotherapy- • Prevention of microbiota associated toxicity • Complication chemotherapy • Improved symbiosis • Improved efficacy prevention • Improved efficacy gastrointestinal • Therapeutic risk of chemotherapy, (e.g. surgical of chemotherapy, function and QOL prediction radiotherapy and site infection) radiotherapy and • Prevention of • Therapeutic immunotherapy • Maintenance of immunotherapy disease progression Unmet need targeting • Prevention of drug major microbiome • Prevention of drug • Oncological • Drug discovery resistance functions resistance outcome prediction

Dynamic gut microbiota diversity and function

Figure 2 | A model for the future analysis and translation of the are providing notable insightsNature Reviewsinto the |mechanisms Gastroenterology through & Hepatology which the oncomicrobiome for improved cancer outcomes. The microbiota is microbiome modifies chemotherapy toxicity and efficacy. Given that highly personalized and niche specific. However, its structure and function treatment-induced dysbiosis probably affects the efficacy of second and is dynamic and it is influenced by all interventions along the modern third-line therapies, longitudinal studies will be critical in fully elucidating multimodal treatment journey. The journey for patients with cancer is the role of the microbiome in this field, and microbiota-augmented clinical subject to substantial inter-individual and intra-individual variation; trials in oncology are now urgently required. There are multiple tools for example, not all patients will undergo surgery. However, sampling through which the microbiota might be modulated for improved clinical methodologies can be standardized, and future microbiome studies should outcomes, which could be deployed at all stages of the treatment journey. seek to sample not only faeces, but tissue and multiple biofluids so that the However, evidence to support their use is variable, and trials for more broad systemic array of microbiome functions can be analysed once stored controversial interventions such as faecal microbiota transplantation (FMT) in biobanks. Metataxonomic (16S ribosomal RNA (rRNA) gene sequencing) are ongoing. However, the microbiota serves as a rich resource for and metagenomics (shotgun sequencing) approaches and other answering many of the unmet clinical needs in oncology and it represents ‘–omics’-based technologies (such as metabonomics and proteomics) a major new avenue in drug discovery in cancer. QOL, quality of life.

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of metabolic and immune functions that these bacteria fat and protein exert their carcinogenic effect through exert on the host, cancer and the drugs used to treat it. upregulated metabolism of choline, branched-chain Moreover, it is clear that many cancer therapies will not amino acids and bile salts and a reduction in SCFA only influence the metabolism of the host, but also that metabolism66. Although diet (for example, one high of the gut bacteria themselves, and the unintended con- in fibre and low in protein and fat) can be an effective sequences of this function remain unknown. This influ- method of chemoprevention for colorectal cancer, its ence is critical as modern chemotherapeutic regimens are role in the modification of chemotherapeutic efficacy commonly multimodal, and the potential for ­unintended remains poorly understood. This lack of knowledge is harm to be caused to patients through this route is sub- in part because the analysis of dietary modification of stantial. Longitudinal drug studies that target host– the gut microbiota during cancer remains challenging, microbiota interactions using a suite of metabonomic, not only because of variability in the adverse effects of metagenomic and metatranscriptomic approaches are chemotherapy (such as nausea and appetite suppres- required to provide meaningful insights into the mech- sion), but also because of nutritional interventions (for anisms of the microbiome, to identify novel drug targets example, enteral nutrition) and surgical diversion (stoma and to reduce their lead times into clinical use62. formation). However, animal data is emerging, and mice fed a diet high in protein, l‑leucine, fish oil and specific Gut microbiota as a biomarker for outcomes. Personal­ oligosaccharides demonstrated a reduced incidence and ized biomarkers are urgently required to help clinicians severity of Pseudomonas aeruginosa translocation during identify dysbiotic states associated with poor or deleteri- cyclophosphamide-induced immune suppression when ous outcomes from chemotherapy, and to identify micro- compared with an isoenergetic control diet67. bial targets suitable for modification. This aim is a major Nutritional modulation of cancer metabolism is also challenge, as these biomarkers must account not only for an emerging therapeutic approach, with fasting68,69, inter-individual variation, but also variation in the expres- carbohydrate restriction70, amino acid restriction sion of bacteria according to tumour stage and pheno­ (­l-asparaginase71, methionine72, serine73, tyrosine and type. For example, the mucosal abundance of strains of phenylalanine74) and ketogenic diets75 under investi- Fusobacterium nucleatum is associated with advanced gation. Some evidence suggests that fasting might also stage and poor outcome in patients with colorectal minimize the adverse effects of some chemotherapies; ­cancer63. The challenge is further exacerbated by system for example, it has been shown to reduce the vomit- complexity and co‑dependent functions of microbial net- ing associ­ated with doxorubicin treatment in dogs76. works and the very large portfolio of enzymatic functions Although these approaches have yet to be extensively carried by multiple different species, exemplified by the studied as diet–microbiota–chemotherapy interactions glucuronidase family. in cancer, data from human obesity and dietary studies However, evidence does suggest that the gut micro- clearly shows that the microbiota has a critical role in biota could act as a predictor of patient response to the modu­lation of cellular metabolism and the disease treatment and that this approach is feasible. Montassier pheno­type through both direct and indirect mech­anisms and colleagues64 found that a diverse gut microbiota is (for example, SCFA, branched-chain amino acid metab- associated with protection against chemotherapy-re- olism and bile acids). Thus, it is almost certain that the lated bloodstream infection. Interestingly, decreases in microbiota are modified by diet and that an altered oral diversity preceded the start of treatment and the authors intake during cancer treatment, in turn, is a critical medi- suggest a microbiota-based predictive risk index model, ator of chemotherapy efficacy. Intriguing evidence from which might be used to stratify patients at risk of com- experiments in FabplCre;Apc15lox/+ mice (an intestine-spe- plications before treatment. Correspondingly, Galloway- cific APC mutant model developing intestinal tumours) Pena et al.65 found that microbial diversity at baseline was treated with irinotecan support this hypothesis. Animals markedly lower in patients with acute myeloid leukaemia that fasted for 3 days were protected from the adverse who developed infectious complications after induction effects of irinotecan, although its efficacy was main- chemo­therapy than those who did not. Furthermore, a tained77. Although the influence of the microbiota was deleterious prognostic impact of reduced microbial diver- not studied in this cohort, it is highly likely that it had a sity over the course of treatment was sustained — patients major role, and more research is required. in this category were more likely to have an infection Specific food supplements have also been shown to be within 90 days after their neutrophil levels had recovered. modifiers of microbiota-driven chemotherapy tox­icity. Ginseng is a popular herbal medicine that is thought to Optimizing chemotherapy via the microbiota have anti-neoplastic therapeutic potential78. The proto­ Current tools designed to modify the gut microbiota panaxadiol group of ginsenosides are metabolized via remain fairly blunt, and almost all have been aimed at the enteric microbiota from compound K79. In vitro reducing the toxicity of chemotherapeutic agents rather studies suggest an enhanced effect of 5‑FU against than improving their efficacy. colorectal cancer cell lines in the presence of ginseng compounds80,81. Moreover, protopanaxadiol promotes Dietary interventions. Much work has been done to the anti-neoplastic effects of 5‑FU in a cell line model, determine the influence of dietary–microbiota inter- providing further evidence that the microbial metabo- actions on human health in IBD, obesity and in cancer lism of nutritional constituents modifies the efficacy of aetiology12. For example, pro-inflammatory diets high in chemotherapeutic targets.

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Ellagic acid, a product of dietary polyphenols found reduced compared with controls92. This finding implies in certain fruits, walnuts and wines, is metabolized by that there are multiple probiotic targets with applica- the gut microbiota to release urolithins82. Urolithin A tions in this field; however, there is an unmet need for is purported to have anti-proliferative effects in human improved methods for treatment stratification as not all colon cancer cells, mediating cell cycle arrest83, and studies have demonstrated clinical benefit. For exam- co‑treatment in vitro with urolithin A potentiates the ple, a combination of Enterococcus faecium M-74 with anticancer effects of 5‑FU and its pro-drug intermediate selenium did not prevent episodes of febrile neutro­ 5’-deoxy‑5‑fluorouridine84. Polysaccharides extracted penia in patients with leukaemia receiving induction or from the ink of the squid Ommastrephes bartramii consolidation chemotherapy93. also altered the intestinal microbiota composition after The use of prebiotics in conjunction with cytotoxic cyclophosphamide administration in mice85. Specifically, drugs (5‑FU, doxorubicin, vincristine, cyclophospha- it resulted in the enrichment of bifidobacteria and a mide, methotrexate and cytarabine) has been studied in reduction in the levels of Bacteroidetes, further suggest- mice94. Dietary supplementation with both oligofructose ing that nutritional modulation of the microbiota during and inulin potentiated the effect of all six drugs, increas- cancer therapy might protect against the adverse effects ing the lifespan of animals with the intraperitoneal form of chemotherapy. of a transplantable liver tumour95. Continuing this theme, an inulin–doxorubicin conjugate maintains therapeutic Probiotics, prebiotics and synbiotics. Good evidence response in vitro at lower doses than doxorubicin alone96. in both animal and human studies support that pro- biotics, prebiotics and synbiotics (preparations that Antibiotics, ecology and synthetic engineering. combine probiotics and prebiotics, which act synergis- Antibiotic therapy is broadly used as a prophylactic tically) have a role in the prevention of mucositis during approach for preventing sepsis during chemo­therapy. chemotherapy, and that they rarely cause sepsis86. Bowen However, almost 27% of bacteria causing infections and colleagues87 found that VSL#3, which contains a during chemotherapy in the USA are resistant to mixture of Streptococcus thermophiles, Bifidobacterium standard prophylactic antibiotics20, and the efficacy breve, B. longum, B. infantis, Lactobacillus paraca- of anti­biotic prophylaxis in cancer and especially dur- sei, L. delbrueckii subsp. Bulgaricus, L. acidophilus ing neutropenia is arguable. Importantly, antibiotic and L. plantarum, reduced diarrhoea and weight loss use in these immunocompromised patients is also an in irinotecan-treated rats, with associated increased unwanted cause of diarrhoea through the potentiation intestinal crypt proliferation and inhibition of apop- of Clostridium difficile97. Conversely, to date, no trials tosis. L. fermentum, L. rhamnosus and B. lactis did not have examined whether antibiotics might preferentially protect against intestinal mucositis in mice delivered improve chemotherapy efficacy, and such approaches ­intraperitoneal 5‑FU88. have largely been used as a method for experimental L. casei, L. rhamnosus and B. bifidum have all demon- modulation of microbiota–host drug interactions26,29. strated that they can attenuate chemotherapy-associated However, there are two interesting concepts that arise diarrhoea in a mouse model through the inhibition of from the clinical use of antibiotics in cancer. Firstly, as it Tnf, Il1b and Il6 mRNA expression89. In a randomized is possible that given bacteria might transmit antibiotic study of 150 patients with colorectal cancer receiving resistance through horizontal gene transfer98, it is equally 5‑FU‑based chemotherapy, L. rhamnosus GG supple- possible that chemo­therapeutic resistance might be trans- mentation also reduced episodes of severe diarrhoea and mitted in a similar fashion, for example, through the abdominal discomfort, compared with guar gum fibre90. transfer of ­β‑glucuronidase or pyrimidine phosphatase Motoori and co‑workers91 performed a randomized functions. Bacteria modify chemoresistance through a study comparing a synbiotic containing 1 × 108 living large number of possible pathways, such as through B. breve strain Yakult and 1 × 108 L. casei strain Shirota lipopolysaccharide-­modulated upregulation of TLR4 per gramme with a control substance containing 1 × 109 receptors99 and, therefore, this finding suggests that the Streptococcus faecalis during neoadjuvant chemotherapy microbiota is a legitimate target­ for the treatment of treatment for patients with oesophageal cancer. Levels of patients with metastatic disease. Secondly, it also sug- Bifidobacterium and Lactobacillus species decreased in gests that there is a role for selective antibiotic use, and the control group compared with the treatment group that retired compounds (no longer in routine clinical after chemotherapy, and concentrations of SCFAs were use) can be repurposed for engineering the microbiota. higher in the latter. Modest improvements in rates of The precedent has been set with amoxapine, a tetra­cyclic severe diarrhoea and febrile neutropenia were observed antidepressant, which was re‑purposed as a selective in the treatment group, but the majority of clinical ­β‑glucuronidase inhibitor for alleviating chemotoxicity47. parameters, including response to chemotherapy, showed A different approach for manipulation of the micro­ no difference between groups. The primary endpoint of biota is to take advantage of ecological engineering, which the study was not stated. is the synthetic manipulation of ecology and ecosystems A small placebo-controlled trial of B. breve strain to deliver a desired change or function. One such drastic Yakult in children undergoing chemotherapy for a method for modification of the ecology of microbiota is variety of malignancies also demonstrated that the faecal microbiota transplantation100. This approach has treatment group experienced fewer episodes of fever yet to be used for the purpose of modulation of chemo- and the frequency of intravenous antibiotic use was therapy, although it is probably only a matter of time as

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data from C. difficile studies suggest FMT might have a or in combination with 5‑FU, was orally administered potentially valuable role101. A note of caution arises from to a syngeneic mouse transplantation model of hepatic disappointing phase II trial results of Seres’s stool-derived colorectal metastases, tumour activity was markedly bacterial spore treatment to prevent recurrent C. difficile reduced and a survival benefit was obtained. This exam- infection102. Moreover, challenges remain in regulation103, ple shows that synthetic engineering now could have a and extra care will be needed in the oncological setting, for role in the optimization of standard chemotherapeutic which the consequences for cancer survival of ­re‑setting approaches in cancer. the microbiota during treatment are not known. Another approach is the engineering of target bac- Conclusions teria to deliver drugs or pro-drugs within target organs Mechanistic evidence is now available to support the and to serve as vectors for gene therapy. The production hypothesis that the gut microbiota plays a major part in of the tumour suppressor bone morphogenetic protein 2 defining both the efficacy and toxicity of chemothera­ by transgenic bacteria is one such example104. Spores of peutic agents. Biological complexity remains a major anaerobic bacteria can also be used for this purpose, barrier to the full elucidation of the multiple pathways as they will reach oxygen-depleted regions within cancers­ through which host–microbial interactions modulate and will germinate. Din and colleagues105 demonstrated clinical outcomes; however, systems medicine prov­ an elegant example of how synthetic engineering of ides a tangible methodology for mapping these host bacteria can be used to improve the efficacy of chemo- oncological–microbiota­ interactions. The implication is therapeutic drugs. They engineered a synchronized lysis that the gut microbiome represents a notable target for circuit based on the quorum sensing feedback loops making chemotherapy safer and for further improving in E. coli, so that the bacteria would lyse at a threshold rates of cancer survival. Clinicians and translational population density to release genetically encoded cargo. scien­tists are likely to make a major contribution to this After quorum lysis, a small number of surviving bac- work and will be integral to delivering future trials that teria reseed the growing population, therefore, leading are essential if the potential of the gut microbiota in this to pulsatile delivery cycles. When the lysis strain, alone field is to be realized.

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