Growing evidence supports the importance of gut microbiota in the control of tumor growth and response to therapy. Here, we select prebiotics that can enrich bacterial taxa that promote anti-tumor immunity. Addition of the prebiotics inulin or mucin to the diet of C57BL/6 mice induces anti-tumor immune responses and inhibition of BRAF mutant melanoma growth in a subcutaneously implanted syngeneic mouse model. Mucin fails to inhibit tumor growth in germ-free mice, indicating that the gut microbiota is required for the activation of the anti-tumor immune response. Inulin and mucin drive distinct changes in the microbiota, as inulin, but not mucin, limits tumor growth in syngeneic mouse models of colon cancer and NRAS mutant melanoma and enhances the efficacy of a MEK inhibitor against melanoma while delaying the emergence of drug resistance. We highlight the importance of gut microbiota in anti-tumor immunity and the potential therapeutic role for prebiotics in this process.

The prebiotics inulin and mucin have been reported to induce Bifidobacterium spp. and Akkermansia muciniphila (). Cultivation of fecal samples with inulin and mucin increased the relative abundance of several species implicated in tumor growth control in the Rnf5mice (). Therefore, in this study, we investigated the effects of inulin and porcine gastric mucin on growth of a subcutaneously implanted tumor and its propensity to resist targeted therapy in syngeneic mouse models. We demonstrate the ability of these prebiotics to elicit changes in gut microbiota composition that play a pivotal role in eliciting effective anti-tumor immunity.

In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota.

Previous reports indicated that the prebiotic inulin increases the relative abundance of Bifidobacteria, Bacteroides, and Akkermansia muciniphila in mice (). Among these, Akkermansia muciniphila is known to reside in the mucin layer of the GI tract, in which it consumes glycan substrates decorating mucin proteins (muc2). Our earlier studies identified microbiota-dependent anti-melanoma immunity in syngeneic Rnf5mice, which was conferred by select bacterial strains that induced tumor infiltration by T cells and dendritic cells (DCs) and inhibited melanoma growth upon their inoculation in GF mice ().

In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota.

Despite their clinical efficacy, checkpoint inhibitors are effective in only a fraction of treated patients. Human fecal microbiota derived from therapy-responsive patients confer treatment responsiveness when transplanted into germ-free (GF) mice (), while a small set of phylogenetically unrelated gut microbiota species was suggested to promote anti-tumor phenotypes. For example, introduction of Bacteroides thetaiotaomicron or Bacteroides fragili to GF mice was sufficient to restore anti-tumor responses via induction of a skewed T1 response (). Our recent study demonstrated that Bacteroides rodentium induced anti-tumor immunity in melanoma and colon cancer models that were subcutaneously implanted in syngeneic C57BL/6 mice (). In another study, the abundance of Akkermansia muciniphila was associated with anti-PD-1 responsiveness in humans and restored an anti-tumor phenotype when co-administered with anti-PD-1 therapy to melanoma patients (). Administration of Bifidobacterium spp. in combination with anti-PD-L1 agents, attenuated tumor growth and promoted anti-tumor immunity in a syngeneic mouse model (). Moreover, human melanoma patients who responded to anti-CTLA-4 (ipilimumab) were found to have gut microbiota enriched in three butyrate-producing bacterial species (), and administration of Enterococcus hirae and Barnesiella intestinihominis improved their response to cyclophosphamide chemotherapy (). An assessment of mice treated with various antibiotics revealed that ampicillin-treated mice retained a simplified microbiota with a potent anti-tumor phenotype (). Isolates from these mice identified 11 strains, enriched in Bacteroides, Parabacteroides, Alistipes, and an uncharacterized Ruminococcaceae, that increased the abundance of CD8interferon-γ (IFN-γ)-producing cells in the gut and potentiated anti-PD-1- and anti-CTLA-4-mediated control of tumor growth. Collectively, these findings point to the importance of gut microbiota in controlling cancer growth and reveal the complex variety of species that can promote anti-tumor immunity.

The gastrointestinal (GI) tract harbors a complex and dynamic population of bacteria, called gut microbiota, that are implicated in the maintenance of health and the onset and progression of disease (). In these roles, gut microbiota affect key components of host physiology and homeostasis, including the development and function of the immune system (). Changes in gut microbiota composition are linked to local and systemic alterations that affect tumor growth, in part through modulation of tissue remodeling, mucosal immunity, and anti-tumor immunity (). Gut microbiota also influence the incidence and progression of colorectal carcinoma () and breast and hepatocellular carcinoma (). The importance of gut microbiota composition in cancer () has been further demonstrated in studies showing the ability of the microbiota to enhance responses to checkpoint inhibitors such as anti-PD-(L)1 antibodies () and anti-CTLA-4 antibodies (). Furthermore, bacterial commensals that were found to be more abundant in the gut of melanoma patients responding to anti-PD-1 therapy (), provided a rationale for performing fecal microbiota transplantation to non-responding patients.

Melanoma remains one of the most aggressive tumor types, mainly because of its propensity to metastasize and resist therapy. Aberrant activation of the mitogen-activated protein kinase (MAPK) pathway has been reported in human BRAF and NRAS mutant tumors, including melanomas, in which they account for more than 70% of genetic changes. Although selective inhibitors to BRAF mutant proteins have been developed, their effectiveness is limited by the frequent emergence of resistance (). Inhibitors of the MAPK pathway, including MEK, have also been developed and are commonly used for the treatment of NRAS mutant melanomas (). The emergence of immune checkpoint therapy has resulted in unprecedented clinical success and offered new therapeutic modalities (). At present, BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) are being tested in several clinical trials, in combination with other therapies, including immune checkpoint inhibitors and gut microbiota modulators ().

To determine whether mucin and inulin in combination have additive or synergistic effects on melanoma growth, we employed two models. In the first, the growth of syngeneic SW1 NRAS mutant melanoma tumors that were subcutaneously transplanted in the syngeneic C3H/HeOuJ mice was attenuated by mucin treatment alone, a response that was enhanced by co-administration of mucin and inulin ( Figure 7 C). In contrast, growth of YUMM1.5 BRAF mutant melanoma tumors that were subcutaneously transplanted in the syngeneic C57BL/6 mice was reduced by treatment with either mucin or inulin alone, but they did not have an additive effect ( Figure S6 ). These findings suggest that tumor genotypes and/or mouse strains affect the ability of prebiotics to attenuate tumor growth, further illustrating the complexity of the mechanisms by which gut microbiota impacts the anti-tumor phenotype.

We next asked whether prebiotic treatment can enhance the efficacy of anti-PD-1 antibody, a commonly used immune checkpoint therapy. YUMM1.5, the BRAF mutant mouse melanoma cell line, is considered a cold tumor, that is, poorly responsive to immune checkpoint therapy (). Administration of anti-PD-1 or the prebiotics reduced the growth of YUMM1.5 tumors in C57BL/6 mice. However, combination treatment with anti-PD-1 plus either inulin or mucin did not further attenuate tumor growth ( Figures 7 A and 7B ), implying that anti-PD-1 and prebiotic therapy may elicit similar or overlapping changes in the immune system.

Data are representative of two independent experiments. Graphs show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA with Bonferroni’s correction.

(C) Growth of SW1 mouse melanoma cells in C3H/HeOuJ mice that received a control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation (control, n = 8; mucin, n = 8; inulin, n = 7; mucin+inulin, n = 9).

(B) Growth of YUMM1.5 tumors in C57BL/6 mice that received 0% or 3% mucin in drinking water starting 14 days before tumor inoculation (control, n = 7; mucin, n = 7; PD-1, n = 10; PD-1+mucin, n = 9). Mice were injected with antibodies as described in (A).

(A) Growth of YUMM1.5 tumors that were subcutaneously transplanted in syngeneic C57BL/6 mice that were fed control chow or chow supplemented with 15% inulin starting 14 days before tumor inoculation (control, n = 7; inulin, n = 7; PD-1, n = 10; PD-1+inulin, n = 9). Mice were injected with control IgG or anti-PD-1 blocking antibody on days 7, 10, 13, and 16 after tumor inoculation.

We next determined the effect of mucin and inulin on gene expression by intestinal epithelial cells (IECs), which have been implicated in the activation of DCs and T cells in vivo (). After treatment of WT C57BL/6 with inulin or mucin for 2 weeks, IECs were isolated from the small intestine and were examined for expression of immune-related cytokines and chemokines. IECs from inulin- or mucin-treated mice exhibited enhanced expression of select genes; thus, although tumor necrosis factor alpha (TNF-α) mRNA levels were elevated in IECs from mucin-treated mice, NOD2, IL-6, and CXCL2 mRNA were increased in IECs from inulin-treated mice ( Figure S5 E). These findings suggest that inulin and mucin differentially enhance the transcription of key immune-activating cytokines and chemokines in IECs, providing a potential mechanism by which alterations of the gut microbiota may elicit anti-tumor immunity.

To further explore the mechanism of action of mucin and inulin on the anti-tumor immune response, we treated murine bone marrow-derived DCs (BMDCs) for 24 h in vitro with mucin or inulin (0.05 or 0.5 mg/mL). As shown in Figure S5 C, expression of the DC activation markers CD40 and CD80, as well as MHC class I and MHC class II, was increased by mucin treatment, but not inulin treatment. In contrast, in vitro treatment of CD8T cells isolated from the spleens of C57BL/6 mice revealed that whereas mucin had a limited effect on T cell activation ( Figure S5 D), inulin treatment increased the production of cytokines, chemokines, and the cytotoxic effector protein granzyme B ( Figure S5 D). These results suggest that inulin and mucin differentially affect the expression of genes involved in activation of antigen-presenting and/or effector functions of DCs and T cells. Given that mucin failed to elicit anti-tumor immunity and tumor growth inhibition in GF mice, we speculate that the effects of mucin and inulin detected in vitro would likely be secondary to the effects of prebiotics on the microbiota in vivo ( Figure S5 B).

To verify the dependency of prebiotic-induced tumor control on gut microbiota, a minimal microbiota (altered Schaedler flora [ASF]) was allowed to colonize GF C3H/HeN mice for 2 weeks to induce immune maturation, and then the mice were initiated on mucin treatment for 2 weeks before injection of subcutaneously transplanted N-Ras mutant SW1 tumor cells. In these mice, mucin treatment failed to attenuate SW1 tumor growth ( Figure S5 B), indicating that mucin-promoted tumor growth control depends on specific gut microbiota. These observations are consistent with our previous demonstration that transfer of feces from Rnf5mice, which harbor microbiota that can limit tumor growth, to WT GF mice elicited an effective anti-melanoma response ().

Among the taxa enriched in gut microbiota of prebiotic-fed mice that control tumor growth are many that mapped in or near Clostridium cluster XIVa, which includes butyrate-producing species. We therefore next tested the effect on tumor growth of individual or combinations of the short-chain fatty acids (SCFAs) butyrate, propionate, and acetate (150 mM alone or 50 mM each in combination) administered in the drinking water starting 2 weeks before melanoma cell injection and for the duration of the experiment thereafter. None of the SCFAs, alone or in combination, reduced the tumor burden ( Figure S5 A), suggesting that production of SCFAs alone is insufficient to affect tumor growth.

Studies of microbiota-mediated control of tumor growth have largely focused on a single or small group of bacterial species and/or species that are over-represented in mice or humans and are positively associated with control of tumor growth. However, in the present study, we detected negative correlations between tumor size and the abundance of multiple phylogenetic clades ( Figure 6 ), which has been difficult to reconcile with earlier studies. Some bacterial strains identified here have not previously been described to be associated with anti-tumor immunity. In addition to Bifidobacteria, Bacteroides, Barnesiella, and Parabacteroides (), which have been implicated in anti-tumor responses, we identified Olsenella, Prevotellamassilia, and Culturomica as additional taxa whose abundance correlates with tumor size. Six members of the Firmicutes phylum, including taxa mapping in or near Clostridium cluster XIVa, were also associated with tumor growth inhibition ( Figure 6 ).

Cladogram representation of taxa enriched in fecal microbiota of mice (control, n = 12; mucin, n = 15; inulin, n = 15) administered mucin (red) or inulin (blue). Data are representative of two independent experiments.

MEKi co-administration altered the relative abundance of 21 phylotype groups (5 increased) in inulin-treated mice and of 15 phylotype groups (6 increased) in mucin-treated mice at sacrifice ( Table S3 ). Analysis of the phylotypes in inulin+MEKi-treated mice compared with inulin-treated mice revealed four groups that negatively correlated with tumor size, based on relative abundance at sacrifice ( Figure 5 F). Of these, Akkermansia muciniphila was robustly enriched, together with Actinobacteria, Bifidobacterium longum, Olsenella profusa, and Parvibacter caecicola. Akkermansia muciniphila has previously been demonstrated to possess anti-tumor properties (). Because Akkermansia muciniphila was also induced in mucin+MEKi-treated mice, enrichment of this phylotype may be insufficient to control MaN-RAS tumor growth. Rather, interactions between other inulin-induced taxa may be required for Akkermansia muciniphila to promote the anti-tumor phenotype. The four phylotype groups that were enriched in mucin+MEKi-treated mice compared with mucin-treated mice, and that negatively correlated with tumor size, may be involved in mucin-promoted control of tumor growth ( Figure 5 G).

Mice treated with doses of MEKi alone that effectively controlled subcutaneously implanted MaN-RAS1 tumor growth in syngeneic C57BL/6 mice showed enrichment of 14 phylotype groups in the gut microbiota, none of which negatively correlated with tumor size ( Table S3 ). Analysis of taxa in tumor-bearing mice fed inulin identified eight phylotype groups enriched in Actinobacteria, Bifidobacterium longum, and two Olsenella spp. ( Table S3 ). Only one phylotype group mapping distantly to Clostridium cellobioparum was negatively correlated with tumor size. Mucin treatment alone resulted in an increase in the relative abundance of 56 phylotype groups featuring diversity of taxa, including Bacteroides, Parabacteroides, Olsenella, and Clostridium. Mucin uniquely increased the relative abundance of five Lactobacillus spp., all of which were positively correlated with tumor size, albeit not to the level of statistical significance. Mucin feeding may thus induce an imbalance between enrichment of phylotypes that correlated positively versus negatively with tumor size, resulting in a failure to control tumor growth ( Table S3 ).

Inulin and mucin treatment before MEKi injection increased the relative abundance of 39 and 23 phylotype groups, respectively ( Figures 5 D, S4 B, and S4C), and the abundance of these groups was negatively correlated with tumor size ( Figures S4 B and S4C). Both inulin and mucin primarily increased the relative abundance of taxa mapping in or near Clostridium cluster XIVa ( Figures 5 D and 5E). Inulin specifically induced six phylotypes related to Bacteroides spp. (primarily Bacteroides acidifaciens), three phylotypes related to Barnesiella spp., and a group related to Parasutterella excrementihominis, the latter of which was not detected following mucin treatment. Inulin also increased the relative abundance of three phylotype groups related to Bifidobacterium spp., whereas only one group was induced by mucin. The genomes of Bacteroides, Bifidobacterium, and Barnesiella spp. encode numerous glycosyl hydrolase activities ( http://www.cazy.org/ ) that support cross-feeding interactions with sugar-fermenting bacteria, particularly Clostridiales spp.

We next determined whether prebiotic supplementation influenced the efficacy of MEKi treatment by examining growth of the N-RAS mutant mouse melanoma cell line MaN-RAS that was obtained from genetically engineered N-Ras melanoma grown in C57BL/6 mice. Syngeneic mice (C57BL/6) were subcutaneously inoculated with tumor cells 2 weeks after the initiation of feeding with inulin or mucin with or without MEKi. In the absence of MEKi, inulin, but not mucin, modestly controlled tumor growth ( Figure 5 A). However, co-administration of MEKi with inulin had an additive effect on tumor growth control, and the emergence of MEKi resistance was delayed compared with MEKi alone ( Figure 5 A), implying that MEKi resistance may be partially overcome by this prebiotic. Consistent with these findings, tumors from mice treated with inulin+MEKi contained elevated numbers of total CD45cells, CD4and CD8T cells, pDCs, and mDCs, and the DCs expressed elevated levels of MHC class I compared with mice treated with MEKi alone ( Figures 5 B and 5C). However, no differences in T cell-mediated cytokine production were detected in tumors from inulin+MEKi-treated mice ( Figure S4 A).

Data are representative of two independent experiments. Graphs show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA with Tukey’s correction (B and C) or by two-way ANOVA with Bonferroni’s correction (A).

(C) Number of tumor-infiltrating DCs and DC subsets per tumor weight (in grams) and expression of MHC class I on DCs in mice treated as in (A) (MEKi, n = 8; MEKi+mucin, n = 7; MEKi+inulin, n = 8).

(B) Number of tumor-infiltrating effector (CD44 hi ) CD4 + and CD8 + T cells and total CD45 + cells per tumor weight (in grams) in mice treated as in (A) (MEKi, n = 7; MEKi+mucin, n = 8; MEKi+inulin, n = 8).

(A) Growth of NRAS Q61K mouse melanoma cells (1 × 10 6 ) (control, n = 11; mucin, n = 9; inulin, n = 9; MEKi, n = 10; MEKi+mucin, n = 8; MEKi+inulin, n = 10) that were subcutaneously transplanted in syngeneic C57BL/6 mice that received control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation. When tumors reached a volume of 10–20 mm 2 , mice were administered MEKi (PD325901, 10 mg/kg) once daily by gavage. Tumor volume was assessed every 4 days.

Analysis of fecal microbiota from MC-38 tumor-bearing inulin- or mucin-treated mice indicated that both prebiotics increased the relative abundance of a similar number of phylotype groups (25 inulin and 21 mucin), of which 7 were common to both prebiotics ( Table S2 ). More than 68% of the phylotype groups induced by inulin mapped to Clostridium cluster XIVa, compared with 33% induced by mucin. Additional analysis demonstrated that the relative abundance of six inulin-specific phylotypes was inversely correlated with MC-38 tumor size ( Figure 4 C), whereas no relationship with tumor size was detected for the phylotype groups induced by mucin.

We next assessed whether mucin or inulin supplementation affects the growth of tumor types other than melanoma. Using the same protocol (prebiotic feeding starting 2 weeks before tumor inoculation), we found that inulin, but not mucin, attenuated the growth of subcutaneously transplanted MC-38 colon cancer tumors in syngeneic C57BL/6 mice ( Figure 4 A) and enhanced the anti-tumor immune response, as reflected by increased MHC class I and MHC class II expression on DCs ( Figure 4 B). No differences in the abundance of CD4or CD8T cells, total CD45cells, DCs, or DC subsets or the production of cytokines were observed in tumors from mucin- or inulin-treated mice ( Figure S3 ). Although both mucin and inulin induced anti-tumor immunity that limited the growth of melanoma tumors, only inulin was able to attenuate colon cancer growth. Thus, the differences in taxa induced by these prebiotics may account for their distinct effects on control of select tumor types.

Data are representative of two independent experiments. Graphs show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA with Bonferroni’s correction (B) or by two-way ANOVA with Tukey’s correction (A).

(C) Boxplot of the relative abundance of the taxa enriched in inulin-treated mice and positively correlated with tumor size (n = 10).

Sequencing of the amplified 16S V3-V4 region followed by computational analysis led to the identification of increased relative abundance of 17 phylotype groups in inulin-treated mice and 2 phylotype groups in the mucin-treated mice that were not present in control mice ( Table S1 ). Inulin increased the relative abundance of taxa that were phylogenetically coherent, 66% of which mapped most closely to members of Clostridium cluster XIVa, primarily Clostridium populeti- and Clostridium saccharolyticum-related taxa ( Table S1 ). Although this cluster consists of numerous butyrate producers, the phylogenetic distance of the phylotypes profiled here makes the butyrate-producing potential of these taxa uncertain. Among the phylotypes that displayed increased relative abundance following inulin treatment, six were negatively correlated with tumor size ( Figure 3 C). Mucin also predominantly enriched taxa with similarity to members of Clostridium cluster XIVa ( Table S1 ); however, none of the phylotypes induced by mucin were negatively correlated with tumor size. These findings suggest that inulin and mucin drive distinct changes in gut microbiota, both of which are capable of inducing anti-tumor immunity.

We next used 16S rRNA amplicon sequencing to profile the fecal microbiota of WT C57BL/6 mice before and 14 days after prebiotic feeding and 20 days after YUMM1.5 tumor cell inoculation, with tumor cells originally obtained from genetically engineered C57BL/6 mice harboring Brafmutation, Pten deletion, and Cdkn2a deletion. Although the bacterial communities at baseline were heterogeneous and generally not well clustered ( Figures 3 A and 3B ), prebiotic feeding resulted in the formation of more highly related communities that were distinct from those in control mice. Furthermore, the bacterial communities underwent additional restructuring following tumor cell inoculation. These data are consistent with recent observations that distal tumor growth results in a reconfiguration of gut microbiota (). Individual phylotype groups (two or more highly related, but not identical, 16S sequences of strains approximating a species) that were altered in the microbiota of control and prebiotic-treated mice were identified but were not further assessed. Thus, only phylotypes that were associated with a specific prebiotic treatment were further studied.

(C) Time course of the relative abundance of the six taxa enriched in inulin-treated mice that negatively correlate with YUMM1.5 tumor size (control, n = 12; inulin, n = 15). Time points are before inulin treatment, before tumor injection, and before tumor collection.

(A and B) Principal-component analysis of all taxa enumerated in fecal microbiota of control and mucin-treated (A) or inulin-treated (B) C57BL/6 mice, examined before prebiotic treatment, before subcutaneous injection of syngeneic YUMM1.5 tumor cells, and before tumor collection (control, n = 12; mucin, n = 15; inulin, n = 15).

Consistent with the elevated abundance of TILs in mucin-treated compared with control C57BL/6 mice, levels of the cytokine interleukin (IL)-1α and chemokine CXCL13 were increased in the sera of mucin-fed mice before tumor cell inoculation ( Figure 2 C), suggesting that the inflammation-promoting effects of mucin were systemic. Strikingly, we found that mucin-treated tumor-xenograft-bearing mice exhibited reduced serum levels of IL-6, IL-1α, IL-10, IL-17A, and IL-23 compared with control animals ( Figure 2 D). High serum levels of IL-6 and IL-17 were previously associated with poor clinical outcome (), whereas reduced IL-1α levels were associated with attenuated tumor growth (). Lower serum levels of the chemokines CXCL1 and CXCL13 were also found in mucin-treated mice compared with control mice ( Figure S2 B), linking levels of both chemokines and cytokines with the anti-tumor response.

Independent support for the activation of T cells in prebiotic-treated mice was obtained using the OVA-specific OT-I transgenic mouse model. WT OT-I CD8CD45.1T cells were transferred to untreated or mucin-treated WT mice harboring B16F10-OVA melanoma tumors, and their frequency in tumor-draining and non-draining lymph nodes was monitored. OT-I CD8T cells were more abundant in the draining lymph nodes of mucin-treated mice compared with control mice ( Figures 2 B and S2 A), pointing to either increased recruitment or increased survival of OT-1 cells in the prebiotic-treated mice. Altogether, these results confirm that prebiotic treatment promotes anti-tumor immunity via effects on both innate and adaptive immune cells.

To identify possible mechanisms for the elevated immune cell infiltration and anti-tumor immunity in prebiotic-treated mice, we examined tumor-xenograft samples grown in syngeneic mouse models for changes in the transcription of immune-associated genes (including chemokines), inflammasome activity, and antigen presentation. Both prebiotics increased the expression of chemokines (CCL4 and CCL8), inflammasome-related genes (TLR3 and TLR7), and antigen presentation-related genes (CD40, Stat1, and ICOS) ( Figure 2 A), suggesting a mechanism by which prebiotic supplementation enhanced the recruitment and activation of immune cells in the tumor microenvironment.

Data are representative of three independent experiments (A and B) or one experiment (C and D). Graphs show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA with Tukey’s correction (A) or by two-tailed t test or Mann-Whitney U test (B–D).

(B) Quantification of CD45.1 + OT-I CD8 + T cells in the tumor-draining lymph nodes (TdLN) and non-draining lymph nodes (ndLN) of C57BL/6 mice (CD45.2 + ) treated with or without mucin and injected with B16-OVA melanoma cells (TdLN, n = 7; ndLN, n = 8). Right dot plots show gating of CD45.1 + CD8 + cells.

(A) qPCR analysis of immune-related genes in subcutaneously transplanted melanoma grown in syngeneic C57BL/6 mice that received a control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation (n = 6).

Inulin is a naturally occurring fructosyl polymer with chain-terminating glucosyl residues. Mucins are highly decorated with polysaccharides composed of various core structures similar to those found in Lewis blood type antigens, including the sugars galactose, fucose, sialic acid, galactosamine, glucosamine, and mannose. To determine whether prebiotics inhibit tumor growth, mucin (3% in drinking water) or inulin (15% w/w in chow) were administered to wild-type (WT) C57BL/6 mice starting 2 weeks before subcutaneous injection of syngeneic melanoma tumor cells (YUMM1.5 cells, 1 × 10cells/mouse) through the remainder of the experiment. Administration of mucin or inulin led to attenuated melanoma tumor growth ( Figure 1 A). To determine whether these changes could be attributed to anti-tumor immunity, we analyzed tumor-infiltrating lymphocytes (TILs) 20 days following tumor inoculation. Compared with control mice, tumors from mucin- or inulin-treated mice were enriched in CD45cells, including effector (CD44) CD4and CD8T cells (e.g., IFN-γ-producing CD4T cells), plasmacytoid DCs, and conventional CD8αDCs ( Figures 1 B–1D). Tumor-resident DCs isolated from inulin- or mucin-treated C57BL/6 mice expressed higher levels of major histocompatibility complex (MHC) class I and MHC class II ( Figure 1 E), implying greater stimulatory capacity, compared with the tumor-associated cells from control mice. These data indicate that prebiotic supplementation induced a shift to a proinflammatory tumor microenvironment associated with a more potent anti-tumor response. The immunomodulatory effects of the two prebiotics were largely overlapping, but not identical, yet had similar effects on tumor control. Antibody-mediated depletion of CD4and CD8T cells in inulin-treated mice ( Figure 1 F) reduced the suppression of tumor growth, pointing to an essential role for CD4and CD8T cells in the inulin-promoted anti-tumor phenotype.

Data are representative of three independent experiments (A–E) or one experiment (F). Graphs show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA with Bonferroni’s correction (A and F) or by one-way ANOVA with Bonferroni's correction (B–E).

(F) Wild-type C57BL/6 mice (n = 12) were fed control or 15% inulin-supplemented chow starting 14 days before subcutaneous (s.c.) injection of YUMM1.5 melanoma cells (1 × 10 6 ). Anti-mouse Thy1.2 or control immunoglobulin G (IgG, 400 μg) were injected two times a week starting 3 days after tumor inoculation (n = 12). Tumor volume was assessed two times a week. FACS analysis revealed >90% depletion of blood CD4 + and CD8 + T cells on day 8 after tumor inoculation.

(B) Quantification of tumor-infiltrating total CD45 + cells and effector (CD44 hi ) CD4 + or CD8 + T cells from mice treated as in (A) (control, n = 9; mucin, n = 10; inulin, n = 10).

(A) Growth of YUMM1.5 tumors that were subcutaneously transplanted in syngeneic C57BL/6 mice. Mice were provided with a control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation (control, n = 12; mucin, n = 15; inulin, n = 15).

Inulin and mucin have been shown to alter the relative abundance of bacterial species that were identified as anti-tumor immunity-promoting bacteria in Rnf5mice, including Parasutterella excrementihominis, Bacteroides rodentium, and Clostridium viride ( Figures S1 A and S1B). We thus tested the effect of inulin and mucin on anaerobic cultivation of 12 fecal samples derived from healthy human subjects. Both prebiotics increased the relative abundance of Bacteroides spp., whereas only mucin increased the relative abundance of Akkermansia muciniphila in most cultures ( Figure S1 C). Surprisingly, inulin, but not mucin, promoted the growth of Bifidobacteria spp. in only two of the cultures.

Discussion

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et al. Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab. Among the phylotypes induced by inulin and mucin are taxa in the butyrate-producing Clostridium cluster XIVa. This result is consistent with an earlier report demonstrating that over-representation of three butyrate-producing taxa was strongly associated with responsiveness to ipilimumab in human melanoma patients (). However, we found that butyrate, propionate, and acetate, alone or in combination, had no effect on melanoma growth, suggesting that production of other SCFAs or microbial products may mediate this microbiota-driven anti-tumor response.

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et al. BRAF and MEK Inhibitors Increase PD-1-Positive Melanoma Cells Leading to a Potential Lymphocyte-Independent Synergism with Anti-PD-1 Antibody. Yue et al., 2018 Yue P.

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et al. Agonist immunotherapy restores T cell function following MEK inhibition improving efficacy in breast cancer. Vella et al., 2014 Vella L.J.

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et al. SHP2 Inhibition Prevents Adaptive Resistance to MEK Inhibitors in Multiple Cancer Models. Increasing attention is being paid to the effects of MEKi, which target a signaling pathway often deregulated in tumors, on immune system components (). MEKi has been shown to impede the growth of tumors in mice while concomitantly promoting the effector phenotype and longevity of tumor-infiltrating CD8T cells (). Furthermore, MEKi can promote tumor immunogenicity in preclinical models of triple-negative breast cancer, in which MEKi in combination with either anti-4-1BB or anti-OX-40 agonist antibodies resulted in superior therapeutic efficacy (). MEKi is also capable of promoting the maturation of DCs, as reflected by enhanced antigen uptake, processing, and cross-presentation to T cells (). Despite effective tumor inhibition by MEKi, resistance to these inhibitors invariably occurs (). Here, we demonstrated that co-administration of inulin reduces the resistance of melanoma to MEKi, pointing to the possible consideration of prebiotics as a means to limit therapy resistance, which remains a crucial unmet clinical need.

Collectively, the results of this study advances our understanding of tumor growth control by gut microbiota, demonstrating that taxa from multiple unrelated phylogenetic groups share the capacity to induce anti-tumor immunity. It is expected that refinement of the specific bacterial strains and metabolites that mediate these phenotypes will not only advance our understanding of the phenomenon but also facilitate the development of therapeutic modalities that could be tested across species.