Effect of microbiome group on immune checkpoint inhibitors in treatment of gastrointestinal tumors

Abstract

In recent years, immune checkpoint blockade (ICB) therapy has become an important treatment strategy for gastrointestinal tumors, however, it only benefits about 1/3 of patients. Since the microbiome has been shown to play an important role in the human body for a long time, a growing number of studies are focusing on its relationship to ICB therapy in cancer, specifically how intestinal microbes affect the efficacy of immune checkpoint inhibitors (ICIs) therapy in patients. On this basis, probiotic interventions, fecal microbiota transplantation (FMT), dietary interventions, and other methods which improve or maintain the structure of the intestinal flora have attracted widespread attention. This article discusses the four aspects of the microbiome, ICB, combined treatment of gastrointestinal tumors, and regulation of gut microbiome. Particularly, the discussion focuses on the contribution of probiotic intervention in improving the therapeutic effect of ICIs to prolong the survival time of patients and reduce the severity of immune-related adverse effects (irAEs).

Introduction

Microorganisms colonize all parts of the human body, with the gut having the highest number of microbes (1). Differences in the diversity and quantity of intestinal microbiota are associated with many diseases, including colorectal cancer (CRC), type 2 diabetes, hepatic steatosis, inflammatory bowel disease, and hypertension among others (2). The host’s lifestyle and medication use greatly affect the intestinal microbiome, which in turn affects the overall health, indicating that regulating the intestinal microflora may improve the health status of an individual (2,3). Immune checkpoint blockade (ICB) is based on the activation mechanism of immune T cells; immune checkpoint inhibitors (ICIs) target immune checkpoints, increase immune suppression, activate T cell activity, and enhance immune response (4). ICIs have been approved for indications in a variety of cancers and multiple combination treatments (5).

Microbiome and gastrointestinal tumors

In defining the microbiome composition, most researchers agree that the microbiota includes all living members that form the microbiome. It does include not only the microbial community but also the “active zone” of microorganisms, which involves structural elements, metabolites, and other molecules that are produced by the microbes. There are about 4×1,013 microbial cells and about 3×103 species of microorganisms in the human body, of which about 97% exist in the colon, about 2%−3% exist outside the colon, and about 0.1%−1.0% are archaebacteria and eukaryotes (6). The microbes in the human intestine are estimated to be composed of more than 2×103 species, including mainly Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (7).

The microbiome has two roles in the development of cancer. On the one hand, some microorganisms can promote the occurrence and growth of cancer (6). For example, one study concluded that Fusobacterium, Bacteroides fragilis (B.fragilis), and Escherichia coli (E. coli) were significantly increased in specimens from CRC patients compared to normal subjects and that these microorganisms are important components of the tumor microenvironment (TME) (7). In another study, it has been shown that human infections with Helicobacter pylori (H. pylori) may cause damage to gastric tissue, leading to atrophic gastritis (AG) and subsequently triggering its progression to gastric cancer (GC) (8). H. pylori is considered to be the main microorganism in the development of GC and seems to be necessary in the early stages of the process (9).

Meanwhile, some microorganisms have immunostimulatory properties and have been shown to have anti-tumor immune effects (6). For example, Roseburia, Lachnospira, and bacteria that can produce short-chain fatty acids (SCFAs) affect the immune response to some extent, thus reducing the incidence of CRC (7). Moreover, another preclinical study found that supplementing experimental mice with Clostridiales can effectively prevent CRC by regulating the body’s immune response, and is more effective than immune blockade in CRC treatment (10).

ICIs for gastrointestinal tumors

ICB therapy, which targets receptor-ligand interactions, has made a breakthrough in cancer treatment (11). Due to the memory characteristics of the immune system, ICB therapy often produces a longer-lasting response than chemotherapy or targeted therapy (12). Immune checkpoints, also known as inhibitory immunoreceptors, are molecules that act as gatekeepers for the immune response. These include but are not limited to the programmed death receptor 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) (12,13). ATTRACTION-2 has demonstrated the benefits of nivolumab in cancer patients in clinical trials and found that this monoclonal antibody can be used for the advanced treatment of GC and gastroesophageal junction (GEJ) cancer (14). Furthermore, the CheckMate649 trial explored the efficacy of nivolumab and ipilimumab in the treatment of advanced gastric, GEJ, and esophageal cancers compared with chemotherapy (15). The study confirmed the efficacy of nivolumab but advised that the treatment of ipilimumab needs further investigation to ensure improved results and reduced adverse reactions (15).

Current studies have found that anti-CTLA-4 therapies are not as effective as anti-PD-1/PD-L1 therapies. PD-1/PD-L1 inhibitors that have been approved for marketing include toripalimab (16), sintilimab (17), camrelizumab (18), tislelizumab (19), penpulimab (20), zimberelimab (21), durvalumab (22), atezolizumab (23), and envafolimab (24), among others, in addition to nivolumab (14). However, the application of ICIs remains limited in two main ways: firstly, the overall response rates remain unsatisfactory, particularly for cancers with low mutational burden (12). Additionally, an increasing number of studies have shown that when widely used in clinical treatment, some adverse reactions to ICB treatment appear, with the most common being immune reaction (12). Common immune-related adverse effects (irAEs) are specific to ICB treatment and usually occur in the later stage of therapy, focusing on the skin, gastrointestinal tract, endocrine system, liver, and lung, among others (25).

Gut microbiome and ICB therapy

Several studies in recent years have demonstrated that gut microbes and their metabolites can regulate anti-tumor immunity and influence the outcome of cancer immunotherapy, particularly ICB therapy (26). However, understanding the mechanisms by which gut microbes influence the efficacy of ICIs is proving difficult and is still being explored (27). It is now proposed that the gut microbiome enhances the role of immune cells, as well as the anti-tumor effects of the body’s immune response, and reprograms immunity in the TME, thus improving the efficacy of ICIs (28). Moreover, the wide variability of gut microbiota in humans also affects cancer progression and the outcome of ICIs (29). Therefore, modulation of the gut flora can enhance the efficacy of ICIs by formulating reasonable microbial-based treatment strategies (28,30).

Effect of gut microbes on ICB efficacy

A growing body of evidence suggests that the microbiota has not only pro-tumor but also anti-tumor effects (31). The microflora can regulate the function of the mucosa and the strength of systemic immune responses, which in turn affects cancer immunotherapy, particularly negative effects on the immune checkpoints PD-1 and CTLA-4 (32). Furthermore, several studies have shown significant differences in the abundance and number of intestinal microbes between responsive and non-responsive patients to ICIs, which also affects the clinical response of patients to ICIs in terms of progression-free survival (PFS), overall survival (OS), and irAEs (33). The effects are summarized in Table 1.

Table 1. Effect of gut microbes on efficacy of ICB.

Monoclonal antibody	Related gut microbes	Cancer	Mechanism	Effects	Ref.
ICB, immune checkpoint blockade; PD-1, programmed death receptor1; PD-L1, programmed death ligand 1; CTLA-4, cytotoxic T lymphocyte-associated protein 4; B.fragilis, Bacteroides fragilis; HCC, hepatocellular carcinoma; GI, gastrointestinal; IL-12, interleukin 12; Th1, T helper 1; DC, dendritic cell; STING, stimulator of interferon genes; PFS, progression-free survival.
PD-1/PD-L1 monoclonal antibody	Lactobacillus, Akkermansia muciniphila and Ruminococcaceae	HCC	/	Improving response rate to anti-PD-1 therapy	(34)
	Faecalibacterium genus	HCC	/	Significantly prolonging PFS	(35)
	Prevotella/Bacteroides ratio	Advanced-stage GI cancer	/	Improving response rate to anti-PD-1/PD-L1 immunotherapy	(36)
CTLA-4 monoclonal antibody	B. fragilis	Multiple types of cancer	Increasing IL-12-dependent Th1 cells to promote DC maturation	Facilitating tumor control while sparing intestinal integrity	(37)
	Bifidobacterium	Melanoma	Manipulating gut microbiota	Reducing immunotoxicity associated with anti-CTLA-4 therapy	(38)
CD47 monoclonal antibody	Bifidobacterium	Colon adenocarcinoma, etc.	Triggering the STING pathway inside DCs	Facilitating local anti-CD47 immunotherapy on tumor tissues	(39)

PD-1/PD-L1 monoclonal antibodies

PD-L1 is expressed in a variety of cells in the body and binds to PD-1 on T cells, resulting in the inhibition of T cell activity, and subsequent inhibition of the immune response (40). In cases of inflammation or carcinogenic damage, PD-L1 is up-regulated, and the body’s immune response is weakened and is unable to attack tumors (41). Monoclonal antibodies against PD-L1 or PD-1 affect their binding and can promote our body’s anti-tumor immunity (41,42). Moreover, numerous studies have confirmed that the intestinal microbiota has a significant influence on both the therapeutic and adverse effects of anti-PD-1 immunotherapy for digestive tract tumors.

In demonstrating the influence of the gut microbiota on ICB treatment in liver cancer patients, Zheng et al. (34) found that the abundance of Lactobacillus, Akkermansia muciniphila, and Ruminococcaceae in the intestinal flora of responders (R, complete or partial response, or stable disease lasting for over 6 months; n=3) was higher than that of patients who did not respond to the treatment (non-responders, NR, advanced disease or stable disease lasting less than 6 months; n=5). The researchers treated eight hepatocellular carcinoma (HCC) patients with anti-PD-1 antibodies and found that three were R while five were NR (34). The dynamic changes and specificity of intestinal bacterial characteristics were analyzed through metagenomic sequencing (34). It was found that throughout the treatment process, R showed a higher abundance and number of bacteria than NR, and the microbial composition in R remained relatively stable, while in NR, Proteobacteria gradually increased to become the dominant bacteria (34). The study further demonstrated the biological significance of particular bacterial strains in anti-PD-1 immunotherapy and indicated that the therapeutic effect in tumor patients could be modified by regulating the composition of intestinal microorganisms (34).

In another recent study, Li et al. (35) found a greater abundance and number of intestinal microbes in the R group in most patients with advanced primary HCC receiving anti-PD-1 immunotherapy in a hepatitis B virus-infected population. Oral samples from 60 patients and stool samples from 55 patients were collected, and the microbiota was found to be less variable in oral samples compared to stool samples (35). Therefore, this study focused on the gut microbiota characteristics (n=55) and concluded that the gut microbiota diversity was significantly higher in HCC patients receiving ICIs (35). Moreover, subjects with a higher abundance of Faecalibacterium had longer PFS and better treatment outcomes after immunotherapy (35).

In a study exploring the effects of the intestinal microbiome on the efficacy of anti-PD-1/PD-L1 in GI cancer patients, Peng et al. (36) found that the microbiome has the potential to become a marker of ICB response among some patients. The researchers studied nearly 100 patients with advanced GI tumors treated with PD-1/PD-L1 blockers and found that a better treatment response correlates with a higher Prevotella/Bacteroides ratio in stool samples (36). In addition, the R group had a greater density of Ruminococcaceae, Prevotella, and Lachnospiraceae, while the NR group had a greater proportion of Bacteroides (36). Furthermore, intestinal bacteria belonging to Eubacterium, Lactobacillus, and Streptococcus, which can produce SCFAs can promote the body’s immune response to attack tumor cells and increase the efficacy of ICB in patients (36).

CTLA-4 monoclonal antibodies

CTLA-4 is another target of ICB therapy, and its ligands can bind to CD28 or CTLA-4 itself, leading to co-stimulatory or co-inhibitory responses, respectively (43). CTLA-4 molecules have higher affinity and can compete with CD28 to bind B7 molecules (CD80 and CD86) on the surface of APC, thereby delivering inhibitory signals leading to T cell inactivation (44). This implies that CTLA-4 inhibits the immune function by suppressing T-cell activity, which naturally weakens the anti-tumor ability of patients, and explains how ipilimumab restores the anti-tumor effect on patients by inhibiting CTLA-4 (45).

Vétizou et al. (37) mainly studied the relationship between the intestinal microflora and the efficacy of CTLA-4 inhibitors. They found that the number of effector CD4+T cells and tumor-infiltrating lymphocytes (TIL) decreased in germ-free (GF) mice or antibiotic-treated mice, resulting in a decrease in the efficacy of anti-CTLA-4 (37). This reduction in anti-CTLA-4 immune efficacy was shown to be associated with a disruption of the intestinal flora structure and a decrease in B. fragilis (37). Further studies have shown that CTLA-4 inhibitor treatment affects the structure of the organism’s microbiome, and that supplementation with B. fragilis induces T helper 1 (Th1) immune responses, increases interleukin-12-dependent Th1 cells to promote dendritic cell (DC) maturation, improves anti-CTLA-4 efficacy, and facilitates tumor control in mice and patients (37).

In another study, Wang et al. (38) noted that a stumbling block in treating cancer with ICIs is that they often lead to severe autoimmunity, most commonly colitis, and the associated toxicity can be severe or even life-threatening. Therefore, the team focused on mitigating the autoimmune response induced by anti-CTLA-4 and other ICIs by modulating the gut microbiota (38). They found that mice treated with vancomycin before the induction of colitis had an earlier and more severe onset after anti-CTLA-4 treatment compared to controls, which was essentially fatal. This suggests that Gram-positive bacteria could alleviate colitis associated with anti-CTLA-4 therapy (38). Furthermore, when a mixture of four Bifidobacterium species was further administered orally before the induction of colitis in mice, it was concluded by comparison that Bifidobacteria could ameliorate vancomycin-induced intestinal ecological imbalance and attenuate the immunotoxicity associated with anti-CTLA-4 treatment (38).

CD47 monoclonal antibodies

CD47 is another immune checkpoint, and Jiang et al. (46) indicated that CD47 is commonly present in the organism, but its expression is increased in some tumor cells. CD47 binds to signal regulatory protein alpha (SIRPα) and instructs macrophages to “do not eat me”, thus weakening phagocytosis. The increased expression of CD47 allows tumor cells to survive by blocking phagocytosis and subsequently decreasing the anti-tumor ability of the body (46). In addition to this, several studies have indicated that anti-CD47 treatment alone does not restore its phagocytic function and that the CD47-SIRPα signaling pathway is also a key immune checkpoint in a variety of cancers (46,47). Some CD47 monoclonal antibodies can enhance phagocytosis in patients by blocking the binding of CD47 to SIRPα, so as to exert anti-tumor effects (48). Xu et al. demonstrated that, in addition to macrophages, CD47-SIRPα also inhibits the function of DCs, and that blocking this pathway promotes the uptake of tumor cells by DCs and facilitates the delivery of tumor antigens (49).

In a study that explored the effects of intestinal flora on the efficacy of CD47 monoclonal antibodies in the treatment of tumors, Shi et al. (39) found that Bifidobacterium could enhance the anti-tumor effects of CD47 blockade. Wild-type mice receiving oral antibiotics and GF mice did not respond to CD47 blockade treatment, whereas when supplemented with Bifidobacterium, the tumor-bearing mice recovered their immune response against CD47, indicating that Bifidobacterium can effectively promote anti-CD47 immunotherapy (39). The researchers propose that after anti-CD47 treatment, Bifidobacterium supplementation stimulates the immune signaling pathway of stimulator of interferon genes (STING) and increases the cross-presentation by DCs to activate downstream immune responses, thus enhancing the anti-tumor effect of CD47 blockade (39).

Pathways of gut microbes affecting efficacy of ICB

One of the ways intestinal flora regulates anti-tumor immunity is through their metabolites, which can spread to the whole body, affect the metabolic status, and strengthen the anti-tumor immune function to a certain extent, thus increasing the efficiency of ICB (50). In addition, it has been shown that intestinal microbes can translocate to the tumor and promote systemic anti-tumor responses (51). The pathways are summarized in Table 2.

Table 2. Pathways of gut microbes affecting ICB therapy.

Variables	Mechanism	Effects	Ref.
ICB, immune checkpoint blockade; SCFAs, short-chain fatty acids; BA, bile acid; PD-L1, programmed death ligand 1; NKT, natural killer T; AhR, aryl hydrocarbon receptor; I3A, indole-3-aldehyde; PFS, progression-free survival; ICI, immune checkpoint inhibitor.
Gut microbial metabolite
SCFAs	Regulating immune cell response and improving the curative effect of PD-L1	Longer PFS	(52)
Inosine	Enhancing the anti-tumor ability of T cells	Enhancing the effect of checkpoint blockade immunotherapy	(53)
BAs	Controlling a chemokine-dependent accumulation of hepatic NKT cells	Inhibiting the growth of liver tumor	(54)
Bacterial translocation
Lactobacillus 　reuteri	Releasing AhR agonist I3A	Promoting ICI efficacy and survival	(55)

Gut microbial metabolite mediated ICB therapy

Microbial metabolites, which are small molecules produced by the intermediates or end products of microbial metabolism, are key factors in the interaction between intestinal microorganisms and the immune system (56). These include molecules such as SCFAs, indole derivatives, and bile acid (BA) metabolites, which have a significant impact on intestinal barrier function and immune response (56). They can act directly on the host to activate immunity or indirectly by modulating the composition and activity of other microorganisms, largely affecting pathogen colonization, intestinal barrier integrity, and host metabolism (57).

SCFAs are the products of indigestible carbohydrates decomposed by intestinal bacteria. The concentrations of acetate, butyrate, and propionate in the body are higher than those of valerate and formate (58). Moreover, SCFAs strongly induce immunoglobulin A (IgA), promote Treg cell differentiation, induce Th1 and T helper 17 (Th17) cells, potentiate CD8+ T cell responses, and promote interleukin-10 (IL-10) production (58). Nomura et al. (52) demonstrated an association between SCFAs and the efficacy of anti-PD-1 treatment for solid tumors. Fecal and plasma samples from 52 patients with solid tumors treated with anti-PD-1 were collected and their SCFAs concentrations after treatment were compared between the R and NR groups (52). It was then concluded that SCFAs concentrations were higher in the R group, and the higher fecal or plasma SCFAs concentrations were associated with anti-PD-1 efficacy and longer PFS (52). Further studies have shown that fecal SCFA concentrations can be increased by dietary changes such as high-frequency intake of high-fiber foods such as green vegetables, cabbage and mushrooms (52).

Inosine, an intermediate product of purine metabolism, also participates in the metabolism of various other substances which affect human health (59). The product can activate the immune response, stimulate metabolic function, and improve the efficacy of immunotherapy for cancer patients (28). McCoy KD’s team determined the role of inosine in altering the immune efficacy in mice with CRC and found that three bacteria, B. pseudolongum, Lactobacillus johnsonii, and Olsenella, significantly enhanced the effects of ICIs (53). The production of inosine by B. pseudolongum promotes the differentiation of initial T cells to Th1 cells, and anti-CTLA-4 increases the production of interferon-gamma (IFN-γ), which further activates Th1 cells and acts as a tumor suppressor (53).

BAs are cholesterol-derived molecules that participate in basic physiological activities, and regulate immune cell function to some extent, thus helping to maintain homeostasis. While most BAs return to the liver via the intestinal-hepatic cycle, another fraction enters the colon where they are used by symbiotic bacteria to produce secondary BAs (60). In some studies secondary BAs have shown immunosuppressive effects, with human intestinal bacteria and corresponding enzymes converting secondary BAs to 3-oxolithocholic acid (3-oxoLCA) and the intestinal enriched metabolite isoLCA, thereby inhibiting Th17 cell differentiation (61). In addition, the secondary BAs 3β-hydroxydeoxycholic acid (isoDCA) increases gene expression of its anti-inflammatory phenotype by acting on DCs, inducing Foxp3 expression, promoting CD4+ T cell differentiation to peripheral Treg (pTreg) cells, and promoting immune escape (60). Ma et al. (54) found that primary BAs induced C-X-C motif chemokine ligand 16 (CXCL16) expression and recruited CXCL16-mediated natural killer T (NKT) cells in mice with liver tumors, which inhibited liver tumor growth. Moreover, antibiotic treatment with vancomycin induced the accumulation of liver NKT cells and reduced liver tumor growth. Additionally, colonized Clostridium metabolized primary BAs to secondary BAs, reversing the accumulation of NKT cells and inhibiting liver tumor growth in mice (54).

Bacterial translocation affects ICB efficacy

A team of researchers provided strong evidence for the presence of microorganisms in paraffin cuts of patients’ tumor tissues (62). Studies using extremely strict aseptic conditions and extensive controlled trials to exclude contamination by exogenous bacteria have found that intra-tumor bacteria are predominantly found in tumor cells and immune cells, and that microbial composition characteristics and diversity correlate with tumor type, patient survival, and response to immunotherapy (62). In addition, in the past few years, researchers have confirmed the phenomenon of bacterial translocation and colonization in solid tumors by studying tumor models in mice (63). The bacteria migrate out of the intestine and translocate to the tumor, activating the intrinsic immune pathway and assisting in activating the systemic anti-tumor response (51).

Similarly, studies have shown for the first time that oral administration of a variety of bacteria directly affects immune cells in tumors by transferring them to tumors outside the gut, thereby improving the efficacy of cancer immunotherapy (55). This study tested the effects of various probiotics on tumor growth in a tumor-bearing mouse model and found that orally administered Lactobacillus reuteri (Lr) could migrate into the melanoma. Its dietary tryptophan catabolite indole-3-aldehyde (I3A), when released in tumors, enhances anti-tumor immune responses and improves the efficacy of ICIs by activating the aryl hydrocarbon receptor (AhR) signaling in CD8+ T cells (55).

Gut microbiota and ICI therapy toxicity

ICB therapy suppresses the immune checkpoint and enhances the anti-tumor function of T cells, which helps treat tumor patients, but at the same time affects the immune microenvironment of the host and destroys the immune balance (64). Almost all organ systems such as skin, pulmonary, liver, gastrointestinal, endocrine, and cardiovascular, may be affected by the activation of the immune response to ICIs treatment (65), which eventually develops into irAEs, including ICI-related colitis (IRC) and diarrhea, which are the main reason for ICI discontinuation (64). It has been further shown that ICB treatment can increase the incidence of diarrhea and colitis, and anti-CTLA-4 treatment results in a greater risk of colitis and diarrhea compared to other ICB treatments (66). In addition, diarrhea and colitis are more common and more severe with combination immunotherapy than with anti-CTLA-4 therapy alone (66). Zhou et al. (64) concluded that alteration and dysbiosis of the gut microbiota are strongly associated with ICI-induced irAEs, particularly IRC, and that antibiotic-induced dysbiosis of the microbiome may increase the risk of IRC. Further studies suggest that metabolites, immune cells, and other factors are related to the increase of adverse reactions and that the microbiome can be regulated to combat irAEs, including methods like probiotic therapy and fecal transplantation (64).

The influence of intestinal microorganisms on the occurrence of irAEs has been confirmed by numerous studies. Research by Mao et al. (67) showed that the flora diversity of patients with severe diarrhea is lower than that of patients with mild diarrhea. This may indicate that the diversity of intestinal flora affects the appearance of irAEs. Moreover, the study confirms the feasibility of intestinal flora as a biomarker for immunotherapy in liver cancer (67). Furthermore, the team investigated the adverse effects of anti-PD-1 monoclonal antibodies for advanced hepatobiliary malignancies in study subjects and performed macrogenomic analysis of their stool samples for diarrhea (immunotherapy-associated colitis) to further explore the relationship between the composition and relative abundance of intestinal flora and irAEs (67). The data showed that patients with severe diarrhea had a better clinical response and longer PFS than those with mild diarrhea (67). A total of 16 species differed significantly between the two groups of patients, most significantly, the relative abundance of Prevotellamassilia timonensis was greater in patients with severe diarrhea compared to those with mild diarrhea, and thus is a potential biomarker for predicting severe immune adverse reactions (67).

Effect of ICB therapy on gut microbiome

While there is no doubt that gut microbes influence the efficacy of immunotherapy, it has also been shown that ICB therapy affects the structure and functions of the gut microbiome. Zheng et al. (34) studied stool samples from eight patients with primary HCC before and after immunotherapy. The composition of the fecal microbiome of the patients before treatment was consistent with that of healthy individuals and composed mainly of Bacteroidetes, Firmicutes, and Proteobacteria. After immunotherapy, changes in intestinal microbial composition were not significant in R, but the fecal microbial composition of NR showed a significant decrease in the abundance of Bacteroidetes and a significant increase in Proteobacteria (34).

ICB therapy also induces gut microbiota translocation. Andrew Y. Koh’s team indicated that immune checkpoint blockade therapy (ICT) induces intestinal bacterial translocation into secondary lymphoid organs and tumors and promotes extraintestinal anti-tumor immunity (68). Through 16S rRNA sequencing and other techniques, the team found that melanoma alone will not cause bacterial translocation, but the use of ICT will lead to bacterial translocation regardless of whether there is a tumor or not (68). It is also proposed that ICT-induced bacterial translocation depends on DCs-induced immune response to promote anti-tumor immunity (68).

Improving immunotherapy by regulating gut microbiota

Although numerous studies have shown that ICB therapy can improve the outcome and prognosis of patients with advanced cancer, leading to breakthroughs in the treatment of solid metastatic malignancies, not all patients benefit from the treatment. With an objective response rate (ORR) of only 10%−30% (28) and with at least 2/3 of treated patients experiencing severe irAEs, better treatment options should be investigated to improve the outcome of ICB treatment (33). Numerous studies have shown that the intestinal microbiome affects anti-tumor immunity to a great extent, thus affecting the treatment response and outcome of tumor patients treated with ICB (69). Overall, these suggest the possibility of improving the efficacy of immunotherapy and reducing the occurrence of irAEs by regulating the structure of the intestinal flora (70). The specific measures are shown in Figure 1.

Figure 1.

Several pathways can modulate the gut microbiota and thus improve immunotherapy. (A) The use of antibiotics may lead to imbalance of intestinal flora, which shortens the PFS and OS of immunotherapy in cancer patients, reduces RR, and makes immunotherapy less effective; (B) FMT can directly modulate the gut microbial structure and thus improve the efficacy of immunotherapy; (C) Supplementation of probiotics regulates the composition of intestinal flora; (D) Diet and lifestyle indirectly change the structure of intestinal flora; (E) Other approaches including bacterial genetic engineering technology, GQD, Shaoyao Ruangan mixture and smectite can also improve the efficacy of immunotherapy by regulating the composition of intestinal flora. PFS, progression-free survival; OS, overall survival; RR, remission rates; FMT, fecal microbiota transplantation; GQD, Gegen Qinlian decoction.

Antibiotics

Given all the relevant studies showing that gut microbial composition strongly influences ICB treatment’s success, the most important approach widely recognized in improving ICB’s efficacy is minimizing antibiotic use in cancer patients preparing for ICB treatment (71). Some literature has suggested that the use of antibiotics can shorten the PFS and OS of cancer patients treated with immunotherapy, and three main factors can explain the changes in intestinal microflora as caused by antibiotics (72). First, the loss of microorganisms weakens immune function. Secondly, antibiotics can have a direct impact on the body (73). Finally, antibiotics can cause the death of epithelial cells (72). Therefore, it can be said that antibiotics affect the efficacy of immunotherapy in many ways.

Ahmed et al. (74) demonstrated that ICB treatment efficacy was affected in subjects treated with antibiotics. The retrospective study involved advanced cancer patients treated with ICIs and compared the therapeutic effects in patients with or without antibiotics (74). The researchers pointed out that patients treated with systemic antibiotics had lower remission rates (RR) and worse PFS than patients who did not receive antibiotics, and explored that the use of antibiotics was the most important reason for reducing RR and PFS (74). In addition, patients on broad-spectrum antibiotics exhibited reduced immunotherapy efficacy and shorter OS duration compared to those on narrow-spectrum antibiotics (74). Overall, their findings suggest that the use of broad-spectrum antibiotics reduces the effectiveness of ICI in treating cancer patients. In contrast, Huang et al. (75) suggested that while the brief use of antibiotics before and after immunotherapy (before or after 2 months) was significantly associated with the efficacy of ICIs, the effect of antibiotic exposure seemed to be eliminated during long-term treatment of ICIs.

Fecal microbiota transplantation (FMT)

FMT can directly modulate the intestinal microbial structure through the transfer of fecal bacteria preparation to the recipient. This is done through endoscopy or oral administration to directly change the composition and re-establish the balance of the intestinal microbiota (76). Furthermore, FMT has become a common treatment for recurrent Clostridium difficile infection (77). Numerous studies in recent years have confirmed that microbial transplantation via FMT can improve the effectiveness and potentially minimize the adverse effects of immunotherapy (78). Li et al. (69) also concluded that GF mice receiving anti-PD-1 R FMT had enhanced anti-tumor immunity and were effective against anti-PD-1 treatment, while those receiving NR FMT were unable to respond to PD-1 treatment.

The effectiveness of FMT in some types of diseases has been recognized by numerous studies, but as it has only been used as a treatment in recent years, its safety is controversial. The structure of the fecal microbiome remains to be studied, and some adverse effects have been observed in the clinical use of FMT, including fever, constipation, diarrhea, and pneumonia (72). It has been shown that two patients developed E. coli bacteremia after receiving FMT in independent clinical trials and that these cases were associated with the same fecal donor, even to the point of death in 1 of the patients (79). Another study collected donor feces from 66 tested individuals, of which 11 cases (17%) were multi-drug resistant (80). Therefore, it is necessary to enhance donor screening to remove harmful bacteria, viruses, and parasites, and improve the safety of FMT treatment.

Probiotic interventions

Probiotics are living microorganisms that can improve health in appropriate concentrations and can regulate the structure of intestinal flora, participate in metabolic activities, and affect host health and disease recovery. These include species such as Bifidobacteria and Lactobacilli among others (81). A growing body of research suggests that microorganisms can influence cancer prevention, treatment, and prognosis by modulating host immune and inflammatory responses (82). Probiotic supplementation is a simple and feasible way to modulate the composition of the microbiota to prevent the emergence and development of cancer, improve the effectiveness of current anti-cancer treatments, and reduce the serious adverse effects of chemotherapy and radiotherapy (82).

To confirm that probiotics can regulate the composition of intestinal microflora in CRC patients and thus influence cancer development, 15 CRC patients in one study were given Bifidobacterium lactis and Lactobacillus acidophilus (83). The results showed that compared with the normal subjects, the number of CRC-related bacteria in the fecal microbial composition of the patients decreased, revealing the ability of probiotics to alleviate intestinal dysbiosis and improve immunity against cancer (83). This study also showed that probiotics such as Bifidobacterium lactis and Lactobacillus acidophilus can potentially contribute to CRC treatment (83). In addition, researchers at Jiangxi Provincial Cancer Hospital investigating the combination of probiotics and anti-PD-1 therapy for various cancer types found that bacteria such as Lactobacillus, Bifidobacterium, and Akkermansia muciniphila improved the effects of PD-1 inhibitors on tumor-bearing mice (69). Furthermore, in studying liver and lung cancer patients treated with probiotics in combination with PD-1 inhibitors, the researchers expected a better prognosis with the combination therapy compared to monotherapy by comparing ORR, PFS, and OS (69).

Dietary intervention and life style

Dietary changes can rapidly alter the structure of the gut microbiome, which can optimize the outcome of ICB treatment. One study suggests that patients should reduce their intake of animal meat and increase their intake of plants, with no less than 30 kinds of plants per week, because researchers have indicated that high fiber intake (>30 g/d) is beneficial in increasing ICI response rates (84). In addition, it has been suggested that ideally, all patients should be referred to a dietitian to match their diet before starting ICIs to achieve better outcomes (84). Meanwhile, to confirm that dietary changes can reduce the incidence of many types of cancer, Reynolds and colleagues analyzed data from more than 200 studies and trials (85). They found that people with a lower dietary fiber intake had higher morbidity and severity of cardiovascular disease, type 2 diabetes, CRC, and breast cancer than those with higher dietary fiber intake (85).

In addition to dietary interventions, activities such as smoking, alcohol consumption, exercise, and sleep also affect the gut microbiota to a certain extent and affect the efficacy of tumor immunotherapy by changing local and systemic immune responses. A large number of literature have shown that drinking can increase the risk of CRC, and abstinence from alcohol can restore the structure and function of the human intestinal barrier (69). Moreover, exercise may reduce the concentration of lactic acid, increase the number of tumor-infiltrating immune cells, and modulate the TME, so it is helpful to the ICB treatment of tumor patients (72). In addition, sleep may lead to changes in the composition of intestinal microflora, short sleep or light sleep can cause an imbalance in the diversity of intestinal microflora, affecting body health and immunotherapy (72).

Other strategies

Bacterial genetic engineering technology is widely used to modulate the gut microbiota or metabolites and improve the anti-tumor response to ICI since genetically engineered drugs have specificity that FMT cannot achieve (28). Using synthetic biology techniques, Canale and other researchers developed an engineered probiotic strain of E. coli Nissle1917 to produce high concentrations of topical arginine (86). The strain can colonize tumors, continuously convert ammonia accumulated in tumors to L-arginine, and increase the number of tumor-infiltrating T cells. Furthermore, it has a significant synergistic effect with PD-L1 blocking antibodies in clearing tumors (86). Overall, engineered microbial therapies can metabolically modulate the TME and thus improve the efficacy of immunotherapy (86).

Numerous studies have proved that some drugs besides probiotics also have certain effects on the intestinal microflora. First, the Chinese herbal medicine Gegen Qinlian decoction (GQD) in combination with an anti-PD-1 monoclonal antibody can be used to treat microsatellite stable (MSS) CRC (72). Meanwhile, the Shaoyao Ruangan mixture leads to a greater abundance of Bacteroides spp. that may limit the development of primary liver cancer (87). Some western drugs, such as smectite, an anti-diarrhea drug, may induce a similar effect (88). Smectite promotes the expansion of probiotics (especially Lactobacillus) in the mouse intestine by promoting the formation of probiotic biofilms on smectite, which in turn inhibits tumor growth, when used alone, and enhances the efficacy of chemotherapy or immunotherapy (88).

Conclusions

Based on the available findings, the microbiome, especially the intestinal microbiome, is closely related to the efficacy of ICB treatment for gastrointestinal tumors. This literature summarizes the changes in the structure of the intestinal microbiota during ICB treatment and observes that some bacteria can significantly improve the efficacy of ICB. Therefore the regulation of the gut flora should be emphasized to provide a healthier microbiota for optimal ICB treatment. Firstly, the use of antibiotics before or during treatment should be avoided or minimized, since it can disrupt the balance of the gut microbiota. Both FMT and probiotic interventions may help to improve the gut microbiota environment. Lifestyle changes such as diet may also cause an imbalance in the gut microbiome, emphasizing that high-quality living habits should be practiced. Most of the microbial anti-tumor mechanisms are still unknown, and elucidating the complex mechanisms between the gut microbiome and ICI treatment proves to be challenging. Since the gut microbiome is highly individualized, the next challenge in improving the efficacy of ICB is to develop individualized diagnostic and therapeutic strategies for cancer patients and to advance the development of precision anti-cancer therapy.