An emerging strategy: probiotics enhance the effectiveness of tumor immunotherapy via mediating the gut microbiome

ABSTRACT

The occurrence and progression of tumors are often accompanied by disruptions in the gut microbiota. Inversely, the impact of the gut microbiota on the initiation and progression of cancer is becoming increasingly evident, influencing the tumor microenvironment (TME) for both local and distant tumors. Moreover, it is even suggested to play a significant role in the process of tumor immunotherapy, contributing to high specificity in therapeutic outcomes and long-term effectiveness across various cancer types. Probiotics, with their generally positive influence on the gut microbiota, may serve as effective agents in synergizing cancer immunotherapy. They play a crucial role in activating the immune system to inhibit tumor growth. In summary, this comprehensive review aims to provide valuable insights into the dynamic interactions between probiotics, gut microbiota, and cancer. Furthermore, we highlight recent advances and mechanisms in using probiotics to improve the effectiveness of cancer immunotherapy. By understanding these complex relationships, we may unlock innovative approaches for cancer diagnosis and treatment while optimizing the effects of immunotherapy.

GRAPHICAL ABSTRACT

KEYWORDS:

• Probiotics
• tumor immunotherapy
• gut microbiome
• tumor microenvironment
• tumor microbiome

1. Introduction

The complex interplay between the gut microbiota and human well-being has recently emerged as a captivating area of research. Tumors arise and progress in individuals with compromised immune function, where the immune system fails to recognize and eliminate abnormal cells. This process is also accompanied by disturbances in the gut microbiota.¹ Numerous studies have shown notable variations in gut microbiota between healthy individuals and cancer patients, with unique microbial signatures observed among various cancer groups. Inversely, the impact of the gut microbiota on the onset and growth of cancer is increasingly clear,^2,3 affecting the tumor microenvironment (TME) and impacting both local and distant tumors (Figure 1). Tumor immunotherapy is an approach to treatment that stimulates the body’s tumor-specific immune responses to harness their inhibitory and cytotoxic functions against cancer cells, which has been rapidly developed and garnered attention due to its high specificity and potential for long-term effectiveness.⁴ However, the major limitation of tumor immunotherapy is that it does not work for all cancer patients. Substantial evidence has demonstrated that among various types of cancer, some individuals may not show significant treatment responses to ICIs and have side effects due to intestinal microorganisms.⁵

Figure 1. The healthy gut and various diseases that may be caused by gut dysbiosis.

Within this context, as beneficial microorganisms, probiotics have drawn attention for their ability to modulate the gut microbiota and hold promise in potentially alleviating various health conditions and diseases.^6,7 Probiotics also modulate intestinal immune function by interacting with various cell types, including IECs, dendritic cells (DCs), T cells, and B cells, influencing their differentiation, activation, proliferation, and secretion.^8–11 In addition, they also play a crucial role in enhancing cancer immunotherapy and providing better inhibition of tumor growth. On the one hand, probiotics can regulate the gut microbiota and immunity, influencing the body’s response to immune inhibitors. On the other hand, probiotics can also translocate to tumor sites and exert their inhibitory effects.^12–15

Therefore, this manuscript explores the interplay between the gut microbiota and the TME. We summarize the disturbance of the intestinal microbiota in cancer patients and potential underlying causes, as well as the microbial biomarkers that can be used for the diagnosis and prognostic assessment of cancers.^16–18 We also investigated how the gut microbiota affects the tumor microenvironment, including the composition of the tumor microbiota and its influence on cancer development. Furthermore, we highlight recent advances in using probiotics to improve the effectiveness of cancer immunotherapy. This comprehensive review aims to provide valuable insights into the dynamic interactions between probiotics, gut microbiota, and cancer. By understanding these complex relationships, we may unlock innovative approaches for cancer diagnosis and treatment while optimizing the effect of immunotherapy.

2. Gut microbiome and probiotics

The gut microbiota, consisting of a large number of microorganisms, has garnered widespread attention for its multifaceted role in human health. These microorganisms, predominantly anaerobic bacteria, are roughly equivalent in number to human cells,¹⁹ with their unique genes exceeding the human genome by approximately 100-fold.²⁰ The gut microbiota contributes to food digestion and nutrient absorption, engages in diverse metabolic processes, and profoundly impacts host immunity maintenance and regulation.²¹ Consequently, overall health needs to maintain the balance of the gut microbiota. Diet, geography, and health status have the most remarkable effects on gut microbiota composition. In general, it remains in dynamic equilibrium; once the balance of the gut microbiota is disturbed, it can cause the host to lose various functions, including barrier function, immunological function, and inflammation, which makes it harder to fight pathogens,²² thereby inducing various diseases, such as inflammatory bowel diseases;^23,24 metabolic disorders, such as obesity and type 2 diabetes;^25–27 autoimmune diseases,^28,29 such as food allergies,³⁰ eczema,³¹ and asthma;³² and even the growth and incidence of malignancies (Figure 1).^33,34 In addition, growing interest in the significance of the gut microbiota in disease states has garnered increasing attention, as it can extend beyond the intestinal barrier and impact primary lymphoid organs and the tumor microenvironment.^35–38 In tumor immunotherapy research, only a subset of participants demonstrates a significant response, implying that the intestinal microbiota is crucial in tumor immunotherapy.

By intervening in the gut microbiota, probiotics can potentially enhance the host’s well-being and improve the host’s health. Probiotics exert their pleiotropic effects to reduce potential disease risks by employing several key mechanisms by competitively excluding harmful microbes, producing beneficial metabolites, including short-chain fatty acids,^39,40 hydrogen peroxide,⁴¹ and bacteriocin,⁴² regulating immunity, and maintaining the integrity of the mucosa.⁴³ Numerous studies have revealed that probiotics can help prevent and treat gastrointestinal diseases,^44,45 chronic metabolic diseases,^46,47 and cardiovascular and cerebrovascular diseases.^48–50 In recent years, some studies have applied probiotics in tumor immunotherapy and found that probiotics can cooperate with tumor immunotherapy to achieve better tumor suppression effects. This also suggests a brighter prospect for using probiotics in treating and preventing diseases.

3. The gut microbiome and cancer

Multiple studies have shed light on the gut microbiota’s function in promoting or inhibiting tumor development.⁵¹ The interaction between microorganisms and cancer is multifaceted. Microbes can contact cancerous tissues directly, influencing their behavior.^52,53 Additionally, microbes could indirectly affect cancer, including the modulation of host physiology or the induction of systemic inflammation by the gut microbiota, affecting tumors’ spread to distant places.^51,54 When dietary or host-released metabolites enter the gut, they undergo biotransformation through microbial catalysis.⁵⁵ These metabolites can reach remote tissues upon absorption and circulation within the host and influence tumor progression.⁵⁶ Both individual microbial species and the overall microbial community contribute to tumor initiation and development. For instance, Helicobacter pylori, initially identified as the causative agent of gastritis, has a significant correlation with gastric cancer epidemiologically.⁵⁷ Gastric cancer risk was considerably decreased following the elimination of H. pylori. Meanwhile, tumors arise and progress in individuals with compromised immune function, where the immune system fails to identify and eliminate abnormal cells;¹ these effects occur alongside disturbances in the gut microbiota.

3.1. Characteristics of the gut microbiome in patients with various types of cancer

Numerous studies have shown notable variations in the gut microbiota composition between healthy subjects and cancer patients. Moreover, within different groups of cancer patients, there may be unique microbial signatures (Table 1).

Table 1. Characteristics of the Gut Microbiota in Patients with Various Types of Cancer.

Cancer type	Gut microbiome	References
Gastric cancer	Patients with gastric cancer had six times the amount of H. pylori in their feces as people without the disease.	^Citation58
	Clostridium XVIII, Veillonella, and Escherichia/Shigella were more prevalent, with the abundance of Bacteroides decreasing in the gastric cancer group.	^Citation59
	Escherichia, Lactobacillus, and Klebsiella were increased in the fecal samples.	^Citation60
	The relative abundance of Veillonella, Megasphaera, and Prevotella increased at the genus level. Lactobacillus salivarius, Streptococcus salivarius, and Bifidobacterium dentium increased at the species level.	^Citation61
	The proportion of probiotic Bifidobacterium was decreased, while the pathogens Streptococcus, Peptostreptococcus, and Prevotella were raised in the gut microbiota.	^Citation62
	Bacteria from the Enterobacteriaceae family are more prevalent, whereas those from the Lactobacillaceae family are less prevalent.	^Citation63
Colorectal cancer	Decrease the abundance in Roseburia and increase in Bacteroides, Escherichia, Fusobacterium, and Porphyromonas genera.	^Citation64,Citation65
	B. fragilis, E. coli, F. nucleatum, S. gallolyticus, and E. faecalis were found to have higher abundance.	^Citation66–70
	Increasingly more fecal samples contained members of the genera Prevotella, Peptostreptococcus, Parvimonas, and Porphyromonas.	^Citation71 , Citation72–74
	C. difficile was also enriched in CRC patients.	^Citation75
	As potential CRC pathogens, E. coli, F. nucleatum, enterotoxigenic B. fragilis, Streptococcus bovis, Peptostreptococcus anaerobius, and E. faecalis have all been observed.	^Citation76
	Genera Bacteroides, Enterococcus, Escherichia, and Clostridium could contribute to CRC development.	^Citation74
Esophageal cancer	Patients with esophageal cancer had more Firmicutes and Actinobacteria, with a decline in Bacteroidetes.	^Citation77
	There are fewer Spirochaetes, Fusobacteria, and Bacteroidetes in the patient’s fecal samples.	^Citation78
Hepatocellular carcinoma (HCC)	The abundance of Streptococcus, Prevotella_9, Faecalibacterium, and Bacteroides was increased.	^Citation79 , Citation80,Citation81
	In fecal samples of HCC patients, the genera Lactobacillus and Streptococcus were enriched, whereas the Akkermansia, Subdoligranulum, Prevotella_2, and Faecalibacterium were reduced.	^Citation82
	Ruminococcaceae, Porphyromonadaceae, and Bacteroidetes were decreased.	^Citation83
	In fecal samples of liver cancer patients, lipopolysaccharide (LPS)-producing genera, such as Klebsiella, increased, whereas butyrate-producing genera, such as Ruminococcus, decreased.	^Citation84
	The presence of GelE-positive E. faecalis can promote the process of liver cancer.	^Citation85
Intrahepatic cholangiocarcinoma (ICC)	Lactobacillus, Alloscardovia, Peptostreptococcaceae, and Actinomyces were enriched in ICC patients.	^Citation86
	Ruminococcaceae, Porphyromonadaceae, and Bacteroidetes were decreased in patients with intrahepatic cholangiocarcinoma.	^Citation87
	Lactobacillus, Actinomyces, Peptostreptococcaceae, Alloscardovia, and Bifidobacteriaceae were higher in the fecal samples of patients.	^Citation86
Cholangiocarcinoma (CCA)	The genera Bacteroides, Alistipes, Muribaculaceae unclassified, and Muribaculum were particularly abundant in the CCA patients.	^Citation88
	Patients had considerably higher Bacteroides, Anoxybacillus, Meiothermus, and Geobacillus levels than controls.	^Citation89
Pancreatic ductal adenocarcinoma (PDA)	Proteobacteria, Fusobacteria, Actinobacteria, and Verrucomicrobia were more prevalent in PDA patients’ guts.	^Citation90
	Patients with PDA had enriched gut populations of Bacteroidetes Firmicutes, Actinobacteria, and Proteobacteria, as well as the genera that make up these four phyla.	^Citation90
	Proteobacteria, Euryarchaeota, and Synergistetes were substantially more prevalent in PDA patients.	^Citation90
	Romboutsia timonensis, F. prausnitzii, Bacteroides coprocola, and Bifidobacterium bifidum were depleted, while Fusobacterium hwasookii, F. nucleatum, V. atypica, and A. omnicolens were enriched in the feces of patients with pancreatic adenocarcinoma.	^Citation91
	Patients with pancreatic adenocarcinoma had higher concentrations of the bacteria V. parvula, Streptococcus species, and V. atypica and lower concentrations of F. prausnitzii.	^Citation92
Breast Cancer (BC)	The increase of Blautia sp. was associated with the stage of breast tumor development.	^Citation93
	Lactobacilli, Enterobacteriaceae, and aerobic Streptococci were higher in premenopausal BC patients’ feces.	^Citation94
	In comparison to healthy women, premenopausal breast cancer patients exhibited a noticeable rise in the presence of Bacteroides, Clostridia, and aerobic Lactobacilli.	^Citation95
	Eubacterium eligens and Roseburia inulinivorans decreased, while increased abundances of Enterococcus gallinarum, Erwinia amylovora, E. coli, Citrobacter koseri, Acinetobacter radioresistens, Actinomyces spp. HPA0247, Shewanella putrefaciens, Salmonella enterica, and F. nucleatum were observed in postmenopausal BC patients.	^Citation96
	Patients with postmenopausal BC showed elevated levels of Ruminococcaceae, Clostridiaceae, and Faecalibacterium and low amounts of Dorea and Lachnospiraceae.	^Citation97
	Patients with postmenopausal BC had higher levels of Clostridiales, and the abundance of Bacteroides decreased in their feces.	^Citation98
Lymphoma	Lactobacillus johnsonii was lacking in lymphoma mice.	^Citation99
Lung cancer	Enterococcus was enriched in the gut microbiota of lung cancer patients.	^Citation100
	Dialister, Kluyvera, Faecalibacterium, Escherichia-Shigella, and Enterobacter were decreased, whereas Bacteroides, Fusobacterium, and Veillonella were notably higher in lung cancer patients.	^Citation101
	Bacillus and Akkermansia muciniphila were enriched and facilitated the growth of lung cancer.	^Citation102
	Eight genera, including Collinsella, Klebsiella, Enterococcus, Bifidobacterium, Lactobacillus, Streptococcus, Escherichia, and Dorea, were increased in the feces of lung cancer patients, yet Prevotella and Coprococcus abundances had a sharp decline.	^Citation103
	At the genus level, three genera, Bacteroides, Fusobacterium, and Veillonella, were higher, while five genera, Escherichia-Shigella, Dialister, Kluyvera, Enterobacter, and Fecalibacterium were decreased in the lung cancer patients.	^Citation104
Non-small cell lung cancer	Rikenellaceae, Enterobacteriaceae, Prevotella, Lactobacillus, Streptococcus, Oscillospira, and Bacteroides plebeius were significantly higher in the NSCLC patients.	^Citation105
	Gut butyrate-producing bacteria exhibited remarkably low levels, including Clostridium leptum, Clostridial Cluster I spp, Ruminococcus, and F. prausnitzii.	^Citation106
Prostate cancer	The abundance of Bacteroides massiliensis increased, while Eubacterium rectalie and F. prausnitzii levels decreased in the gut of people with prostate cancer.	^Citation107
Bladder cancer	Cancer patients had an enrichment in genera Fusobacterium, Actinobaculum, Facklamia, Campylobacter, Veillonella, and Streptococcus, while Corynebacterium was decreased in the gut.	^Citation108
	Thermoactinomycetaceae was enriched, and the abundance of Sphingobacteriaceae was significantly decreased in cancer patients.	^Citation109
	Prevotella and Clostridium cluster XI were decreased in the gut.	^Citation110
Epithelial ovarian cancer (EOC)	The beneficial bacteria Actinobacteria, Coprococcus, Bifidobacterium, and Ruminococcus were decreased, while Bacteroides, Peptostreptococcus, Proteobacteria, Fusobacteria, Prevotella, and Gardnerella were observed to had significant enrichment of in epithelial ovarian cancer patients.	^Citation111
	The genus Akkermansia was markedly reduced.	^Citation112
Cervical cancer	In the intestines of patients with early-stage cervical cancer, there is a decrease in Bacteroides, Bifidobacterium, Haemophilus, Faecalibacterium, Blautia, Clostridium, Roseburia, Ruminococcus, Gemmiger, Lachnospira, Unclassified Lachnospiraceae, and Unclassified Clostridiales, and a stimulation of various other genera including Prevotella, Streptococcus, Varibaculum, Peptoniphilus, Actinobaculum, Finegoldia, Dialister, WAL_1855D, Peptostreptococcus, Anaerococcus, and Corynebacterium.	^Citation113
	There were increases in the phylum Proteobacteria and genera Parabacteroides, Escherichia Shigells, and Roseburia.	^Citation114

With a troubling rise in occurrence among young adults over the past 20 years, colorectal cancer (CRC) has risen to the third most common cancer among all cancers worldwide.¹¹⁵ Numerous studies have illuminated the complex interaction between the gut microbiota and the emergence of CRC. Moreover, certain members of the microbial population have been linked to colorectal cancer. Animal models used in functional investigations have identified several microorganisms, including Escherichia coli, Enterococcus faecalis, Fusobacterium nucleatum, and Bacteroides fragilis, and demonstrated their potential carcinogenic roles in colorectal cancer development.¹¹⁶ B. fragilis can produce B. fragilis toxin (BFT), which triggers inflammatory responses and the abnormal proliferation of intestinal cells.⁶⁶ Enterotoxigenic B. fragilis causes colonic signal transduction and activates transcription-3 (Stat3) activation and T helper 17 (Th17) cell responses, thereby promoting colon tumorigenesis.¹¹⁷ Patients with colorectal cancer have been found to have more B. fragilis than healthy controls. Furthermore, F. nucleatum, which is more prevalent in the feces of people with CRC, is known to adhere to colorectal cancer cells. This bacterium might aid cancer growth by promoting inflammation and inhibiting the immune response.^118,119 In particular, the F. nucleatum can stimulate TLR4 signaling to encourage the development of tumors.^67,120 Extracellular superoxide and hydrogen peroxide produced by E. faecalis have been shown to cause DNA damage in colonic epithelial cells.⁶⁷ Additionally, E. faecalis can produce cytotoxic necrotizing factor 1 (CNF1), which results in the growth and spread of cancer cells.¹²⁰ Additionally, E. coli and Streptococcus gallolyticus both promote CRC development.^68,69 Polyketide synthase-positive (pks+) E. coli-derived colibactin, a small-molecule genotoxin, can induce double-strand DNA breaks (DSBs), causing DNA damage in colonic epithelial cells to facilitate CRC progression.^121,122 In addition to these bacterial species that promote colorectal cancer, the intestinal microbial community structure of colorectal cancer patients also has unique characteristics that are significantly different from those of healthy people. Human studies have revealed that CRC patients have increased species richness in the gut microbiota, with decreases in Roseburia and increases in the Bacteroides, Escherichia, Fusobacterium, and Porphyromonas genera.^64,65 Another study demonstrated that the genera Prevotella, Peptostreptococcus, Parvimonas and Porphyromonas were increased in the fecal samples of CRC patients.^71–74 Among them, Peptostreptococcus anaerobius, which belongs to the genus Peptostreptococcus, is a CRC-enriched bacterium that promoted carcinogenesis by activating the TLR2 and/or TLR4 pathways in mouse models.¹²³ Moreover, Clostridium difficile was also enriched in CRC patients.⁷⁵

These unique characteristics can be used as biomarkers for early diagnosis. Yu and her team employed seven bacterial species, including B. fragilis, F. nucleatum, Parvimonas micra, Porphyromonas asaccharolytica, Prevotella intermedia, Thermanaerovibrio acidaminovorans, and Alistipes finegoldii, as biomarkers for early-stage colorectal cancer diagnosis. They achieved an area under the receiver-operating characteristics curve (AUC) of 0.80; when combined with clinical information, the accuracy was further enhanced to an AUC of 0.88.¹²⁴ Additionally, Ma et al. developed an innovative prediction model that utilized single nucleotide variant (SNV) sites within the gut microbiota to diagnose early CRC. This prediction model contained 22 single-nucleotide variant sites (SNVs), primarily derived from Eubacterium rectale and F. nucleatum, and the model demonstrated high accuracy in distinguishing between disease and healthy cohorts (AUC = 73.08%~88.02%). Furthermore, when tested via a meta-analysis that included patients with other metabolic disorders, the model exhibited excellent specificity.^23,125

Esophageal cancer has long been regarded as one of the most prevalent cancers in the world, with common symptoms including difficulty swallowing, which can lead to indigestion.¹²⁶ Park et al. shed light on a potential link between gram-negative bacteria expressing lipopolysaccharides and enhanced inducible nitric oxide synthase expression (iNOS). This may lead to a reduction in esophageal sphincter relaxation, which ultimately contributes to gastroesophageal reflux disease (GERD) and is linked to an increased risk of esophageal cancer.¹²⁷ In the context of esophageal cancer, distinct alterations in the gut microbiota have been observed. Compared to healthy individuals, patients with esophageal cancer often have lower levels of Bacteroidetes and higher amounts of Firmicutes and Actinobacteria.⁷⁷ Other research has also shown that individuals with esophageal cancer have lower Bacteroidetes, Fusobacteria, and Spirochaetes in their feces.⁷⁸

Amino acids, bile acids, short-chain fatty acids, and choline are essential signaling molecules and metabolic substrates that influence liver function.^128,129 Intestinal barrier dysfunction allows intestinal-derived bacteria and microbiota metabolites, particularly lipopolysaccharides, to induce CXCL1 expression in hepatocytes in a TLR4-dependent manner and increase CXCR2+ PMN-MDSCs. The increase in PMN-MDSCs could induce liver cells to form an immunosuppressive environment and lead to the occurrence of liver cancer.¹³⁰ In fecal samples of hepatocellular carcinoma (HCC) patients, the genera Streptococcus, Lactobacillus Prevotella_9, Faecalibacterium, and Bacteroides were enriched, whereas Akkermansia, Subdoligranulum, Prevotella_2, and Faecalibacterium were reduced. These bacteria are all involved in BA synthesis.^79–82,131 Meanwhile, in fecal samples of liver cancer patients, lipopolysaccharide (LPS)-producing genera, such as Klebsiella, increased, whereas butyrate-producing genera, such as Ruminococcus, decreased.⁸⁴ Research has shown that the presence of GelE-positive E. faecalis can promote the progression of liver cancer. This bacterium expressed metallopeptidase GLE, which increased intestinal permeability, led to an increase in plasma lipopolysaccharides and activation of hepatocyte TLR4-MYD88 proliferation signaling, and finally promoted the development of liver cancer.⁸⁵

Based on these microbes, eight genera, including Faecalibacterium, Lactobacillus, Klebsiella, Citrobacter, Dorea, Ruminococcus Gnavus group, Veillonella, and Burkholderia Caballeronia Paraburkholderia, were proposed to be used in the gut microbiota-based model, and high accuracy (AUC >0.90) was obtained when distinguishing patients with liver cancer from healthy controls.¹³² Zhang et al. employed only the three genera mentioned above, Faecalibacterium, Burkholderia Caballeronia Paraburkholderia, and Ruminococcus-1, as biomarkers to differentiate between cholangiocarcinoma (CCA) patients and healthy subjects.⁸⁸ Han et al. employed Enterococcus, Lactobacillus, and Escherichia as biomarkers to distinguish between groups with and without lung cancer and achieved an accuracy of 0.81.¹⁰³

Chronic inflammation is intimately related to cancer development, and the gut microbiome may impact pancreatic tissue through the inflammatory process, thereby promoting the growth and spread of pancreatic cancer. In patients with pancreatic ductal adenocarcinoma (PDA), comparative investigations of the gut microbiota have revealed that at the phylum level, Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia are more prevalent in these patients’ guts.⁹⁰ During the inflammatory process, changes in gut permeability occur, which enable the translocation of intestinal microorganisms, particularly those belonging to the Proteobacteria phylum, which migrate to the pancreas and accumulate there. In turn, Toll-like receptor 2 and Toll-like receptor 5 are consequently activated in the pancreas and trigger downstream immune suppression. This combined effect and genetic risk factors promote the development of PDA.^90,133 Additionally, another study indicated that pancreatic adenocarcinoma patient feces contained higher concentrations of the bacteria Fusobacterium hwasookii, Veillonella atypica, F. nucleatum, and Alloscardovia omnicolens. At the same time, Bacteroides coprocola, Faecalibacterium prausnitzii, Romboutsia timonensis, and Bifidobacterium bifidum were deficient.⁹¹ Similarly, another pancreatic adenocarcinoma patient study confirmed a decrease in F. prausnitzii. Concurrently, Veillonella parvula, V. atypica, and Streptococcus species were more abundant in the intestines.⁹²

Breast cancer predominantly affects females, with a significant increase in breast cancer risk observed during menopause. Hormone levels undergo substantial fluctuations in women before and after menopause, accompanied by significant alterations in the gut microbiota. Consequently, compared to healthy individuals, there are distinct characteristics in the gut microbiota of premenopausal and postmenopausal breast cancer patients. In premenopausal breast cancer patients, more Enterobacteriaceae, Lactobacilli, and aerobic Streptococci were present compared to healthy individuals.⁹⁴ Additionally, there was an observable increase in the presence of Bacteroides, Clostridia, and aerobic Lactobacilli.⁹⁵ However, postmenopausal breast cancer patients had a decrease in Bacteroides abundance and an increase in Clostridiales abundance.⁹⁸ At the species level, postmenopausal breast cancer patients exhibited a decreased abundance of Roseburia inulinivorans and Eubacterium eligens, while Acinetobacter radioresistens, E. coli, Salmonella enterica, Erwinia amylovora, Citrobacter koseri, Shewanella putrefaciens, Enterococcus gallinarum, Actinomyces spp. HPA0247 and F. nucleatum had an increased abundance.⁹⁶ The reason behind these differences is widely believed to be linked to fluctuations in estrogen levels, which have been linked to the development of breast cancer. Possible mechanisms include, on the one hand, the hypothesis that estrogen metabolism can be influenced by gut bacteria. For postmenopausal women in particular, elevated estrogen levels are a significant risk factor for breast cancer.¹³⁴ Certain gut microbiota can also induce 16α-hydroxylation of estrogen, which raises the risk of developing breast cancer.¹³⁵ On the other hand, a high-fat diet may cause the gut microbiota of patients to create steroidal chemicals that are linked to cancers. These steroids may influence estrogen levels or affect breast tissue, which could promote the growth of tumors.¹³⁶ Furthermore, breast cancer incidence and inflammation are tightly related. The gut microbiota regulates mucosal and systemic immune responses, impacting cancer cells and the surrounding environment.¹³⁷ This influences obesity status, breast cancer risk, and endogenous and exogenous metabolism.¹³⁸

These findings highlight the intricate interplay between specific gut microbiomes and the development of colorectal cancer, thereby offering potential avenues for further research and therapeutic interventions.

3.2. The gut microbiome and the tumor microenvironment (TME)

Although the gut microbiota has been identified as a pivotal biomarker and modulator in cancer development and treatment response, recent research has provided evidence that the gut microbiota has a crucial impact on the TME and impacts both local and distant tumors (Figure 2). One notable example is pancreatic adenocarcinoma, a cancer typically associated with poor survival rates. Research conducted in 2019 on pancreatic adenocarcinoma patients revealed that long-term responders, specifically those surviving for over five years, exhibited a more diverse tumor microbiome than short-term survivors. Notably, some of the microbes within the tumors originated from the gut, which may communicate through the pancreatic duct and may be impacted by the regulation of gut microbial communities.¹³⁹ This underscores the role of gut microbiota in the tumor microenvironment, a role further supported by experiments involving fecal microbiota transplantation (FMT). FMT from long-term survivors effectively slowed tumor development in pancreatic adenocarcinoma mice and bolstered their immune cell activity.¹⁴⁰ Many studies have also reinforced the importance of local microbial communities as essential constituents of the TME. This significance is particularly evident in cancers developing at mucosal locations such as the lungs, skin, and gastrointestinal tract.^141–144 Notably, the bacterial populations residing in tumors are unique to certain tumors.^145,146

Figure 2. The crosstalk between gut microbiome and TME.

3.2.1. Characteristics of the tumor microbiome in various types of tumors

Microbiomes are present in various tumor tissues and play a vital role in tumor development, metastasis, and treatment. Within the context of cancer development, microbes within the tumor microenvironment can be a double-edged sword. On the one hand, they can facilitate tumor development and spread through mechanisms such as inducing DNA mutations, activating oncogenic pathways, fostering chronic inflammation, and promoting complement system activity and metastasis.¹⁴⁷ On the other hand, these tumor-associated microbes may also contribute to inhibiting tumor growth or enhancing the immune system’s response. They can either strengthen or weaken the antitumor immune response, induce different immunotherapeutic effects and outcomes, and impact long-term survival rates.^147,148 Notably, recent research by Nejman et al. analyzed seven different cancer types and revealed distinct microbial compositions within tumors and cells across various cancer types.¹⁴⁸ Numerous studies have also found that different tumor types exhibit unique microbial community structures in tumors such as those associated with colorectal, lung, and breast (Table 2).^148–150

The phyla Firmicutes and Bacteroidetes were the most predominant in colorectal cancer tissues.¹⁴⁸ Yachida et al. found that the species F. nucleatum and Solobacterium moorei showed a progressive rise from the early to late stages of carcinogenesis in colorectal tumors.^72,151 In addition, enterotoxigenic B. fragilis and E. coli both had a high abundance of tumors, and the colonization of tumors by E. coli expressing the genomic island polyketide synthase (pks+ E. coli) leads to an increase in tumor multiplicity and invasion.^68,152–154

Lung cancer is the most prevalent cancer, with the highest occurrence and fatality rates. Research has shown that both healthy and diseased lungs are not sterile environments. Tumor tissues harbor unique microbial communities. For instance, Laroumagne et al. detected E. coli, Haemophilus influenzae, Enterobacter, and Staphylococcus enrichment in lung cancer tissues.¹⁵⁵ Lee et al. identified phyla such as Firmicutes and TM7 and genera including Veillonella and Megasphaera that were notably elevated in lung cancer tissues.¹⁵⁶ Another study revealed specific microbial compositions in lung cancer tumor tissues, including Streptococcus, Acinetobacter, Thermus, Megasphaera, Granulicatella adiacens, Enterococcus, Prevotella, Rothia, Brevundimonas, Propionibacterium, Enterobacter, E. coli and Legionella.^157–159 Research has indicated that the Actinobacteria and Firmicutes phyla showed a higher abundance in lung cancer tissues than in healthy individuals.¹⁶⁰ The genera Staphylococcus, Escherichia, and Bacillus were the characteristic markers of the lung cancer group.¹⁶¹ Discrepancies in detection results may be attributed to tumor subtypes, sampling locations, different stages of the disease, and various detection methods. Yu et al. found that the abundance of the Thermus genus was increased and related to advanced-stage cancer.¹⁴³ Yan et al., using quantitative PCR methods, confirmed the significant alterations in Neisseria, Capnocytophaga, and Veillonella among lung cancer patients.^156,162,163 In addition, in small-cell carcinoma (SCC), the genera Klebsiella, Comamonas, Rhodoferax, Acidovorax, and Polaromonas are commonly reported.¹⁶⁴

In pancreatic ductal adenocarcinoma, at the phylum level, the bacterial phyla Proteobacteria (45%), Bacteroidetes (31%), Firmicutes (22%), and Actinobacteria (1%) were most prevalent in human pancreatic ductal adenocarcinoma (PDA) samples. Proteobacteria dominate the microbiome of pancreatic adenocarcinoma,⁹⁰ and these bacteria can induce T-cell anergy in a Toll-like receptor-dependent manner, thereby hastening the advancement of tumors. Among them, Gammaproteobacteria were detected in 76% of PDAC patients.¹⁶⁵ At the genus level, the genera Pseudomonas and Elizabethkingia were predominant in human PDA samples.⁹⁰ Bifidobacterium pseudolongum was also detected in the pancreas.⁹⁰ Additionally, another study indicated that F. nucleatum was enriched in PDAC cohorts.¹⁴⁸ Riquelme et al. found that Pseudoxanthomonas, Streptomyces spp., and Saccharopolyspora were enriched in patients with longer survival rates. In contrast, Bacillus clausii was increased in pancreatic adenocarcinoma (PDAC) patients with shorter survival.¹⁴⁰

In cholangiocarcinoma tumors, a total of 266 species, including those of Clostridiales, Sphingomonadales, Pseudomonadales, Burkholderiales, Bacillales, and Xanthomonadales, were detected in intrahepatic cholangiocarcinoma.¹⁶⁶ There was also an increase in H. pylori, Helicobacter hepaticus, and Helicobacter bilis.^167,168 The potential mechanism underlying these changes is that gram-negative bacteria affect hepatocytes to create a microenvironment that is immunosuppressive by inducing CXCR2+ PMN-MDSC accumulation through TLR4-dependent CXCL1 production, thereby promoting the development of liver tumors.¹⁶⁹

Breast cancer (BC) is one of the most prevalent malignancies in women. Numerous studies have identified microorganisms important for breast tumor development, progression, and prognosis. Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia were enriched in breast tumor tissues.¹⁷⁰ Thompson et al. also confirmed that Firmicutes, Actinobacteria, and Proteobacteria were increased, and the species Mycobacterium phlei and Mycobacterium fortuitum were abundant in breast tissues as well.¹⁷¹ In the four BC subtypes, Proteobacteria and Actinomyces were also confirmed to be significantly enriched.¹⁷² According to Xuan et al., Methylobacterium radiotolerans was enriched, while Sphingomonas yanoikuyae was decreased in breast tumor tissue at the genus level.¹⁷⁰ Many studies have found that F. nucleatum is enriched and accelerates the development and metastasis of breast tumors.^148,173,174 In malignant breast tissue samples, the genus Fusobacterium was found to have a high accumulation.¹⁷⁴ F. nucleatum can promote aggressive tumor behaviors through Treg lymphocytes in a chemokine-dependent manner, particularly involving CCL20; this bacterium is thereby associated with an unfavorable prognosis.¹⁷⁵ B. fragilis was also detected in cancerous breast tissues.¹⁷⁶ Urbaniak et al. indicated that Comamondaceae, Bacteroidetes, Enterobacteriaceae, Bacillus, and Staphylococcus were enriched in BC cancer patients.¹⁷⁷ Meng et al. showed that the genus Propionicimonas and families Methylobacteriaceae, Caulobacteraceae, Nocardioidaceae, Rhodobacteraceae, and Micrococcaceae were enriched in BC tissues.¹⁷⁸ Additionally, the genus Agrococcus, which is connected to malignancy, and the relative abundance of the family Bacteroidaceae were reduced.¹⁷⁸

Table 2. Characteristics of the microbiomes in various types of Tumors.

Cancer type	Intratumoural microbiome	References
Intramucosal carcinomas	Atopobium parvulum and Actinomyces odontolyticus abundance dramatically increased at an early stage of multiple polypoid adenomas and intramucosal carcinomas. Moreover, the number of F. nucleatum continues to rise into the late stages of cancer.	^Citation72
CRC	From the early to the late stages of carcinogenesis, the species Solobacterium moorei and F. nucleatum showed a considerable increase in abundance.	^Citation72,Citation151
	Enterotoxigenic B. fragilis (ETBF) has a high abundance in tumors.	^Citation152
	Colonizing tumors by pks+ E. coli leads to an increase in tumor multiplicity and invasion.	^Citation68,Citation153,Citation154
	F. nucleatum was observed to have a significant concentration in CRC cancerous tissue.	^Citation179 , Citation180,Citation181
	The abundance of phyla Firmicutes and Bacteroidetes were dominant in colorectal tumors.	^Citation148
Lung cancer	High levels of Haemophilus influenzae, E. coli, Staphylococcus, and Enterobacter were shown in lung cancer tissues.	^Citation155
	Significant alterations in Neisseria, Capnocytophaga, and Veillonella were confirmed among lung cancer patients.	^Citation156,Citation162,Citation163
	Streptococcus, Acinetobacter, Thermus, Megasphaera, Granulicatella adiacens, Enterococcus, Prevotella, Rothia, Brevundimonas, Propionibacterium, Enterobacter, E. coli and Legionella were identified in tumor tissues.	^Citation157 , Citation158,Citation159
	Acidovorax is more prevalent in the tissue of squamous cell carcinoma.	^Citation164
	The Actinobacteria and Firmicutes phyla were enriched in the lung cancer tissue.	^Citation160
	Phyla such as Firmicutes and TM7, as well as genera Veillonella and Megasphaera, were notably elevated in lung cancer tissues.	^Citation156
	Genera Klebsiella, Comamonas, Rhodoferax, Acidovorax, and Polarmonasare were more commonly discovered in SCC.	^Citation164
	The genera Staphylococcus, Escherichia, and Bacillus were the characteristic markers of the lung cancer group.	^Citation161
	Thermus genus abundance was increased and related to advanced-stage cancer.	^Citation143
Pancreatic cancer	Pseudoxanthomonas, Streptomyces spp., and Saccharopolyspora were enriched in patients with longer survival rates. Meanwhile, Bacillus clausii increased in pancreatic adenocarcinoma (PDAC) patients with shorter survival.	^Citation140
	Proteobacteria dominated the microbiome of pancreatic adenocarcinoma.	^Citation90
	F. nucleatum enriched in PDAC cohorts.	^Citation148
	Gammaproteobacteria were detected in 76% of PDAC patients.	^Citation165
	Proteobacteria (45%), Bacteroidetes (31%), Firmicutes (22%), and Actinobacteria (1%) were most prevalent in human pancreatic ductal adenocarcinoma (PDA) samples. At the genus level, the genera Pseudomonas and Elizabethkingia were predominant in PDA samples.	^Citation90
	B. pseudolongum was detected in the pancreas.	^Citation90
Melanoma	Fusobacterium and Trueperella were enriched in melanoma tumors.	^Citation182
	In tumors of anti-PD-1 immunotherapy responders, Clostridium was more predominant, while Gardnerella vaginalis was enriched in nonresponders’ tumors.	^Citation183 , Citation184,Citation185
Esophageal cancer	F. nucleatum in esophageal cancer tissues promoted aggressive tumor behaviors and was correlated with a poor prognosis.	^Citation175
Liver cancer	Bacteroides and Bifidobacterium were detected in the tumor lesion, which was linked to hepatocellular carcinoma caused by HBV.	^Citation148,Citation186,Citation187
	The phyla Proteobacteria and Actinobacteria were dominant in the TME of HCC.	^Citation188
	Pseudomonadaceae was decreased, while the family Rhizobiaceae and genus Agrobacterium were markedly elevated in tumor tissues, which had a linear relationship with patients’ chances of surviving primary liver cancer.	^Citation189
	Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were the most prevalent bacterial phyla in malignancies.	^Citation189
Intrahepatic cholangiocarcinoma (ICC)	A total of 266 species, including those of Burkholderiales, Pseudomonadales, Xanthomonadales, Bacillales, Clostridiales, and Sphingomonadales, were detected in intrahepatic cholangiocarcinoma.	^Citation166
Cholangiocarcinoma (CCA)	There was an increase in three Helicobacter species, H. Pylori, H. Bilis, and H. Hepaticus.	^Citation167,Citation168
Breast cancer (BC)	B. fragilis was detected in cancerous breast tissues.	^Citation176
	A high accumulation of the genera Fusobacterium was observed in the microbiome of malignant breast tissue samples.	^Citation174
	F. nucleatum is enriched and accelerates the development and metastasis of breast tumors.	^Citation148,Citation173
	Comamondaceae, Bacteroidetes, Enterobacteriaceae, Bacillus, and Staphylococcus were enriched in BC cancer patients.	^Citation177
	The genus Propionicimonas, family Methylobacteriaceae, Caulobacteraceae, Nocardioidaceae, Rhodobacteraceae, and Micrococcaceae were enriched in BC tissues.	^Citation178
	The genus Agrococcus, which is connected to the emergence of malignancy, and the relative abundance of the family Bacteroidaceae was reduced.	^Citation178
	Firmicutes, Actinobacteria, and Proteobacteria, were increased, and the species Mycobacterium phlei and Mycobacterium fortuitum were also abundant in breast tissues.	^Citation171
	In the four BC subtypes, Proteobacteria and Actinomyces were also confirmed to be significant enrichment.	^Citation172
	Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia were enriched in breast tissue.	^Citation170
	Methylobacterium radiotolerans was enriched, while Sphingomonas yanoikuyae decreased in breast tumor tissue at the genus level.	^Citation170
Gastric cancer	Persistent infection with H. pylori has been determined to be a significant factor in the progression of gastric cancer.	^Citation190
Esophageal cancer	Intratumoural F. nucleatum was detected and could affect the malignant behavior of esophageal cancer.	^Citation191
Prostate cancer	Staphylococcus saprophyticus and Vibrio parahaemolyticus were significantly decreased, and Shewanella and Microbacterium were enriched.	^Citation192
	The most common bacteria were Propionibacterium acnes spp., which were strongly related to prostate tissue inflammation.	^Citation193
	Family Enterobacteriaceae, specifically the genera Escherichia and Propionibacterium acnes, were dominant in tumors, with a relative abundance of 95%.	^Citation194
	Staphylococcus spp exhibited an increased abundance in tumors.	^Citation195
	More than 40 distinct bacterial genera were identified and dominant in the tumors, including Pseudomonas, Escherichia, Acinetobacter, and Propionibacterium.	^Citation196
	The phyla Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes were predominant in tumors.	^Citation197
Oral squamous cell carcinoma (OSCC)	The genus Peptostreptococcus was detected, and higher levels of Peptostreptococcus were correlated with long-term survival.	^Citation198
	The intratumoral microbiota was dominated by bacterial species that belong to the Fusobacterium and Treponema genera.	^Citation199

3.2.2. The tumor microbiome and tumor development

The tumor microbiome, which accounts for approximately 25% of all cells in the tumor environment (including immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, etc.), influences tumor development and treatment through various mechanisms.²⁰⁰ These mechanisms include inducing DNA damage and mutations, thereby directly promoting tumorigenesis by increasing mutagenesis,^145,201,202 activating oncogenic signaling pathways, and enhancing cell proliferation and transformation.^203,204 Several studies have pointed to members of the Enterobacteriaceae family, which produce colibactin, a DNA-damaging toxin that induces senescence in epithelial cells. Senescent cells activate signaling pathways that ultimately promote the proliferation of neighboring cells unaffected by colibactin damage, thus inducing tumor formation.²⁰⁵ The pks+ E. coli, a bacterium known to cause DNA damage, leads to distinct mutational features in organoids.²⁰⁶ Another group of bacteria capable of inflicting DNA damage includes enterotoxigenic B. fragilis, which produces a toxin called fragilysin. This zinc-dependent metalloproteinase promotes the cleavage of epithelial cell E-cadherin. This cleavage releases β-catenin, activating cell proliferation-regulating transcription factors and facilitating the proliferation of colon epithelial cells.²⁰⁷ Furthermore, F. nucleatum, with its antigen adhesin A (FadA), encourages the development of CRC through the E-cadherin-calcium-catenin-Wnt-β-catenin signaling pathway.²⁰⁸ H. pylori contributes to CRC through the induction of inflammation and by regulating mucosal cell growth and proliferation via critical intracellular signaling pathways.²⁰⁹

Additionally, the tumor microbiome can also break down and metabolize antitumor drugs, reducing their effectiveness and leading to drug resistance.²¹⁰ For instance, Ravid Straussman’s research has demonstrated that microbes within tumors can directly metabolize gemcitabine, leading to chemotherapy resistance in pancreatic cancer. Gemcitabine resistance was mediated by intratumoral Gammaproteobacteria and Bifidobacterium pseudolongum in mouse models.^165,211 Moreover, certain bacteria, including intratumoral Mycoplasma hyorhinis and species of Proteobacteria, can also metabolize gemcitabine into 2,2-difluoro deoxyuridine, rendering the drug inactive and causing resistance.¹⁶⁵ Using ciprofloxacin to eliminate bacteria could overcome this resistance in mouse models.

The tumor microbiome can also modulate the host immune system, thereby influencing immune cell activation and polarization and altering the immune milieu within the tumor microenvironment.²¹² For instance, intratumoral bacteria can activate the functionality of antitumor T cells by producing immunogenic molecules or stimulating interferon signaling.²¹³ Certain intratumoral microbes can affect the synthesis and secretion of cytokines such as IL-6 and TNF-α.²¹⁴ Numerous studies indicate that the intratumoral microbiota may impact cytokine production, thereby inducing proinflammatory responses that subsequently activate the NF-κB or STAT3 pathway to promote tumor development.^215,216 Triner and colleagues showed that intratumoral bacteria induce the production of IL-17, which promotes B-cell infiltration and tumor development.²¹⁷ Additionally, Toll-like receptors, including TLR4 and TLR5, are closely connected to the interactions between tumors and microorganisms (including the gut and intratumoral microbiota).²¹⁸

4. The gut microbiome and tumor immunotherapy

4.1. Cancer immunotherapy strategies

Tumor immunotherapy is an approach to treatment that stimulates the body’s tumor-specific immune responses, either actively or passively, to harness their inhibitory and cytotoxic functions against cancer cells.⁴ It offers advantages such as specificity, efficiency, and minimal harm to healthy tissues. Unlike conventional treatments such as surgery, targeted therapy, and chemotherapy, immunotherapy does not directly kill cancer cells.²¹⁹ Instead, it mobilizes immune cells within the body capable of recognizing tumors, enhances the body’s immune system’s capabilities, and relies on them to indirectly eliminate and control cancer.²²⁰ This method has fewer side effects, making it a safe and effective alternative. Immunotherapy encompasses various approaches, including immunotherapy checkpoint inhibitors, adoptive cellular therapy (CAR-T, TIL, NK, and CIK/DC-CIK), cancer vaccines, molecular targeted therapies, and immunomodulators such as cytokine therapy (Figure 3).⁴

Figure 3. Cancer Immunotherapy Strategies included immunotherapy checkpoint inhibitors, adoptive cellular therapy, cancer vaccines, and cytokine therapy.

Immunotherapy checkpoint inhibitors (ICIs) act based on the following principles: Immune checkpoint molecules are crucial for regulating the onset and magnitude of immune responses, preserving self-tolerance and autoimmune responses, and reducing tissue damage.²²¹ These checkpoints are expressed on immune cells and inhibit their functions, thereby preventing an effective antitumor immune response and allowing tumors to evade the immune system.²²² Key immune checkpoint molecules associated with cancer are PD-1, Tim-3, CTLA-4, and LAG-3, with the most extensively researched checkpoint inhibitors being PD-1/PD-L1 and CTLA-4 inhibitors.⁴ Their primary function is to impede the interaction between tumor cells expressing immune checkpoints and immune cells, thus thwarting the inhibitory impact of tumor cells on immune cells.

Adoptive cellular immunotherapy (ACI) acts via the following mechanisms: Adoptive cellular immunotherapy transfers immune cells possessing antitumor activity, encompassing specific and nonspecific responses, into individuals with cancer. Immune cells can directly eliminate or trigger the body’s immune response to eradicate tumor cells.²²³ Cell-based immunotherapy has always been one of the most active areas in cancer biotherapy. This therapy is particularly suitable for patients with compromised immune function, leading to reduced immune cell numbers and impaired function, especially for patients with hematological or immunological malignancies. (1) Chimeric antigen receptor T-cell (CAR-T) therapy involves extracting immune lymphocytes from the patient’s blood and genetically engineering them. This modification allows T cells to recognize tumor cell antigens while enhancing their antitumor activity. Then, the modified T cells are reintroduced into the host, specifically targeting and killing cancer cells.²²⁴ (2) Tumor-infiltrating lymphocyte (TIL) therapy involves isolating lymphocytes infiltrating tumor tissue and selecting those with specific anticancer properties.²²⁵ After activation and expansion, these selected lymphocytes are reintroduced into the patient’s body to recognize and target tumor cells specifically. TILs consist of T cells that directly infiltrate tumor tissue, which makes them suitable for solid tumors. (3) Natural killer (NK) cell therapy is based on the following principles: NK cells function as the body’s initial line of defense against cancer and infections and have stronger and more efficient capabilities in killing tumor and virus-infected cells than other immune cells.²²⁶ By isolating and expanding from tumor tissue and then reintroducing them into the patient’s body, tumor cells can be killed. (4) Cytokine-induced killer (CIK) cell therapy is based on the following principles: CIK cells are novel, highly proliferative immune cells with cytotoxic properties and some immunological characteristics. These cells express CD3 and CD56 membrane proteins, resembling NK cells with powerful antitumor activity typical of T lymphocytes.²²⁷

Tumor vaccines are a therapeutic strategy for boosting the immune system’s capacity to combat cancer. These vaccines may contain tumor-associated antigens to assist the immune system in recognizing and targeting cancer cells. Notably, Provenge (Sipuleucel-T) is currently the world’s first and only tumor vaccine approved by the U.S. Food and Drug Administration (FDA).²²⁸ Dendritic cell (DC) vaccines have also made significant breakthroughs in many clinical trials.²²⁹

Molecular targeted therapy focuses on reversing malignant biological behaviors at the molecular level, targeting processes that can lead to cellular transformation, such as cell signaling pathways, oncogenes, tumor suppressor genes, cytokine receptors, antitumor angiogenesis, and suicide genes.²³⁰ This approach aims to control tumor cell growth and achieve complete tumor regression. This approach offers high selectivity at the molecular and cellular levels by efficiently and selectively targeting tumor cells while minimizing damage to normal tissues. Molecular targeted therapy drugs can be broadly categorized into the following two types: monoclonal antibodies and small molecules. Monoclonal antibodies are a key component of biological targeted therapy.

Immunoadjuvants are adjunctive therapies that enhance the activity of the immune system to combat cancer better. These adjuvants can be used in conjunction with other immunotherapy methods.²³¹

Cytokine therapy takes advantage of immune modulators that can help strengthen the immune system’s response against certain tumors. Commonly used cytokines in antitumor immunotherapy include IL-2, IL-12, INF-γ, and TNF.²³²

4.2. The gut microbiome and tumor immunotherapy

In recent years, tumor immunotherapy has developed rapidly and garnered attention due to its high specificity and possible long-term effectiveness.⁴ The most extensively researched immunotherapy strategies are PD-1/PD-L1 and CTLA-4 immune checkpoint inhibitors. Immunotherapy can precisely identify and target tumor cells without causing significant harm to healthy cells. This treatment has shown potential efficacy in various cancer types, including melanoma,^233,234 lung cancer,²³⁵ colorectal cancer,²³⁶ and lymphoma.²³⁷ Some patients achieve long-term clinical responses after immunotherapy and maintain resistance to tumors even after the treatment process.²³⁸ However, the major limitation of tumor immunotherapy is that it does not work for all cancer patients. Some individuals may not exhibit a significant response to treatment. A study conducted in Israel involved 10 individuals with advanced-stage melanoma whose cancer had continued to progress despite prior treatment with a checkpoint inhibitor.²³⁹ Substantial evidence demonstrates that intestinal microorganisms have the potential to impact clinical responses to ICIs and their side effects in a variety of cancer types.⁵

There is clear evidence indicating that the gut microbiota plays a crucial role in shaping the immune system, is closely related to innate and adaptive immunity, and can impact the clinical responses and adverse effects of immune checkpoint inhibitors in various cancer types.^240,241 Components within the gut microbiota can modulate the host’s antitumor immune response.^184,242 Several studies have indicated the role of the gut microbiota in regulating the effectiveness of immune checkpoint blockade therapy.¹⁸⁵ Recent research by a Canadian team explored the potential of FMT from cancer-free donors to prevent immunotherapy resistance in individuals who had not previously received such treatment. Twenty patients with advanced melanoma were employed. Three individuals responded completely after FMT and immunotherapy, 13 partially responded, and three had persistent disease.²⁴³ Additionally, FMT was utilized to evaluate the impact of gut microbiota by transferring fecal microbial communities obtained from responders and nonresponders into a germ-free melanoma model mouse. Intriguingly, researchers found that even without using anti-PD-L1 drugs, transplanting fecal microbiota from responders could lead to tumor shrinkage. A synergistic effect was observed when fecal microbiota transplantation was combined with anti-PD-L1 treatment. However, the combination had no antitumor response in nonresponders.²⁴³ In another study, seven melanoma donors who had achieved a durable response to immunotherapy were employed. Another 16 patients with metastatic melanoma, whose cancer had progressed following prior immunotherapy, were enrolled for FMT with the donors’ feces. Among the 16 recipients, six exhibited a positive response, with three achieving a complete response.²⁰⁰ In melanoma patients who were resistant to anti-PD-1 therapy, FMT from individuals who had responded effectively managed to reinstate the tumor’s sensitivity to PD-1 blockade. This led to beneficial alterations in immune and microbial profiles within both the gut and the tumor microenvironment.^244,245

Distinct features were observed in the responsive and nonresponsive melanoma patients’ gut microbes. Patients who responded to treatment exhibited elevated levels of unidentified species from the Ruminococcaceae and Faecalibacterium families, along with Ruminococcus bicirculans and Barnesiella intestinihominis. Conversely, nonresponders displayed enrichment in Adlercreutzia equolifaciens, Bacteroides thetaiotaomicron, Bifidobacterium dentium, and unidentified species from the Mogibacterium genus.²⁴⁶ However, Matson et al. discovered a strong connection between the clinical response and commensal microbial composition. Furthermore, they identified that E. faecium, Collinsella aerofaciens, and Bifidobacterium longum were common in fecal samples from melanoma patients who positively responded to immunotherapy therapy. Transplanting fecal samples from responding patients into germ-free mice enhanced T-cell responses and increased the effectiveness of anti-PD-L1 therapy.¹⁸⁴ A parallel study delved into the gut microbial populations of melanoma patients and discovered that the abundance of Ruminococcaceae species was linked to clinical responses to checkpoint inhibition.¹⁸³ McCulloch et al. proposed that an undesirable gut microbiota promotes systemic inflammation and resistance factors that affect immunity and the response to immunotherapy. They also noted that the high abundance of Streptococcus species was associated with treatment-related adverse events and a shorter progression-free survival.²⁴⁷ In other research studies, the antitumor effects of CTLA-4 blockade depended on specific Bacteroides species, such as B. thetaiotaomicron and B. fragilis.²⁴² In contrast, the presence of the Bifidobacterium genus was positively linked to the effectiveness of PD-L1 ligand PD-1 blockade.²⁴⁸ Additionally, the presence of A. muciniphila in stool samples positively correlated with clinical responses to ICIs in both patients and mouse models.¹⁸⁵

5. Probiotics regulate the immune system by mediating the gut microbiome

Probiotics are a category of beneficial, active microorganisms that can regulate the gut microbiota and the immune system through multiple mechanisms. (ⅰ) Probiotics can inhibit the colonization and proliferation of pathogenic bacteria in the gut through competitive exclusion, the production of antimicrobial substances, including peptides, bacteriocins, and butyrate,^249,250 and modification of the gut environment. Probiotics can increase the abundance of microorganisms in the gut that produce short-chain fatty acids (SCFAs).²⁵¹ These SCFAs, essential metabolic byproducts of gut microbes, influence immune cells’ metabolism and signal transduction. (ⅱ) Probiotics play a role in maintaining gut permeability and enhancing intestinal barrier function. They achieve this by promoting mucus secretion, increasing tight junction proteins, or reducing intestinal permeability. Research has demonstrated that Lactobacillus rhamnosus GG (LGG) can directly interact with intestinal epithelial cells (IECs) and preserve the integrity of the epithelial barrier.²⁵² Studies have shown that probiotics induce goblet cells to secrete mucins and inhibit pathogen adhesion.²⁵³ Lactobacillus plantarum BMCM12 can secrete extracellular proteins that weaken pathogen adhesion, safeguarding the intestinal barrier.²⁵⁴ (ⅲ) Probiotics also modulate intestinal immune function by interacting with various cell types, including IECs, DCs, T cells, and B cells, by influencing their differentiation, activation, proliferation, and secretion. Probiotics engage with IECs or immune cells associated with the lamina propria through Toll-like receptors. This interaction leads to the release of different cytokines and chemokines, activating both innate immune responses and cytokine release by T cells and stimulating mucosal immune cells.^255,256 Research has indicated that probiotics entering the gut can stimulate the production of IgA antibodies.²⁵⁷ Oral intake of probiotics effectively increases the number of IgA+ cells in the intestinal lamina propria, thereby reinforcing and sustaining immune surveillance in mucosal areas distant from the gut and promoting the maturation of humoral immune mechanisms.^255,258 Probiotics can also augment the number of macrophages and DCs in the lamina propria and enhance their functionality over a specific timeframe. One study demonstrated that specific Lactobacillus probiotic strains activated an in vitro inflammatory response in macrophages by synthesizing proinflammatory mediators, including cytokines and reactive oxygen species (ROS), and were involved in signaling pathways such as nuclear factor-kappa B (NF-kB) and Toll-like receptor 2 (TLR2).²⁵⁷

Probiotics can also impact the host’s immune response by altering the composition and diversity of the gut microbiota. Probiotics can increase the abundance of beneficial bacteria. Treatment with a mixture of Bifidobacterium can modify the gut microbial community, significantly increasing the population of other probiotic species, such as Lactobacillus, Kosakonia, and Cronobacter. These beneficial bacteria can produce metabolites, including SCFAs, which favor immune regulation. Furthermore, the altered symbiotic community enhances the adaptability of mitochondria and intestinal Tregs’ metabolic and inhibitory functions mediated by IL-10. This contributes to maintaining regional immune homeostasis in patients under conditions of CTLA-4 blockade.²⁵⁹ Treg cells are a key mechanism through which probiotics exert their beneficial effects. The mechanism by which probiotics regulate and alleviate inflammatory diseases and atopic dermatitis in neonates and infants was associated with the induction or expansion of Tregs and the manipulation of mucosal DCs to promote regulatory function.^260,261 Furthermore, another study has indicated that bacterial strains from Clostridia clusters IV, XIVa, and XVIII play a pivotal role in Treg development in healthy mice, protecting against pathogens. Additionally, species within the Clostridia group, including F. prausnitzii, may promote Treg differentiation by producing SCFAs.

Beyond this, the next-generation probiotics (NGPs), such as the human commensal bacteria F. prausnitzii or A. muciniphila, have emerged as promising candidates. These strains may offer better adaptability to the gut, are being utilized to alleviate obesity, inflammatory bowel disease (IBD), and cancer, and investigate disease mechanisms.²⁶² Research has shown that the polysaccharide A (PSA) produced by F. prausnitzii can modulate the host’s immune system in terms of both health and disease. PSA also plays a critical role in developing the mammalian immune system and activating CD4+ T cells.²⁶³ F. prausnitzii can enhance the effects of tumor immunotherapy and reduce its side effects.²⁴² Additionally, the presence of A. muciniphila can improve the clinical efficacy of PD-1 inhibitor treatment in patients with NSCLC.²⁶⁴

6. Probiotics enhance tumor immunotherapy via mediating the gut microbiome

The gut microbiota plays a pivotal role in developing and maturing the host’s innate and adaptive immune systems.²⁶⁵ Given the adaptable characteristics of the microbiome, adjusting the microbiota has recently become an appealing approach with the potential to address resistance to tumor immunotherapy in patients. On the one hand, the FMT strategy can be used to transplant the fecal microbiota of responders. On the other hand, increasing research focuses on the regulatory effects of probiotics on the gut microbiota and their promoting effect with tumor immunotherapy (Figure 4).

Figure 4. The primary mechanism of probiotics enhances the effectiveness of tumor immunotherapy.

Routy et al. pinpointed that A. muciniphila could mediate the connection between immunotherapy and treatment response. They found that orally administering A. muciniphila after fecal microbiota transplantation with feces from nonresponders reinstated the effectiveness of PD-1 blockade through an interleukin-12-dependent mechanism. This process facilitated the recruitment of CCR9+CXCR3+CD4+ T lymphocytes into epithelial tumors.¹⁸⁵ Preclinical oral probiotics in mice showed promising results in melanoma and bladder cancer studies. Administration of Bifidobacterium enhanced tumor control significantly. This approach nearly eradicated the tumors when combined with CD274 (PD-L1) blockade. The main contributing factors to success were the improved function of DCs and the recruitment of CD8+ T cells to the tumor microenvironment.²⁴⁸ Le Noci et al. demonstrated that Lactobacillus rhamnosus could counteract immunosuppression and hinder the implantation of lung tumors. Moreover, tumor metastases were reduced when both antibiotics and probiotics were employed. These collective results suggest that the microbiota in the local environment plays a crucial role in the development of lung cancer by influencing the local immune response.²⁶⁶ Supplementation with Lactobacillus pentosus Probio-M9 probiotics promoted the production of beneficial metabolites, such as butyric acid, in the gut by increasing beneficial microbes such as Lactobacillus and Bifidobacterium. In particular, this bacterium accumulated beneficial metabolites, including α-ketoglutarate, N-acetylglutamine, and pyridoxol derived from blood sources, enhancing the anti-PD-1 immune therapy response.¹⁴

In the context of colon cancer, a different study examined the impact of intratumor microbiota on CD47-based cancer immunotherapy. Shi et al. orally administered Bifidobacterium and discovered that Bifidobacterium from the colon accumulates within tumor sites and enhances local anti-CD47 treatment through the STING pathway.²⁶⁷ Additionally, Iida et al. demonstrated that administering Alistipes shahii through oral gavage restored the immunotherapeutic response against colon tumors in mice previously treated with antibiotics.²⁶⁸

In melanoma, C57 mice were orally administered a combination of commensal Bifidobacterium, including B. breve and B. longum, through gavage. This treatment promoted antitumor immunity and enhanced the effectiveness of anti-PD-L1 therapy.²⁴⁸ A recent study published in Cell introduced the only known probiotic capable of homing to tumors and exerting synergistic effects with immunotherapy. Lactobacillus reuteri (Lr) was found to migrate to and colonize melanoma tumors readily and released the dietary tryptophan-derived metabolite indole-3-carbaldehyde (I3A), which stimulated the production of interferon-gamma by CD8+ T cells, leading to the killing of cancer cells and thereby enhanced the efficacy of ICIs. The antitumor immune response induced by I3A secreted by Lr relies on the AhR receptor found on CD8+ T cells.¹⁵

7. Conclusion and outlook

This article explores the relationship between gut microbiota and cancer from the perspectives of gut microbial communities and tumors. We summarized the disruption of gut microbiota, particularly in cancer patients, delving into the underlying mechanisms behind this imbalance. Additionally, the microbial biomarkers that appear in cancer diagnosis and prognosis were addressed. Furthermore, we delved into how the gut microbiota affected the tumor microenvironment, including the composition of tumor-associated microbial communities and their influence on cancer development. By untangling the intricate relationships among microbes, the tumor microenvironment, and cancer cells, valuable insights can be gained for potential cancer treatments, including targeted and personalized therapies, to maximize the efficacy of anticancer treatments. In addition, we highlighted recent advancements in using probiotics to enhance the effectiveness of cancer immunotherapy.

Probiotics are a type of beneficial live microorganism that benefits human health. They can regulate intestinal function, enhance immune system function, and inhibit the growth and spread of tumor cells. In recent years, numerous studies have shown that probiotics can synergize with immune checkpoint inhibitors (such as PD-1, PD-L1, or CTLA-4 antibodies) in various ways to modulate the immune responses of cancer patients, enhancing the effectiveness and tolerance of immunotherapy. On the one hand, probiotics can regulate cancer patients’ gut microbiota and immune microenvironment by promoting the secretion of immune factors at the tumor site, thus influencing the body’s response to immune inhibitors and alleviating the treatment side effects caused by chemotherapy. On the other hand, probiotics can also migrate to tumor sites and exert their inhibitory effects, including stimulating the secretion of immune cell factors and producing antitumor substances such as I3A. This comprehensive review offers valuable insights into the dynamic interactions between gut microbial communities, probiotics, and tumors.

Despite promising research results, the relationship between cancer immunotherapy and probiotics still requires further investigation to clarify their efficacy and safety. Research on the promoting effects of probiotics on cancer immunotherapy continued to be a hot topic in human health. Probiotics could emerge as a novel adjuvant for cancer treatment and be combined with other drugs or therapeutic approaches to improve cancer patient’s prognosis and quality of life. Furthermore, by maintaining a healthy gut microbiome, probiotics might have contributed to the prevention of tumor development or the slowing of tumor progression, providing new avenues for early tumor intervention. Additionally, different types of tumors and patients may respond differently to probiotics, emphasizing the importance of personalized treatment and optimizing the potential of immunotherapy strategies in the future.

Abberivation

TME	=	Tumor microenvironment
ICIS	=	Immune checkpoint inhibitors
I3A	=	Indole-3-carbaldehyde
CRC	=	Colorectal cancer
BFT	=	B. Fragilis toxin
CNF1	=	Cytotoxic necrotizing factor 1
pks+	=	Polyketide synthase-positive
DSBS	=	Double-strand DNA breaks
AUC	=	Receiver-operating characteristics curve
SNV	=	Single nucleotide variant
INOS	=	Inducible nitric oxide synthase expression
GERD	=	Gastroesophageal reflux disease
HCC	=	Hepatocellular carcinoma
LPS	=	Lipopolysaccharide
ICC	=	Intrahepatic cholangiocarcinoma
CCA	=	Cholangiocarcinoma
PDA	=	Pancreatic ductal adenocarcinoma
PDAC	=	Pancreatic adenocarcinoma
ICC	=	Intrahepatic cholangiocarcinoma
BC	=	Breast Cancer
EOC	=	Epithelial ovarian cancer
FMT	=	Fecal microbiota transplantation
SCC	=	Small-cell carcinoma
OSCC	=	Oral squamous cell carcinoma
FADA	=	Antigen adhesin A
ACI	=	Adoptive cellular immunotherapy
CAR-T	=	Chimeric antigen receptor T-cell
TIL	=	Tumor-infiltrating lymphocyte
NK	=	Natural killer
CIK	=	Cytokine-induced killer
DC	=	Dendritic cell
FDA	=	U.S. Food and Drug Administration
SCFAS	=	Short-chain fatty acids
LGG	=	Lactobacillus rhamnosus GG
IECS	=	Intestinal epithelial cells
ROS	=	Reactive oxygen species
NF-kb	=	Nuclear factor-kappa B
TLR2	=	Toll-like receptor 2
Lr	=	Lactobacillus reuteri

Credit authorship contribution statement

Shuaiming Jiang: Conceptualization, Writing – original & editing, Visualization. Wenyao Ma: Writing – original & editing, Visualization. Chenchen Ma: Data curation, Investigation, Visualization. Zeng Zhang: Investigation, Visualization. Wanli Zhang: Conceptualization, Writing – review & editing. Jiachao Zhang: Conceptualization, Writing – review & editing, Funding acquisition.

Acknowledgments

This research was supported by the National Natural Science Foundation of China [32222066] and the Hainan Province Science and Technology Special Fund under Grant [GHYF2023001].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the National Natural Science Foundation of China [32222066] and the Hainan Province Science and Technology Special Fund under Grant [GHYF2023001].