The entire variety of microorganisms that inhabit our bodies is called the microbiota. Bacteria and viruses, fungi and mysterious archaea have settled not only in the intestines, but also in various parts of our bodies. It is difficult to believe that they can regulate appetite or change the way we think. But it is even more difficult to imagine that the microbiota can influence malignant cell transformation and the development of cancer. The relationship between microbiota and tumors is so complex, contradictory, and confusing that it holds no fewer mysteries than the plot of the most exciting detective story. But scientists have already been able to get to know some representatives of the microbiota well enough to understand who is our friend and who is our enemy. Let's try to figure this out.
It has long been known that certain microorganisms can cause malignant cell transformation. In 2024, more than 2,500,000 new cases of cancer associated with infectious agents were identified worldwide. There are many examples of microorganisms associated with carcinogenesis: oncogenic types of human papillomavirus can lead to cervical cancer, hepatitis B and C viruses are associated with liver carcinoma, and Helicobacter pylori bacteria are associated with stomach cancer. Their appearance in the body is highly likely to lead to cancer.
But our normal microbiota is not left out either. Microorganisms that normally inhabit our bodies have many independent and diverse effects on tumors. And although the composition of the microbiota is diverse (it includes viruses, bacteria, and archaea), this article will focus on the influence of bacteria on malignant cell transformation.
Bacteria in the body settle wherever they want. Most of these tiny inhabitants are found in the large intestine, where their number reaches 1011 per ml of intestinal content. The number of bacteria on the skin is no less impressive — about 1011 per m2. At the same time, the microbiota is surprising not only in its abundance but also in its diversity. Even on the cornea of the eye, about 221 different species of bacteria have been found.
By colonizing our bodies, the microbiota protects us from pathogenic microorganisms and helps maintain homeostasis. In addition, bacteria can accompany the mechanisms of carcinogenesis. Some microorganisms help tumors survive, while others, on the contrary, betray them to immune cells. Why is their influence so important? After fatal mutations occur in a cell and it acquires “immortality,” its further fate largely depends on the microenvironment, which, in addition to normal cells of the surrounding tissue, fibroblasts, and immune cells, also contains bacteria. They make up the tumor microbiota, which, along with the intestinal microbiota, influences the growth and development of malignant cells. Let's take a closer look at this.
Cells within cells: bacteria living in tumors
Intratumoral microbiota
Bacteria are part of the tumor microenvironment, with most of them located intracellularly. This conclusion was reached by scientists from the Weizmann Institute in Israel based on a study of 1,526 samples of seven different types of tumors and the normal tissue surrounding them. Samples of melanoma, glioblastoma, breast, lung, ovarian, colon, and pancreatic tumors were studied.
Although the set of microorganisms in each tumor was different and depended on its properties (for example, response to therapy or sensitivity to hormones), common patterns were identified for each type.
Can we say based on this data that the bacteria found caused the tumor to develop? No. Most likely, the microorganisms colonized an existing tumor. The conditions created by malignant cells proved to be an attractive niche for microbes to live in. For example, microorganisms that can break down chemicals in tobacco smoke (nicotine, anthranilate, toluene, and phenol) were found in the lung tumors of smokers. Bacteria capable of doing this belong to the Proteobacteria, Actinobacteria, and Cyanobacteria types. blood supply to the tumor (increased angiogenesis is one of the key signs of cancer), low oxygen concentration, immunosuppressive environment (allowing tumor cells to escape immune surveillance), and the presence of a nutrient substrate — all of these factors probably make a tumor a good place for microorganisms to live. So why wouldn't bacteria settle down next to such convenient neighbors?
How do microbes get into tumors?
A study of pancreatic tumor samples provides an interesting answer to the question of where bacteria come from in tumors. It turns out that the tumor microbiome is about 25% similar to the gut microbiome, about 20% of the bacteria are the same as those living in the surrounding normal tissue, and the rest are unique. But as of today, there's no exact data showing how bacteria actually migrate into tumors. However, this does not prevent us from making plans for the future. If the connection between the intestinal and tumor microbiota is confirmed, we will be able to control the composition of the latter by introducing new inhabitants into the intestine. We will only need to wait until the microorganisms make their way in search of a new place to live.
Impact on treatment outcomes
Surprisingly, the composition and diversity of the intratumoral microbiome can affect a patient's life expectancy after treatment. At the M. D. Anderson Cancer Center at the University of Texas, researchers found that the predominance of Pseudoxanthomonas, Saccharopolyspora, and Streptomyces taxa in tumors in patients with ductal adenocarcinoma of the pancreas is associated with better outcomes after surgery. What is the reason for this? It is believed that their presence has a positive effect on the activation of immune cells and the infiltration of the tumor by cytotoxic CD8+ T cells. These are the very T lymphocytes that play a key role in antitumor defense due to their ability to directly destroy malignant cells.
Unexpected connections: tumor — intestinal microbiota
We remember that part of the intratumoral microbiota coincides in composition with the intestinal microbiota. But the influence of intestinal bacteria on carcinogenesis does not end there. The gut microbiota can have both a systemic effect, affecting tumors in distant organs, and a local effect, directly affecting the cells of the intestine. In addition, the balance of the gut microbiota determines the functioning of the immune system, which monitors all cells in the body.
Let's start with a simple and straightforward statement: the main function of the immune system is to protect our body. When a pathogenic bacterium or virus enters the body, the immune system must fight it off. But how did it happen that our intestines are inhabited by an entire army of bacteria that live seemingly unnoticed and coexist peacefully with their host? The joint evolution of humans and their microbiota has led to a situation where one cannot exist without the other. We provide microorganisms with a comfortable habitat and substrates for nutrition, and in return, they perform many difficult tasks: they synthesize vitamins and even some neurotransmitters that we need, they expand our diet by breaking down fiber that cannot be digested in the small intestine, and they provide so-called colonization resistance — they compete with pathogenic microorganisms and prevent them from colonizing the intestine. In addition, the balance and adequate development of innate and acquired immunity depend on the diversity of the intestinal microbiota.
However, the microbiota can only live without suppression by the immune system if it follows strict rules and does not leave the space allocated to it—the intestine. To maintain such a delicate balance, “negotiations” between the microbiota and the immune system are conducted not directly, but through the intestinal barrier that separates the intestinal cavity from the internal environment of the body. It consists of several layers: the first is a layer of mucus containing antimicrobial peptides, glycoproteins, and secretory immunoglobulin A (IgA); the second is a single layer of epithelial cells (enterocytes) connected by tight intercellular contacts; the third is a subepithelial layer containing cells of innate and adaptive immunity, as well as blood vessels.
This multi-level system is necessary for the microbiota to exist in the intestine without harming the host, and for immune cells to distinguish between their own and foreign cells and not attack friendly bacteria. If the balance is maintained, intestinal bacteria work for the benefit of humans and provide antitumor protection mainly through the synthesis of short-chain fatty acids and modulation of immune system components.
Problems begin when the balance is disturbed: dysbiosis occurs and the permeability of the intestinal barrier increases. Under such conditions, the likelihood that the intestinal microbiota will contribute to malignant cell degeneration increases. Its influence on carcinogenesis is exerted through the maintenance of chronic inflammation, cell damage, and the synthesis of metabolites.
Intestinal bacteria and antitumor protection
Short-chain fatty acids are one of the products of the metabolism of anaerobic gut microbiota, which are responsible for maintaining the homeostasis of the colonic mucosa. Butyrate, propionate, and acetate perform this important function through their ability to modulate the local immune response and maintain the integrity of the intestinal barrier. Short-chain fatty acids stimulate the differentiation of regulatory T cells, the expression of tight junction proteins, and the secretion of mucin (MUC2). In addition, they are able to protect the intestinal mucosa by influencing cytokine secretion.
Cytokines are special protein molecules that allow immune cells to “communicate” with each other and direct each other's actions. Among them are pro-inflammatory cytokines, which promote the development of an inflammatory response, and anti-inflammatory cytokines, which suppress the immune response. Both are important in carcinogenesis, as we will see below.
The protective mechanism of butyrate is associated with increased production of the anti-inflammatory cytokine interleukin-18 (IL-18), which is the result of activation of the GPR109a receptor on the surface of enterocytes (intestinal cells). This cytokine, by influencing the secretion of IFN-γ, activates cells of the monocyte-macrophage series, which enhances the antibacterial and antitumor immune response. IL-18 also regulates the secretion and availability of another cytokine, IL-22, which is necessary for the regeneration of the intestinal mucosa. Thus, butyrate affects the secretion of anti-inflammatory cytokines necessary for adequate epithelial regeneration and maintenance of mucosal integrity.
Butyrate metabolism differs in normal and tumor cells. In normal cells, butyrate performs an energetic function: it is metabolized to acetyl-CoA and participates in the tricarboxylic acid cycle. In tumor cells, despite aerobic conditions, energy is obtained through glycolysis, during which glucose is metabolized to lactate, and the tricarboxylic acid cycle is practically not involved (Warburg effect).
Thus, butyrate remains unused in malignant cells. Accumulating inside cells, it acts as an inhibitor of histone deacetylase. The function of this enzyme is to “cut off” acetyl radicals from histones (proteins associated with DNA). This leads to a change in the conformation of chromatin: it transitions to a condensed state, and the reading of information from DNA ceases. Butyrate, being an inhibitor of this enzyme, prevents chromatin condensation, regulating the expression of certain genes. Thus, through epigenetic mechanisms (non-genomic mechanisms that affect gene activity without affecting the structure and integrity of the genome), butyrate controls genes involved in proliferation (growth) and apoptosis (cell death), suppressing the growth of tumor cells.
Short-chain fatty acids are formed as a result of the breakdown of dietary fiber by bacteria. By inhibiting histone deacetylase, butyrate is able to suppress the proliferation of tumor cells (epigenetic regulation). It also promotes the differentiation of naive CD4+ T cells into regulatory T cells. Butyrate induces the secretion of TGF-β by epithelial cells. By binding to the GPR109a receptor on the surface of epithelial cells, it stimulates the secretion of the anti-inflammatory cytokine IL-18. In addition, butyrate stimulates the production of IL-10 by dendritic cells and macrophages.
Bacteria belonging to the Faecalibacterium and Roseburia genera are capable of producing butyrate. In patients with colorectal cancer, the number of these bacteria is reduced, which apparently affects the permeability of the intestinal barrier and the persistence of inflammation in the intestinal mucosa.
Finally, the intestinal microbiota is capable of activating CD8+ T cells, which play a key role in antitumor defense. Scientists from Japan have identified 11 strains of microorganisms that stimulate the activation of IFN+ CD8+ T cells. At the same time, the number of cytotoxic T cells increased not only in the intestine, but also in some other organs. This systemic effect is likely due to the circulation of metabolites (mevalonate and dimethylglycerol) produced by the intestinal microbiota. The migration of CD8+ T cells from the intestine to distant organs is unlikely, since the cells found in the intestine and those found in other organs are phenotypically different.
Intestinal bacteria — participants in carcinogenesis
Increased intestinal barrier permeability is closely associated with inflammation, which can begin either in the intestine itself (as a result of an imbalance between harmful and beneficial bacteria) or in other organs if microorganisms begin to travel.
Translocation (movement) of microbiota or associated lipopolysaccharides through the intestinal barrier to other organs, such as the pancreas, provokes inflammation and ultimately contributes to the development of cancer. The first link in triggering an immune response is the activation of special pattern-recognition receptors (PRRs) responsible for recognizing molecular structures characteristic of microorganisms (e.g., lipopolysaccharides). These receptors include Toll-like receptors (TLRs), Nod-like receptors (NLR), and others. TLR are the most studied. They are exposed on the membranes of immune cells, fibroblasts, epithelial cells, and tumor cells. The activation of TLR leads to the secretion of pro-inflammatory cytokines, whose main function is to maintain inflammation. Proinflammatory cytokines are key players in carcinogenesis, which is associated with chronic inflammation, enhanced angiogenesis, and the creation of a microenvironment that promotes tumor cell invasion and metastasis. For example, high expression of IL-11 in kidney cancer cells is associated with poor patient survival prognosis.
The inflammatory response is dangerous because the cells involved in this process secrete growth factors, proangiogenic factors (vascular growth factors), reactive oxygen species, and other substances necessary for the existence of tumors.
Interestingly, interactions between the microbiota and Toll-like receptors can lead to both tumor development and regression. This is because the outcome of TLR activation depends on the balance of incoming signals and the adequacy of the force of the initiated process. Therefore, the activation of the same molecules can lead to opposite results. Thus, stimulation of TLR 2,3,4,5,7/8,9 can contribute to antitumor protection. At the same time, stimulation of TLR 2,4,7/8 can increase the resistance of tumors to chemotherapy and accelerate their proliferation.
Another key factor in carcinogenesis is DNA damage. If a cell fails to repair its DNA structure, this damage can trigger the process of malignant transformation. Some intestinal inhabitants are genotoxic, meaning they have the ability to damage genetic material. For example, Escherichia coli (a permanent resident of the intestine) and Klebsiella pneumonia produce colibactin, which can cause double-strand breaks in the host's DNA. The CDT toxin produced by some Proteobacteria has a similar effect.
Another way microorganisms affect tumors is through the action of metabolic products. For example, in colorectal cancer tumor cells, the expression of the enzyme ornithine decarboxylase, which is responsible for the metabolism of polyamines, is enhanced. Although polyamines are necessary for the functioning of normal cells, their increased synthesis contributes to carcinogenesis. Difluoromethylornithine (DFMO) is a drug that can inhibit this enzyme. Why was its use ineffective as monotherapy? After taking inhibitors, the synthesis of endogenous polyamines did indeed stop. But it turned out that their deficiency was completely compensated for by exogenous (from outside, from food) compounds. The bacteria that make up the tumor microenvironment are also capable of synthesizing polyamines. That is why the tumor continued to grow.
Are the bacteria living in the mouth dangerous?
Another “densely populated” area of our body, besides the intestines, is the oral cavity. And even the most squeamish among us have wondered more than once how many bacteria are in saliva... Scientists have also thought about this. It has been established that the number of bacteria in saliva reaches 109. A special database, the Human Oral Microbiome Database, was created to systematize the microbiota of the oral cavity. To date, it contains over 700 species of microorganisms. Among them are those that participate in the carcinogenesis of tumors of the head and neck, lungs, colon, and pancreas.
The mechanisms that allow oral bacteria to influence carcinogenesis are common to microbiota of any location. These include chronic inflammation, suppression of apoptosis, and synthesis of carcinogenic substances.
In the oral cavity, chronic inflammation is mainly caused by anaerobic pathogenic bacteria of the species Porphyromonas, Prevotella, and Fusobacterium. Their presence is associated with high concentrations of cytokines IL-1β, IL-6, IL-17, IL-23, TNF, and matrix metalloproteinases MMP-8 and MMP-9. All of the cytokines listed are pro-inflammatory, and, as we remember, they contribute to maintaining the conditions necessary for the growth and development of tumor cells.
Metalloproteinases are enzymes that play an important role in normal physiological processes such as embryonic development and morphogenesis. They are capable of breaking down various components of the extracellular matrix and basement membrane — collagen, elastin, laminin, and others. Tumor cells also know how to use these enzymes for their own purposes: with the help of metalloproteinases, they destroy components of the basement membrane and separate from neighboring cells. If tumor cells are not bound together, they can migrate and penetrate neighboring tissues much more easily, i.e., their ability to metastasize increases. In addition, metalloproteinases can enhance angiogenesis, which is extremely important for tumor growth.
One of the key signs of cancer is resistance to cell death. Apoptosis is a form of programmed cell death in which a cell that has accumulated harmful mutations and can no longer perform its functions self-destructs without adverse consequences for the body. However, tumor cells acquire the ability to become insensitive to death signals during malignant degeneration. They are aided in this by the bacterium Porphyromonas gingivalis, which can infect tumor cells and stimulate the transmission of anti-apoptotic signals via the Jak1/Akt/Stat3 pathway. In addition, the bacterium produces cysteine proteases — gingipains — which activate matrix metalloproteinase-9. Activation of this enzyme leads to the degradation of the basement membrane structures, as a result of which the cells acquire the ability to migrate.
Hardly any of us would deny ourselves the pleasure of “chewing on something.” That is why microbes in the oral cavity will always find something to feed on. They do not sit idle, but actively metabolize. What? Alcohol, for example. Using the enzyme alcohol dehydrogenase, microorganisms convert alcohol into acetaldehyde, which is a chemical carcinogen. Some types of streptococci are capable of this — S. gordonii, S. mitis, S. oralis, S. salivarius, S. sanguinis, fungi of the genus Candida, and bacteria of the genus Neisseria.
From history to new discoveries
Special interest in the microbiota emerged in 2007, when the US National Institutes of Health launched the Human Microbiome Project. The project revealed that the number of microbial genes in our bodies exceeds the number of human genes by about 100 times. This means that only 1% of our bodies consist of our own genes. It became clear that microorganisms affect us much more than we think. However, our understanding of the role microorganisms play in the development of certain tumors remains incomplete. We have already discussed the general mechanisms by which bacteria in different parts of the body influence malignant cell transformation. Next, we will examine how bacteria affect specific tumors. As an example, we will look at the two most studied tumors: colorectal cancer and breast cancer.
Colorectal cancer
Colorectal cancer ranks fourth among the causes of cancer mortality worldwide. In 45% of patients, colorectal cancer is associated with the presence of the bacterium F. nucleatum in the intestine.
Fusobacterium nucleatum is an anaerobic Gram-negative bacterium that is a commensal inhabitant of the oral cavity. It is classified as a microorganism associated with various forms of periodontitis. In addition, it is known for its ability to leave the oral cavity and reach various distant organs through the bloodstream. F. nucleatum is found in tumors of the breast, pancreas, and colon, where it appears to play a significant role in carcinogenesis. F. nucleatum also possesses unique virulence factors — the adhesins FadA, Fap2, and RadD. It is able to travel through the bloodstream thanks to the FadA adhesin, which allows the bacteria to attach to the epithelial cells of blood vessels. The receptor for FadA is vascular endothelial cadherin (VE), an intercellular interaction molecule that binds cells together. FadA binds to VE cadherin, causing it to move, which increases the permeability of the endothelium and allows the bacteria to enter the bloodstream. Traveling through the bloodstream, F. nucleatum enters the intestine, where it contributes to the development of colorectal cancer through several mechanisms: invasion, colonization, and secretion of pro-inflammatory cytokines.
In addition to adhering to endothelial cells, the FadA protein allows the bacteria to attach to both normal intestinal epithelial cells and tumor cells. F. nucleatum binds to epithelial cadherin (or E-cadherin), a protein that permeates the cell membrane and allows cells to stick together. This activates the Wnt/β-catenin pathway, which normally regulates cell proliferation and differentiation, genetic stability, and apoptosis. Aberrant activation of this pathway leads to the accumulation of β-catenin in the nucleus and promotes the activation of oncogenes such as c-Myc and cyclin D1. In addition, the activation of pro-inflammatory genes creates a microenvironment that promotes the development of colorectal cancer.
Another outer membrane protein, Fap2, belongs to the lectin family, i.e., proteins capable of binding to carbohydrates on the surface of cells. With its help, F. nucleatum attaches to the polysaccharide Gal-GalNAc, which is overexpressed on the surface of colorectal cancer cells. This explains why bacteria seem to gather around the tumor and colonize it much more actively than the surrounding normal tissue. In addition, the Fap2 adhesin allows F. nucleatum to stimulate the production of pro-inflammatory cytokines in tumor cells — IL-8 (chemokine and neutrophil activator) and CXCL1 (chemokine). This creates conditions around the tumor that enhance the metastasis process.
While in the intestine, F. nucleatum interacts with other microorganisms surrounding the tumor. The RadD protein can act as a mediator in communication between F. nucleatum and other bacteria, which contributes to the formation of multispecies biofilms. As a result, conditions favorable for the colonization of F. nucleatum are formed.
The presence of F. nucleatum in colon tumors is associated with a poor prognosis for patient survival. This suggests that the composition of the intestinal microbiota should be taken into account when treating patients with colorectal cancer and that its effect on the course of the disease should be studied in more detail.
Breast cancer
The mammary gland has its own microbiota, and the development of cancer leads to a significant change in its composition.
On average, one in eight women worldwide will develop breast cancer. This insidious disease is often associated with a genetic predisposition (mutations in the BRCA1 and BRCA2 genes) and hormonal factors, namely the effect of estrogen on the female body. The intestinal microbiota also contributes to the risk of developing breast cancer.
The intestinal estrobolome is a collection of intestinal bacterial genes whose products are capable of metabolizing estrogen. Bacteria possessing these genes are capable of influencing estrogen levels in the blood.
In the human body, estrogen metabolism occurs in the liver, where a conjugation reaction takes place, i.e., the attachment of sulfuric acid (sulfation) or glucose (glycosylation) to the hormone. This process converts estrogen into a less active and more polar compound that can be excreted from the body. Conjugated estrogen enters the intestines along with bile.
A diet rich in sugars and fats but low in fiber promotes the growth of bacteria in the intestines that possess the enzyme β-glucuronidase. In addition to breaking down sugars, this enzyme is responsible for the deconjugation of estrogen, which makes it possible for it to be reabsorbed and returned to the bloodstream. Bacteria in the intestinal microbiota that possess β-glucuronidase include members of the genera Collinsella, Edwardsiella, Alistipes, and Bacterioides.
A healthy diet rich in plant foods, fiber, and whole grains, on the other hand, promotes the growth of “good” microbiota. Bacteria of the Firmicutes, Bacteroidetes, and Actinobacteria types are capable of enhancing estrogen metabolism, reducing its content in the body. Thus, by regulating the diet, it is possible to influence the composition of the microbiota and reduce the risk of breast cancer.
Another way to fight cancer
Today, there is no doubt that the microbiota has many diverse effects on tumors. Data from various experiments complement each other and gradually form a complete picture. But the path to understanding how to manage the microbiota for the benefit of humans has only just begun. First, it is necessary to determine which taxa of microorganisms are associated with positive effects and whether they can be used for therapeutic purposes. Second, it is necessary to understand which methods will effectively change the composition of the microbiota. Currently, a method of fecal microbiota transplantation is used, in which stool from a healthy donor is introduced into the patient's intestine. This method is already used in oncohematology to normalize the intestinal microbiota in patients after bone marrow transplantation. But whether it will be effective as a therapy for cancer remains to be seen. Another approach is to follow a high-fiber diet to create the conditions necessary for the reproduction of beneficial microbiota in the intestine. This option can be considered as a preventive measure not only for cancer, but also for many other diseases associated with microbiota. The search for new methods of changing the composition of the microbiota continues: the use of fragments of bacterial cell walls or individual metabolites that can have a beneficial effect could make the procedure more accessible and effective.
Perhaps in the future, changing the composition of the microbiota could become the sixth method of fighting cancer, along with surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy.