Comprehensive retrospect and future perspective on bacteriophage and cancer

Abstract

Background

Researchers gradually focus on the relationship between phage and cancer.

Objective

To summarize the research hotspots and trends in the field of bacteriophage and cancer.

Methods

The downloaded articles were searched from the Web of Science Core Collection database from January 2008 to June 2023. Bibliometric analysis was carried out through CiteSpace, including the analysis of cooperative networks (country/region, institution, and author), co-citations of references, and key words.Visual analysis of three topics, including gut phage, phage and bacteria, and phage and tumor, was conducted.

Results

Overall, the United States and China have the most phage-related research. In terms of gut phage, the future research directions are “gut microbiome”, “database” and “microbiota”. The bursting citations explored the phage-dominated viral genome to discover its diversity and individual specificity and investigated associations among bacteriome, metabolome, and virome. In terms of phage and bacteria, “lipopolysaccharide” and “microbiota” are future research directions. Future research hotspots should mainly concentrate on the further exploration and application of phage properties. As for phages and tumors, the future research directions should be "colorectal cancer", "protein" and "phage therapy". Future directions are likely to focus on the research on phages in cancer mechanisms, cancer diagnosis, and cancer treatment combined with genetic engineering techniques.

Conclusion

Phage therapy would become a hot spot and research direction of tumor and phage research, and the relationship between phage and tumor, especially colorectal cancer (CRC), is expected to be further explored.

Introduction

As one of the major threats to global public health security, cancer is currently considered as a genetic disorder caused by both exogenous and endogenous factors. Smoking, alcoholism, high-fat diet, and other bad lifestyle habits are closely related to the incidence of cancer [1,2,3]. In addition, biological factors, such as virus and bacteria, are also believed to be powerful promoters of the development of cancer especially gastrointestinal cancer [4, 5]. Due to the improvement of medical care and the popularity of early cancer screening, the mortality of melanoma and other cancers has decreased significantly in recent years [6]. However, the incidence of some cancers, including breast and uterine cancer, is on the rise, and the population of colorectal cancer (CRC) patients is becoming younger [6, 7]. Despite the rapid development of current cancer treatment methods, it is still difficult to effectively reduce the high mortality rate of cancer.

As an important member of the human viriome, bacteriophage (phage) has the effect of invading bacteria. The gut is rich in phages, about 30 billions of which enter the body every day through the gut epithelium [8]. Benler et al. identified 3,738 phage genomes out of 5,742 human gut metagenomes and discovered that they were involved in lipid biosynthesis pathways [9]. Phages can indirectly affect human health by infecting bacteria. Phage can change the immunity of gut mucosa, affect the production of interferon-gamma (IFN-γ), and is associated with the aggravation of colitis [10].

Bacteria play a “double-edged” role in the progression of cancer. According to the different effects on cancer, bacteria can be divided into cancer-promoting bacteria, cancer-suppressing bacteria, and cancer-related bacteria whose role is not clear yet. Bacteria and their metabolites can damage cellular DNA, interfere with host signal transduction pathways, and directly contribute to the development of cancer. In addition, the body's immune and metabolic functions can be disturbed by bacteria, which indirectly promotes the occurrence of cancer. The common cancer-promoting bacteria, such as Helicobacter pylori has been reported to induce inflammatory response and signal transduction process, leading to gastric mucosal disorder, chronic gastritis and cancer [11]. Recently, Clostridioides difficile strain, as new members of cancer-promoting bacteria, have been found to be significantly enriched in colon tissue and continuously secrete toxins TcdB to promote the occurrence of CRC [12]. On the other hand, different strains of the same bacteria may have opposite effects on cancer. The “double-edged” role of Escherichia coli in cancer has been widely reported. For example, E. coli with pks island can encode the production of colibactin, induce cell double-stranded DNA break and local inflammatory response in the body to promote the development of cancer [13, 14]. Butler et al. reported that uropathogenic E. coli reduced cellular MYC levels, significantly inhibited tumor growth, and prolonged survival in mouse tumor models of bladder and colon cancer [15].

Phage roles in at least some cancers have also been demonstrated. Phages are associated with CRC and may influence cancer progression by altering the bacterial host community. Geoffrey D Hannigan et al. analyzed gut phage structure and corresponding flora changes through 16S rRNA gene, fecal whole shotgun metagenomic sequencing, and purified virus metagenomic sequencing technology. Further studies found that intestinal phages were mainly mild phages, and proved that the phage community was related to CRC, which might affect cancer progression by changing the bacterial host community [16].

However, phages may also be used as tools against cancer. Phages might be used as tools for antimicrobial therapy and vaccine development [17]. The application of phages in the development of tumor vaccines can be divided into two modes, namely the direct use of phages as tumor vaccines and the use of phages to mediate specific immune stimulation against tumor cells. Phage surface antibodies can specifically recognize tumor cell antigens, providing potential strategies for tumor targeted therapy and tumor imaging [18, 19] (Fig. 1). Abnormal collagen fibers in cancer tissues, such as degeneration and degradation, lead to changes in the extracellular matrix. The remodeling of extracellular matrix may inhibit the anti-tumor immune response by acting on the tumor microbiome [20, 21]. Phages have been genetically engineered to image abnormal collagen in lung adenocarcinoma cells, which contributes to the imaging diagnosis of cancer [22]. Additionally, phage display technology is a highly efficient gene expression screening technology. Expressed specific foreign proteins or peptides on the phage surface is also expected to be applied in cancer imaging and molecular diagnosis. Rafael da Fonseca Alves et al. used phage display technology to select biotin-C3 and biotin-H2 peptides as biometric molecules to develop a biosensor that can distinguish serum samples from patients with breast cancer and those with benign breast disease [23]. Moreover, Xue Dong et al. built a personalized cancer vaccine platform based on M13 phage that is loaded with tumor antigens by electrostatic adsorption [24]. It was found that phage-based tumor vaccine could prevent tumor, enhance anti-tumor immune response, and inhibit tumor growth in both primary and metastatic mice almost without toxic side effects [24].

Fig. 1

Application of phage in the treatment and prevention of cancer. Phages have strong targeting ability and carrier potential, so they are mainly applied in five aspects, including cancer immunotherapy, cancer imaging, cancer vaccine, gene therapy, and drug delivery system

Phages have the ability to target bacteria and little toxic side effects, and can be used as carriers to deliver cytokines, tumor killer factors and chemotherapy drugs. Phages are therefore favored by immunotherapy and gene therapy. Paladd Asavarut et al. used the coat protein of tumor-targeting phages as a drug carrier to develop a transparticle system for cancer immunotherapy and gene delivery [25]. In xenografted tumor mouse models, the system delivered the cytokines tumor necrosis factor-α (TNF-α), interleukin 12 (IL12), interleukin 15 (IL15) and their encoded transgenes, aiming to achieve significant enrichment of anti-tumor cytokine at the tumor site and inhibition of tumor cell growth in a safe and efficient manner. Phages successfully circumvent the "cytokine storm" and are more economical cytokine carriers compared with specific antibodies, which indicates the great potential of phages for cancer immunotherapy. The application of nano delivery vector to the improvement of the tumor targeting ability is expected to provide a new method for improving the tumor killing ability of chemical drugs. Wang et al. produced mixed micelles assembled with polyethylene glycol-phosphati-dylethanolamine (PEG-PE) conjugate and a pancreatic cancer cell (PANC-1) specific phage protein (P38 and L1) loaded with the water-insoluble anticancer drug paclitaxel (PCT). This PCT-loaded targeting bacteriophage micelle had a better ability to target PANC-1 cells, and the cytotoxicity is significantly improved [26]. Phage-guided irinoconamil granules also significantly improved the therapeutic efficiency of first-line chemotherapy for CRC and inhibited the growth of the cancer-promoting bacterium Fusobacterium nucleatum in mouse models of spontaneous CRC tumors [27].

In the era of big data, bibliographic analysis is a popular approach to select many information documents to get the research hotspot and trend in a certain field. At present, bibliometric analysis is widely used in biomedicine, ecological environment, information science, and other fields [28, 29]. In recent years, many experimental studies on the development of phage and tumor vaccine and tumor therapy have been carried out in vitro and in vivo, and these preliminary research data highlight the potential value of clinical application of phage in anti-cancer therapies. However, there is a lack of bibliometric analysis focusing on gut phage and cancer to explore research trends in this area. In this study, CiteSpace software was used to conduct a bibliometric analysis of gut phage and cancer, which provided a comprehensive perspective on the research trend of phage and cancer. Considering that the study on phage is hierarchical, three topics in turn, including gut phage, phage and bacteria, and phage and tumor, were visually analyzed. The research progress was described from the perspective of dynamic change and evolution, and it would play an important guiding role for current researchers to explore the hot spots in this field.

Methods

Data sources

Web of Science (https://www.webofknowledge.com) is an important database of global academic information, and it contains over 13,000 authoritative and high-impact academic journals around the world, covering natural sciences, engineering technology, biomedicine, social sciences, arts and humanities [30,31,32]. In this study, Web of Science was used as a database to conduct three searches on gut phage, phage and bacteria, and phage and tumor. The search strategy is as follows: (1) The gut phage corresponds to TS = (bacteriophage* OR phage*) AND TS = (intestinal* OR intestine* OR bowel* OR gut*). (2) The phage and bacteria corresponds to (TI = (Bacteriophage* OR phage*) AND TS = (Gastrointestinal Microbiome* OR Gut Microbiome* OR Gut Microflora OR Gut Microbiota* OR Gastrointestinal Flora OR Gut Flora OR Gastrointestinal Microbiota* OR Gastrointestinal Microbial Community OR Gastrointestinal Microbial Communities OR Gastrointestinal Microflora OR Gastric Microbiome* OR Intestinal Microbiome* OR Intestinal Microbiota* OR Intestinal Microflora OR Intestinal Flora OR Enteric Bacteria OR Lactobacillus* OR Bifidobacterium* OR Escherichia coli*)) NOT TS = ("phage display"). (3) The phage and tumor corresponds to (TS = (neoplasm* OR tumor* OR neoplasia* OR cancer* OR malignant neoplasm* OR malignancy OR malignancies OR benign neoplasm*) AND TS = (bacteriophage* OR phage*)) NOT TS = ("phage display").

Visualized analysis

CiteSpace (https://citespace.podia.com/) is a bibliometric analysis software developed by JAVA [33]. The software supports the visual exploration of new trends and new dynamics of scientific development, with various functions, including research hot spot analysis, research frontier detection, research evolution path analysis, etc.

The parameters of CiteSpace (6.2 R1) were set as follows: The time slices of phage, phage and bacteria, and phage and tumor were chosen from January 2008 to June 2023, the time slicing parameters were set to 1 year, and the criteria were chosen (g-index, g² ≤ k Σ_i≤gc_i, k ∈ Z⁺, k = 25). The included documents were visually analyzed by CiteSpace in terms of country/region, institution, author, reference, and keyword.

A visual analysis of the cooperation network for countries/regions, institutions and authors reflects the degree of cooperation among various units. Reference co-citation analysis is presented through network map, timeline graph and reference burst. If two or more papers are jointly cited by one or more later papers, the co-citation relationship is established, and the co-citation of references can measure the degree of correlation between articles. Cluster analysis, time zone analysis and burst analysis constitute visual analysis for keywords.

In the upper left corner of the generated figure, N represents the number of nodes, E refers to the number of connections, and density is the network density. Q value is the value of clustering module, and more than 0.3 is considered as significant clustering structure effect [34]. The silhouette score ranges from -1 to 1, and a higher score indicates a greater internal homogeneity.

The flowchart of the study is shown in Additional file 1: Fig. S1.

Results

Research tendency

A total of 443 articles were selected focusing on gut phage from January 1, 2008, to June 5, 2023. The deadline for article selection was June 2023, and the whole year of publications in 2023 were not complete, so all line charts covered 2008 to 2023 only. Since 2013, the annual number of articles on gut phage had an upward trend, and the number of articles from 2021 to 2022 had the largest increase (Additional file 1: Fig. S2 A).

From January 1, 2008, to June 30, 2023, a total of 630 articles on phage and gut bacteria were selected. In the two periods of 2010–2012 and 2017–2022, the annual number of publications on phage and bacteria showed an increasing trend. Moreover, 2017 is an important time node, as the number of articles in the field decreased in 2016–2017, while the number of articles surged in 2017–2018 (Additional file 1: Fig. S2 B).

Compared with the other two topics, the number of articles on phage and tumor is relatively small from January 1, 2008, to June 21, 2023, with 141 articles. From 2014 to 2019, the number of annual publications on phage and tumor increased steadily. Furthermore, 2020 is also an important time node, because the number of articles declined in 2019–2020, while the number of articles surged in 2020–2021 (Additional file 1: Fig. S2 C).

Collaboration network

Gut phage

The articles on gut phage for this study were collected from 59 countries/regions, with a network density of 0.0479 (Fig. 2 A). From 2008 to 2023, the United States of America (USA), China, and France were the most frequent publishers, with 173 (39.05%), 63 (14.22%) and 43 (9.71%) articles, respectively (Table 1). The number of articles in the USA had a significant advantage, with a half-life of 10.5, which indicated that the USA had achieved excellent results in the quantity and quality of articles in the field of gut phage research. Additionally, Switzerland, Canada, USA, etc. tended to conduct collaborative research with other countries/regions. For example, Switzerland had international cooperation with 8 countries/regions, such as Russia, while Canada had international cooperation with 5 countries/regions, such as PRC.

Fig. 2

Cooperative network analysis of gut phage. A Cooperation network map for countries/regions. B Cooperation network map for institutions. C Cooperation network map for authors

Table 1 Top 5 of most productive countries,regions on gut phage

A total of 295 institutions and 436 authors were screened for visual analysis. Harvard University (29 articles), UDICE -French research universities (29 articles), University of California System (28 articles), University College Cork (25 articles), Universite Paris Cite (20 articles) were the top 5 most frequently published institutions (Fig. 2 B). Most institutions were universities. Hill, Colin,Ross (12 articles), R Paul,Shkoporov (10 articles), Andrey N (7 articles), Debarbieux, Laurent (7 articles) were the top 4 most frequently published authors (Fig. 2 C).

Phage and bacteria

A total of 58 countries, including USA, China and Canada, were collected from January 1, 2008, to June 20, 2023 (Fig. 3 A). The USA ranked first with 193 articles (31.64%), followed by China with 92 articles (15.08%), and Canada with 48 articles (7.87%) ranked third. There were 16 important countries/regions with high centrality, including France, Canada, England and USA. Some developed countries, including the USA, Canada, Russia and France, started research earlier and carried out extensive international cooperation. For example, Canada had cooperation with 7 countries/regions, such as Finland, Poland and Chile. In terms of number, centrality and half-life of articles, USA and China occupied a dominant position in the quantity and quality of articles published in this field (Table 2).

Fig. 3

Cooperative network analysis of phage and bacteria. A Cooperation network map for countries/regions. B Cooperation network map for institutions. C Cooperation network map for authors

Table 2 Top 6 of most productive countries/regions on phage and bacteria

There were 331 nodes, 512 connections, and a network density of 0.0094 for institutions (Fig. 3 B). Ten institutions, including The Centre National de la Recherche Scientifique (CNRS), University of California System, and University of Guelph, played an important connecting role in the network of institutional cooperation. Among the 10 institutions in the forefront of annual article production (Table 3), 5 belonged to France (50%), 2 to the USA (20%), 2 to Poland (20%), and 1 to Finland (10%).

Table 3 Top 7 of most productive institutions on phage and bacteria

There were 471 nodes, 475 connections, and a network density of 0.0043 for authors (Fig. 3 C). The productive authors include Wegrzyn Grzegorz, Bloch Sylwia, and Wu Vivian C H, and 14 others with at least 5 articles.

Phage and tumor

Articles on phage and tumor from 36 countries/regions, such as USA and PRC, were collected (Fig. 4 A). The USA held the top spot with 48 articles, followed by PRC with 36 articles and England with 16 articles (Table 4). Notably, the USA had an important influence in the network of national cooperation network, so it played a bridging role. What’s more, based on publication frequency and half-life, USA and China served as trailblazers in the field of phage and tumor research.

Fig. 4

Cooperative network analysis of phage and tumor. A Cooperation network map for countries/regions. B Cooperation network map for institutions. C Cooperation network map for authors

Table 4 Top 5 of most productive countries/regions on phage and tumor

There were 240 nodes, 510 connections, and a network density of 0.0178 for institutions (Fig. 4 B). Among them, a total of 7 institutions, including Imperial College London, Harvard University and Polish Academy of Sciences, published at least 5 articles.

There were 372 nodes, 903 connections, and a network density of 0.0131 for authors (Fig. 4 C). Hajitou Amin was the most productive author with considerable article quality and conducted extensive cooperation with other authors. Hajitou Amin had an early insight into the ability of phages to target tumors, and used them as vectors to deliver tumor necrosis factor, CRISPR-Cas9 transgene boxes, etc., for immunotherapy and gene delivery of various cancers such as melanoma [25, 35, 36].

Reference co-citation and research hotspot

Gut phage

There were 668 nodes, 1,414 connections, and a network density of 0.0063, and 13 clusters were presented, including #0 eggerthella lenta, #1 virus-bacteria linkages, #2 phage resistance, #3 human gut, #4 phage translocation, #5 vancomycin-resistant enterococcus faecalis, #6 correlation, #7 crassphage, #8 carrier state lifecycle, #9 probiotic bacteria, #10 microbiome, #11 campylobacter jejuni, and #12 symnioses (Fig. 5 A). #9 probiotic bacteria and #11 campylobacter jejuni started early, while #0 eggerthella lenta, #1 virus-bacteria linkages, and #2 phage resistance have received attention in recent years (Fig. 5 B).

Fig. 5

Reference co-citation analysis of gut phage. A Reference clustering map. B Reference co-citation time diagram. C The top 25 co-cited references with the strongest burst intensity

Totally, 6 citations burst starting in 2021 and beyond (Fig. 5 C). These citations explored the phage-dominated viral genome to discover its diversity and individual specificity and investigated potential associations among bacteriome, metabolome, and viriome [37,38,39,40]. For example, the citation (strength = 8.13) by Ann C Gregory et al. described a human enterovirus database (GVB) of 1,986 individuals from 16 countries, which confirmed the individual specificity of phages and revealed the shift in viral diversity from infancy to old age[39].

Phage and bacteria

The top 13 clusters were presented from 740 co-cited articles, including #0 bacteriophage, #1 jumbo phage, #2 shiga toxin, #3 feces, #4 human gut microbiome, #5 bacteriophage t4, #6 d6, #7 virulence, #8 h7, #9 genome analysis, #10 microbiota, #11 diarrhea, and #12 peptidoglycan hydrolase (Fig. 6 A). Among them, #1 jumbo phage, #4 human gut microbiome, #7 virulence, and #8 h7 were expected to attract more investment in future research. Additionally, #0 e. coli o157:h7 and #5 bacteriophage t4 started early with relatively profound research foundation (Fig. 6 B).

Fig. 6

Reference co-citation analysis of phage and bacteria. A Reference clustering map. B Reference co-citation time diagram. C The top 25 co-cited references with the strongest burst intensity

The timeline of the top 25 co-cited articles on phage and bacteria with the strongest bursts was shown in Fig. 6 C. The citation written by Alejandro Reyes et al. had the strongest burst strength (6.45) and focused on the concept of gut virome early [41]. Articles that might reveal the potential future research hotspots (burst end = 2023) mainly concentrated on the further exploration and application of phage properties. First, the current research on the characteristics of phages seemed to be more focused on the study of its survival mechanism and comparison between individuals, including its mechanism of targeting host and avoiding attack [42, 43], as well as the high individual specificity of the phage-dominated virome [37], which provides the premise of multi-disciplinary application of phages. Besides, based on the impacts of phage on gut bacteria and even gut microbiome, the application of phages was expected to attract more attention, such as the treatment of bacterial infection and food safety problems [44,45,46].

Phage and tumor

There were 465 nodes, 1,199 connections, and a network density of 0.0111 for citation on phage and tumor (Fig. 7 A). The top 13 clusters included #0 esophageal diseases, #1 ge11, #2 melanoma, #3 temozolomide, #4 crispr-cas9, #5 nanoparticle, #6 cec, #7 coli phagelysate, #8 combined therapy, #9 aptamers, #10 hpv vaccine, #11 immune checkpoint inhibitor, and #12 collagen. #2 melanoma, #3 temozolomide, #8 combined therapy, and #9 aptamers started early with a relatively in-depth study while #5 nanoparticle and #7 coli phagelysate appeared recently and could be the focus of the future research (Fig. 7 B).

Fig. 7

Reference co-citation analysis of phage and tumor. A Reference clustering map. B Reference co-citation time diagram. C The top 25 co-cited references with the strongest burst intensity

A total of 7 citation burst started in 2021 and lasted until the search deadline, which had a certain directional effect on future research hotspots (Fig. 7 C). The potential of phages in the diagnosis and treatment of cancer such as CRC has been emphasized, and the changes of intestinal mucosa have been revealed to be related to phages [5, 10, 16, 47,48,49]. For example, the citation by Lasha Gogokhia et al. (strength = 2.11) revealed that phages promoted mucosal IFN-γ through a TLR9-dependent pathway, which enhanced mucosal immunity and exacerbated the occurrence of colitis [10]. Rebekah M. Dedrick et al. reported for the first time a case study (strength = 2.07) of the use of engineered phages in human treatment of drug-resistant mycobacterium abscess with clinical improvements such as liver function recovery, which strongly suggested the potential of effective combination of phage and genetic engineering technology in clinical practice [49].

Keyword co-occurrence and burst

Gut phage

The top 13 clusters were presented from 394 keyword nodes (Q = 0.7519), including #0 phageome, #1 community, #2 antibiotic reststance, #3 dynamics, #4 enterococcus, #5 shiga toxin, #6 escherichia coli, #7 volunteers, #8 ulcerative colitis, #9 capsid proteins, #10 alignment, #11 in vitro, and #12 animal models (Fig. 8 A).

Fig. 8

Keyword analysis of gut phage. A Keyword clustering map. B Keyword time diagram. C The top 25 keywords with the strongest burst intensity

Some keywords, such as “escherichia coli”, “identification” and “therapy”, appeared early, while others, such as “gut virome”, emerged in recent years (Fig. 8 B). The top 25 keywords with the strongest citation bursts were presented in Fig. 8 C. “Phage therapy” (strength = 3.46), “viral community” (strength = 3.76), “infection” (strength = 3.19), etc. were the hot spots of early research, while “gut microbiome” (strength = 2.94), “database” (strength = 3.83), “microbiota” (strength = 3.12), “expansion” (strength = 2.89), and “mice” (strength = 2.18) would be the future research hot spots.

Phage and bacteria

The top 13 clusters were presented from 413 keyword nodes (Q = 0.6972), including #0 operon, #1 antimicrobial resistance, #2 phage therapy, #3 rna polymerase, #4 shiga toxin, #5 bacteriophage t4, #6 biocontrol, #7 outbreak, #8 gut microbiota, #9 escherichia coli, #10 mutants, #11 antagonistic coevolution, and #12 mung bean seeds (Fig. 9 A). “Escherichia coli” had the highest frequency of 177, ranking the first keyword, followed by identification with a frequency of 81, and “bacteriophage” and “phage” with a frequency of 61.

Fig. 9

Keyword analysis of phage and bacteria. A Keyword clustering map. B Keyword time diagram. C The top 25 keywords with the strongest burst intensity

Moreover, “escherichia coli”, “identification”, “protein”, “phage”, etc. were the earliest hotspot keywords since 2008, while “antimicrobial resistance (amr)”, “stability” and “1st step transfer dna” had emerged recently (Fig. 9 B). Among the keywords of high burst intensity, the keywords of burst beginning in 2021 or later were noticed, including “alignment” (strength = 2.93), “lipopolysaccharide” (strength = 2.6), “microbiota” (strength = 2.4), “human gut” (strength = 2.34), “escherichia coli 0157” (strength = 2.34), and “gut microbiota” (strength = 2.93) (Fig. 9 C).

Phage and tumor

There were 322 keyword nodes in total, and 13 clusters are presented (Q = 0.7077) including #0 gene therapy, #1 hpv vaccine, #2 virome, #3 short-wavelength infrared imaging, #4 cancer, #5 cancer gene therapy, #6 nanoparticle, #7 m13 bacteriophage, #8 carbohydrates, #9 biomaterials, #10 microbial network, #11 phage idiotype vaccination, and #12 immune checkpoint inhibitor (Fig. 10 A).

Fig. 10

Keyword analysis of phage and tumor. A Keyword clustering map. B Keyword time diagram. C The top 25 keywords with the strongest burst intensity

Key words, including “delivery” (frequency = 19), “cancer” (frequency = 19), “expression” (frequency = 17), “cells” (frequency = 15), “therapy” (frequency = 12) and “colorectal cancer” (frequency = 18), had deep research foundation, and still are the topic of the current study (Fig. 10 B). Among the keywords with high burst intensity with the burst start time of 2021 and beyond, “colorectal cancer” (strength = 3.7) ranked first, followed by “proteins” (strength = 2.84) and “phage therapy” (strength = 3.7) (Fig. 10 C).

Discussion

Phages, a major member of the human viriome, can specifically infect bacteria and have been found to be involved in the progression of tumors in recent years. This study used CiteSpace to explore three topics, including phage, phage and bacteria, and phage and tumor. Through the analysis of research trends, cooperation networks, reference co-citations, and keywords, the development of these three fields from 2008 to 2023 was found, and the research trends and hot topics in this field were presented.

The results proved that the annual publication number of the three fields was basically on the rise. The annual number of publications on phage and tumor decreased in 2020, which could be due to the outbreak of the COVID-19 pandemic. The upward trend could be attributed to the rapid development of artificial intelligence, bioinformatics, and sequencing methods, which provided strong technical support for researchers. The construction of virus database depends on the acquisition of phage sequences. At present, third-generation sequencing technology has been popularized in recent studies for its advantages of long read length and high precision [50]. In this study, it was found that "sequences" was the early research focus on gut phage, and "database" was a potential future research hotspot. The results revealed that the development of sequencing technology has promoted the shift from fragmented sequence collection to systematic database construction [51]. With the support of bioinformatics and artificial intelligence, the information resources in virus database are expected to exert their application potential. Menglu Li et al. collected over 3,000 phage-host pairs and their protein sequences and used deep convolutional neural networks to construct prediction models for phage-host interactions [52]. This model was expected to contribute to the development of phage therapy and provide suitable phage selection for personalized treatment.

The collaborative network analysis of countries/regions, institutions, and authors in the three fields of gut phage, phage and bacteria, and phage and tumor illustrated that the USA and China occupied an important position in each field. For countries stronger in various fields, early research time and extensive international cooperation are prerequisites for taking a leading position. Notably, half of the top 10 productive institutions in the field of phage and bacteria were from France, which suggested the strong research background of France and provided directions for future research collaborations in this field.

Reference co-citation and keyword analysis provided a comprehensive perspective for the interpretation of research trends and hot spots. “Phage therapy”, appeared in two topics including gut phage, and phage and tumor as a keyword with the high citation burst. Phage therapy is self-limiting, and once the host bacteria are eliminated, the number of phages decreases sharply. Moreover, when the host bacteria spread, it can be expanded autonomously and actively, so it can be administered in small doses. On the one hand, phage therapy offers a solution to wean ourselves off antibiotics, which may contribute to tackle antibiotic resistance. Little et al. reported the successful case of phage in the treatment of Mycobacterium chelonae infection for the first time, and the tested patient had excellent clinical results and developed neutralizing antibodies against the phage, without the bacterial resistance to the phage [53]. Another case of phages treating Mycobacterium abscess in the lungs was also groundbreaking, which provided a reference for solving bacterial infections in organs. Jerry A Nick et al. found that the ability of phage to dissolve Mycobacterium abscess was optimized by genetic engineering, and the drug-resistant bacterial infection disappeared successfully after intravenous infusion of the engineered phage [54].

As for the cancer, this study found that “colorectal cancer” was a recent hot spot in the field of phage and tumor. CRC is the third most common malignant tumors with high mortality, low five-year survival rate and heavy disease burden [7, 55]. In 2023, there were expected to be 153,020 new CRC cases and 52,550 deaths in the USA [7]. Phage differs in abundance and diversity between healthy people and CRC patients, and thus, as a biomarker, it has great application potential in the diagnosis of CRC. Siyuan Shen et al. included 317 metagenomic sequencing samples and identified that 5 phages significantly enriched in CRC, including Peptacetobacter hiranonis Phage, Fusobacterium nucleatum animalis 7_1 Phage, Fusobacterium nucleatum polymorphum Phage, Fusobacterium nucleatum animalis 4_8 Phage, and Parvimonas micra Phage through the random forest model (AUC = 0.8616) [56]. Given the interaction between phages and bacteria, phages can indirectly affect the progression of CRC through the regulation of CRC-related bacteria. In the process of CRC development, phage was found to be correlated with butyrate producing bacteria, including Eubacterium rectale, Eubacterium cellulosolvens, and Butyrivibrio proteoclasticus [57]. The changes of bacterial community structure mediated by phages play an important role in the development of CRC [16]. Phages can enhance the body's immune response to mitigate the effects of cancer-promoting bacteria on CRC. The M13 phage attached with silver nanoparticles was designed to eliminate symbiotic Fusobacterium nucleatum, with an inhibitory anti-tumor immune response, and activate antigen-presenting cells. In situ CRC mouse models, the survival rate of mice was significantly increased and the anti-tumor immune response was enhanced [58]. In addition, the powerful carrier potential and targeting ability of phages are also applied in CRC [27, 59, 60]. The efficiency of phage drug delivery is affected by gastrointestinal environmental factors, such as PH, temperature, stomach acid, etc., thus leading to premature degradation of phages [61,62,63,64]. With the rapid development of materials science, hydrogels, liposomes, electrospinning fibers, etc., are used for embedding and delivery of phages to protect the vitality of phages during gastrointestinal transport, aiming to achieve targeted release and accurate quantification [65,66,67,68].

Increasing evidence confirms the close relationship between phage and tumor, which suggests the value of phages in cancer prevention and treatment. Phage preparation is a heterologous protein for humans, and may cause transient allergic reactions such as fever, shortness of breath, and wheezing when taken orally, topically, by aerosol inhalation, or intravenously. The exponential proliferation of phages is a significant advantage of phage therapy. Under the right conditions, each phage will produce several hundred progeny phages in a lysis cycle. Therefore, the therapeutic effect can be achieved with a small amount of bacteriophage preparation. Phages themselves may carry virulence genes or drug-resistance coding genes, which may increase host virulence or produce novel pathogenic strains through general or specific transduction mechanisms. However, by modifying phages with molecular biological methods, the toxin genes can be eliminated. Phage targeting prevents damage to other organs. Therefore, phage preparation has little self-toxicity. However, phage therapy has not been widely used in clinical practice due to its own limitations, such as narrow antibacterial spectrum, possible allergic reactions, and drug resistance after frequent use. Moreover, the function of phage genes is still largely unknown. Through high-throughput sequencing technology, bioinformatics analysis, combined with multi-omics analysis, it is expected to accelerate the decoding of phage gene function puzzles. The keyword “proteins” had a high burst intensity in phage and tumor in this study. At present, most research focuses on phage display technology to screen specific peptides/proteins for application in cancer diagnosis and treatment. Phage gene coding products, such as lyase, are easy to be edited by genetic engineering technology, and do not proliferate, which provides a new direction for tumor phage therapy, but few studies have reported their discovery in the field of tumor. Phage lysin, as an "early protein", is a novel antimicrobial agent in recent years, and its host selectivity was reported [69]. With the gradual understanding of the function of phage genes, it is expected that the role of phage gene coding products in the field of cancer will be further studied, and their practical clinical application value will be explored.

In this study, in order to explore the relationship between phage and tumor from a dynamic and comprehensive perspective, CiteSpace was used to visually analyze gut phage, phage and bacteria, and phage and tumor in sequence, but with certain limitations. First, the exclusion of non-English articles may lead to the omission of valuable articles. Moreover, articles were only from the Web of Science, so this also leads to incomplete retrieval of articles. In addition, given the high content of phages in the gut virome, the topic will be narrowed down in the future to study the relationship between phage and gut disease, such as CRC, to provide a more granular perspective.

Conclusion

In this study, three topics, including gut phage, phage and bacteria, and phage and tumor were visually analyzed, and the research trends and hotspots in the three fields were provided. From a dynamic and comprehensive perspective, the role and application prospect of phages in cancer, especially CRC, were interpreted, which provided a new direction for future research.