Bidirectional crosstalk between the epithelial-mesenchymal transition and immunotherapy: A bibliometric study

ABSTRACT

Immunotherapy has recently attracted considerable attention. However, currently, a thorough analysis of the trends associated with the epithelial-mesenchymal transition (EMT) and immunotherapy is lacking. In this study, we used bibliometric tools to provide a comprehensive overview of the progress in EMT-immunotherapy research. A total of 1,302 articles related to EMT and immunotherapy were retrieved from the Web of Science Core Collection (WOSCC). The analysis indicated that in terms of the volume of research, China was the most productive country (49.07%, 639), followed by the United States (16.89%, 220) and Italy (3.6%, 47). The United States was the most influential country according to the frequency of citations and citation burstiness. The results also suggested that Frontiers in Immunotherapy can be considered as the most influential journal with respect to the number of articles and impact factors. “Immune infiltration,” “bioinformatics analysis,” “traditional Chinese medicine,” “gene signature,” and “ferroptosis” were found to be emerging keywords in EMT-immunotherapy research. These findings point to potential new directions that can deepen our understanding of the mechanisms underlying the combined effects of immunotherapy and EMT and help develop strategies for improving immunotherapy.

KEYWORDS:

• Bibliometrics
• EMT
• immunotherapy
• VOSviewer
• CiteSpace
• therapeutic strategies

Introduction

Since the discovery of the fundamental role of the immune system in cancer therapy,¹ immunotherapy has become a powerful clinical technique for cancer treatment, significantly influencing the development of therapeutic strategies and the prospective identification of tumors.^2,3 Advancements in the development of immunotherapy drugs and the implementation of combined treatment strategies have resulted in increased anticancer efficacy against several types of cancer.^4–6 A variety of diverse cancer immunotherapies, including checkpoint inhibitors, peptide vaccines, dendritic vaccines, chimeric antigen receptor T cells, and oncolytic viruses, exist.^7,8 However, in some cases, the toxicity of cancer immunotherapy, tumor immunosuppression, or immune evasion results in clinical immunotherapy failure.⁹ Currently, the focus of most studies is on elucidating the molecular mechanisms of immunotherapy, which could help develop novel therapeutic strategies and improve our understanding of the underlying conditions for personalized treatment. For example, a novel ferroptosis molecule, FANCD2, which was found to be strongly correlated with PD-L1 and the tumor microenvironment, holds promise as a prognostic factor in mRNA vaccine therapy for pancreatic adenocarcinoma; this finding could serve as a reference for understanding tumor immunity regulation and guiding vaccine development efforts.³

To date, several tumor-related pathways, including WNT signaling,¹⁰ PI3K/AKT signaling,¹¹ and epithelial-mesenchymal transition (EMT),¹² have been identified. Among them, EMT plays a pivotal role in inducing immunosuppression triggered by the EMT-IF Snail.^13,14 EMT is the process whereby epithelial cells lose epithelial surface markers, such as E-cadherin, and acquire mesenchymal properties, such as high levels of fibronectin, vimentin, and N-cadherin expression.^15,16 This dynamic process regulates embryonic development and wound healing under physiological conditions. As shown in Figure 1(a), the dysfunction of the EMT process is associated with cancer metastasis, drug resistance,^17–19 and immunosuppression.^20,21 The bidirectional regulation of EMT and PD-L1 signaling reportedly improves PD-1/PD-L1 therapeutic resistance, and EMT-targeted therapy combined with PD-L1 inhibitors could represent a synergistic strategy for preclinical or clinical trials.^22,23 As an inducer of EMT, TGF-β inhibits cancer immunity by mediating T helper 2 cells. Blocking the TGF-β pathway in CD4+ cells reshape the tumor microenvironment and suppresses cancer progression, which provides a foundation for the suppression of cancer microenvironments.²⁴ TGF-β type 2 stimulates TGF-β signaling via extracellular vesicles originating from tumors, thereby inducing EMT. TGF-β type 2 induces the activation of SMAD3 to cause CD8+ T-cell exhaustion, resulting in a poor immunotherapy efficacy. There is a need to identify the mechanisms underlying T-cell exhaustion and immunosuppression in breast cancer in order to identify a novel target for immunotherapy against breast cancer. Moreover, a prognostic model based on EMT-related genes could be used to predict the efficacy of immunotherapy, with the most of immune checkpoint genes being expressed at higher levels among high-risk groups. Furthermore, differences exist in the immune cells between high- and low-risk individuals, confirming the clinical value of EMT-related signatures for predicting the HCC immune microenvironment.²⁵ Thus, exploring the role and mechanism of EMT in immunotherapy could provide novel insights into both the development of therapeutics targets and the identification of biomarkers to estimate prognosis.

Figure 1. Potential molecular mechanisms of EMT involving in immunotherapy (a). Procedure of data research and collection (b).

The ubiquitousness of the internet makes it extremely easy to access large volumes of primary scientific research. However, the vast number of available publications necessitates the adoption of a simple and efficient to obtain the desired information. Bibliometric analysis is a scientific and quantitative method based on the statistical analysis of publications and the frequency of citations.²⁶ This method facilitates the effective collection and analysis of a large number of publications, encompassing complex datasets from scholars in the field, thereby facilitating trend characterization within the literature. Compared with traditional reviews, bibliometric analysis provides objective and statistically significant data for further analysis by researchers. In this study, a bibliometric analysis was conducted to explore research trends in EMT-immunotherapy, aiming to identify specific trends and track the research profiles of different academic groups across countries, institutions, authors, and journals. The summary of a large number of references also allows for the creation of knowledge maps. In addition, the temporal analysis of the frequency of the references cited and the keywords selected across the research field in recent years helps to reveal emerging research hotspots.²⁷

Despite its advantages, a bibliometric analysis of EMT-immunotherapy research has not yet been performed. Therefore, in this study, we used bibliometric analysis to construct a scientific knowledge map and provide an overview of current hotspots in the field of EMT-immunotherapy using various tools, including CiteSpace, VOSviewer, R-tools, Bibliometrics Online Analysis Platform, and Microsoft Excel. The three main approaches were as follows: (1) an analysis of key information, including annual publications, geographical distribution, institutions, journals, and authors, to determine the current status of the field and the leading contributors, thereby providing a foundation for potential collaboration between institutions and authors; (2) sorting of the most cited references and top citation keywords in order to define the structure of EMT-immunotherapy research and build a knowledge base. The collection of co-cited references highlights the connections between related studies, while the top citation keywords highlight the fundamental research concepts in the field; (3) identification of research hotspots and emerging trends through a timeline of references and keyword outbreak analyses. The temporal distribution and magnitude of these references reflect the changes in the field over time. Keywords indicate potential research trends, and keyword outbreak analyses facilitate the identification of popular research topics.

The aim of this study was to provide a comprehensive summary of existing studies in the field of EMT-immunotherapy research and highlight emerging topics within these studies to investigate the evolution of the field and predict the development of future studies.

Materials and methods

The results retrieved on EMT in the field of immunotherapy were analyzed according to the number of publications and citations, the distribution of countries and organizations, journals, authors, references, and keywords through four tools (CiteSpace, 6.2.R4; VOSviewer 1.6.16, R-software v4.1.3, Bibliometrics Online Analysis Platform and Microsoft Excel).

CiteSpace is a bibliometric and visual analysis tool that is frequently used to visualize networks and identify prominent trends and pivotal points. In this study, these were verified in collaboration with domain experts, who are also the authors of pivotal research articles in the field of interest. CiteSpace was used to analyze and visualize the geographical distribution, authors, journals, dual-map of journals, and temporal evolution of references of the relevant literature. In the resulting schematic representation, the circumference of each node denotes the number of documents, while the outermost ring in each node (colored in red) indicates its centrality significantly, which represents importance in the network. The time span was set to 1999–2022, with default settings for years per slice and other parameters. The practical implications of this CiteSpace analysis include the identification of challenges and opportunities in the research field.

VOSviewer is an easy-to-use software program that is well suited to visualizing scientific landscapes by network/overlay/density patterns in the linlog/modularity method. Each node in the map represents a specific parameter, wherein the node size is related to the weighing of the attributes (e.g. production of publications, frequency of occurrence, or citation frequency). In addition, the strength of a node is represented by an index of the total link strength (TLS), wherein the number and width of the TLS represent the relationship between two nodes. In this study, VOSviewer was used to identify productive countries and institutions and to create keyword co-occurrences for identifying research trends and hotspots.

Web-based tools for bibliometric analysis are useful for visualizing the co-authorship of countries and institutions. The cooperation network among countries presents the most active collaborative countries worldwide. The “bibliometrix” package in R contains comprehensive bibliometric analysis tools, which when combined with bibliometric analysis, can be used to achieve knowledge structure and network construction. In this study, these were used to analyze the geographical distribution of publications and cumulative occurrence of journals. Microsoft Excel is the most fundamental data processing tool, which was used for importing data and creating tables.

Research ethics

Ethical approval was unnecessary because the data in this study were retrieved from open sources, and no animal experiments or human tissues were used in our research.

Statistical analysis

CiteSpace was used for authors, journals, timeline perspective of references, citation burst by country, references, and keyword analysis. The size of a node is associated with the citation frequency, and the links represent co-occurrence relationships. The citation burst reflects the criticality of each term in certain studies. For country and keyword analyses, VOSviewer was used to display the overlay visualization maps and time-dependent overlay visualization maps. GraphPad 9.5 was used for descriptive statistical analysis of institutions and to generate diagrams.

Results

Trends in annual growth

A total of 1,302 publications were obtained from the WOSCC. Worldwide trends in publications and total citations for EMT-immunotherapy research are shown in Figure 2. The results indicate that research on EMT-immunotherapy has increased rapidly over the last five years. The number of articles increased in 2018 before sharply increasing again from 2019 to 2022. The number of publications peaked in 2022, and a sustained increase in EMT-immunotherapy research is expected in the near future. Additionally, the set of all articles has been cited 40,667 times, with an average of 1,694.45 yearly citations and 31.09 average citations per publication.

Figure 2. Global trend of publications and citations on EMT-immunotherapy research from 1999 to 2022.

Countries and regions

Articles related to immunotherapy and EMT have been published in 74 countries. An analysis of the geographical distribution showed that relevant research articles were mainly published in Asia, followed by North America (Figure 3(a)). A list of the top 10 most productive countries is provided in Table 1, with annual publications from 1999 to 2022 depicted in Figure 3(b). Among the latter, China was the most productive country (49.07%, 639), followed by the United States (16.89%, 220). Publications from the United States received the highest total citations (19,028). Moreover, the highest average citation rate (86.49 times) occurred in the United States.

Figure 3. Analysis of cooperation among countries. (a) Geographical distribution map of countries. (b) Annual publication of top 10 countries. (c) The international collaboration’s visualization map of countries. (d) lization and time-dependent overlay visualization map of countries.

Table 1. Top 10 productive countries related to EMT and immunotherapy.

Rank	Country	Counts	Percentages	SCP	MCP	MCP-Ratio	TLS	Total citation	Average citation paper
1	CHINA	639	0.490783	565	74	0.116	137	10382	16.25
2	USA	220	0.168971	154	66	0.3	240	19028	86.50
3	ITALY	47	0.036098	34	13	0.277	45	2991	63.64
4	JAPAN	40	0.030722	33	7	0.175	15	2195	54.88
5	FRANCE	38	0.029186	20	18	0.474	68	3151	82.92
6	INDIA	35	0.026882	18	17	0.486	50	814	23.26
7	GERMANY	29	0.022273	20	9	0.31	55	1966	67.79
8	SOUTH KOREA	25	0.019201	21	4	0.16	24	1216	48.64
9	IRAN	24	0.018433	10	14	0.583	45	1331	55.46
10	SPAIN	23	0.017665	18	5	0.217	38	2817	122.48

Single country publication (SCP) represents the number of publications coauthored by authors of the same nationality, while Multiple country publication (MCP) indicates the number of publications coauthored by authors from different countries. Therefore, MCP represents the level of global collaboration. According to their MCP rate values, India, France, and Germany demonstrate relatively active international collaboration. Further country cooperation analysis showed that researchers in China are collaborating with their counterparts in various countries, among which the United States and France are the most significant partners (Figure 3(c)). Additionally, the time-dependent overlay map and network map showed collaboration among 36 countries that published more than five papers (Figure 3(d)). The thickness of the lines between nodes is representative of the total link strength (TLS), which is the level of global co-authorship. The network map shows that the United States has the highest TLS value, followed by China. A time-dependent overlay map showed that some countries have conducted this research in the past two years, suggesting that research in this field is attracting global attention. By country, the strongest citation bursts occurred in the United States, with a corresponding burst length of 24.23 from 2007 to 2018 (Figure 4(a)). Taken together, these results demonstrate the vital role of researchers and institutions in the United States in the field of EMT-immunotherapy research.

Figure 4. The strongest citation bursts of country. The strongest citation bursts refer to a variable change dramatically in short period.

Contributions of institutions

According to the VOSviewer analysis, 1,777 different organizations were found to be involved in immunotherapy and EMT-related research, among which 105 met the threshold of more than five papers (Figure 5(a,b)). Fudan University, Central South University, and Shanghai Jiao Tong University were the top three institutions in terms of the number of publications. Central South University, Chinese Academy Medical Sciences & Peking, Sichuan University, Islamic Azad University, University of Tehran, and others were found to represent the emerging institutions in the field of EMT-immunotherapy.

Figure 5. Organizations analysis in EMT-immunotherapy research. (a,b) Overlay visualization and time-dependent overlay visualization map of organizations. (c) immunotherapy research.

The top 10 most productive research institutions are shown in Figure 5(c) and Table 2. The majority of these institutions were found in China, wherein the majority of publications were from Fudan University, followed by Central South University, and Shanghai Jiao Tong University. The University of Texas MD Anderson Cancer Center in the United States had the largest number of citations (n = 3,836), while Shanghai Jiao Tong University had the largest TLS (TLS = 45). These results indicate that organizations in China contribute the most to EMT-immunotherapy research, while the University of Texas MD Anderson Cancer Center in the United States has the greatest influence on this topic.

Table 2. The top 10 productive institutions ranked by the number of publications.

Affiliation	Articles	country	TLS	Total citation	Average citation per paper
FUDAN UNIV	104	China	29	959	9.22
CENT SOUTH UNIV	90	China	19	278	3.09
SHANGHAI JIAO TONG UNIV	78	China	45	1304	16.72
SUN YAT SEN UNIV	77	China	25	307	3.99
UNIV TEXAS MD ANDERSON CANC CTR	71	USA	24	3836	54.03
ZHEJIANG UNIV	60	China	25	382	6.37
ZHENGZHOU UNIV	56	China	26	362	6.46
NANJING MED UNIV	55	China	15	267	4.85
SICHUAN UNIV	55	China	11	194	3.53
HUAZHONG UNIV SCI AND TECHNOL	54	China	17	348	6.44

Journal analysis

Publications related to EMT-immunotherapy included contributions from 429 citation journals and 5,043 co-cited journals. The cumulative occurrence of the top five most cited journals is shown in Figure 6(a). Although Frontiers in Immunotherapy was the most popular journal before 2019, the number of publications in Cancers has greatly increased since then. As shown in Figure 6(b), the overlay visualization of citation journals revealed 56 journals that met the inclusion criteria. The relevant citation journals were classified into four clusters, with clusters in red and green being the two dominant groups, among which Cancers (19.34%), Frontiers in Oncology (15.38%), and Frontiers in Immunotherapy (14.2%) were the most popular. Regarding the reference values of annual impact factors, Cancer Letters, Frontiers in Immunology, and Oncoimmunology can be considered authoritative journals in the field. Judging by both document number and impact factors, Frontiers in Immunology can be considered the most influential journal. The overlay visualization of the cited journals showed four different journal clusters: (i) the cluster in green, which was mainly dominated by Cancer Research, Clinical Cancer Research, and Nature; (ii) the cluster in blue, which was dominated by Oncogene and Journal of Biological Chemistry; (iii) the cluster in red, which mainly included PlosOne, Oncotarget, and Molecular Cancer; (iv) the cluster in yellow, had few journals (Figure 6(c)). The dual-map overlay map presented in Figure 6(d) shows the distribution of topics in related journals.²⁸ The citing journals are displayed on the left side of the map, while the cited journals appear on the right side. Colored paths represent citation relationships. There are two colored paths, orange and green, on this map. This implies that the research studies published in journals related to Molecular/Biology/Immunology and Medicine/Medical/Clinical fields were mainly cited by journals related to Molecular/Biology/Genetics. These results suggest that EMT-immunotherapy research focuses on both basic research and clinical medicine. Among the top 10 popular journals, 60% were JCR Q1 and 80% had impact factors (IF)>5. Although eight of the cited journals were JCR Q1, seven of the cited journals had IF values greater than 10. Moreover, 70% (7/10) of the most-cited journals were from the United States and 30% (3/10) were from the United Kingdom (UK) (Table 3).

Figure 6. Distribution of source journals. (a) The annual growth of top five productive journals. (b) The overly visualization of citation journals. (c) The overly visualization of cited journals. (d) The dual-map overlay map of journals.

Table 3. Top 10 citation journal and co-cited journal related to EMT-immunotherapy research.

Citation journals	Count	IF	JCR	Total citation	Cited journals	Countries	IF	JCR	Total citation
Cancers	83(19.34%)	5.2	Q2	1206	Cancer research	USA	11.2	Q1	5274
Frontiers in oncology	66(15.38%)	4.7	Q2	854	Clinical cancer research	USA	11.5	Q1	3432
Frontiers in immunology	61(14.2%)	7.3	Q1	897	Nature	UK	64.8	Q1	3067
International journal of molecular sciences	37(8.6%)	5.6	Q1	481	Cell	USA	64.5	Q1	2744
Frontiers in cell and Developmental biology	24(5.59%)	5.5	Q1	248	P natl acad sci usa	USA	11.1	Q1	2386
Oncoimmunology	23(5.35%)	7.2	Q1	909	Oncotarget	USA	N/C	N/C	2292
Frontiers in genetics	21(4.89%)	3.7	Q2	28	Oncogene	UK	8	Q1	2145
Frontiers in Pharmacology	21(4.89%)	5.6	Q1	165	Plos one	USA	3.7	Q2	2126
Cancer letters	19(4.43%)	9.7	Q1	741	Cancer cell	USA	50.3	Q1	1885
Aging-us	16(3.72%)	5.2	Q2	68	Nat commun	UK	16.6	Q1	1792

Author and reference analysis

The dataset included 8,155 authors and 60,625 co-cited authors. The analysis of data regarding authors and cited authors contributes to the identification of core authors and the main cooperative relationships in the field. A summary of the most productive and most cited authors is provided in Table 4. Considering the influence and H-index value in this field, the results indicate that Jean Paul Thiery in Singapore and Alberto Mantovani in the UK are authoritative and highly cited authors. The connections between the authors and cited authors were visualized using CiteSpace.

Table 4. The top 10 most productive authors related to EMT-immunotherapy.

Rank	Citation author	Documents	Citations	TLS	H-index	Cited-author	Citations	TLS	H-index
1	Palena, claudia	17	998	30	38	Hanahan, d	228	5443	109
2	Chouaib, salem	15	1102	26	27	Zhang, y	186	4683	89
3	Schlom, jeffrey	12	863	27	68	Thiery, jp	167	3235	106
4	Janji, bassam	8	629	22	43	Liu, y	161	4484	78
5	Noman, muhammad zaeem	8	778	25	36	Mantovani, a	161	4860	186
6	Thiery, jean paul	8	584	20	106	Wang, y	161	4289	104
7	Liu, yu	7	49	4	53	Kalluri, r	156	4095	115
8	Wang, xin	7	67	2	32	Yang, j	143	3703	142
9	Ashrafizadeh, milad	6	236	26	49	Jemal, a	141	2099	136
10	Bonavida, benjamin	6	399	0	70	Li, j	131	3306	67

The top 10 EMT-immunotherapy studies that attracted the most attention from scholars are shown in Figure 7(a) and Table 5. The most notable were three original articles authored by Raghu Kalluri et al.²⁹ Yutaka Kawakami et al.³⁰ and Liao Wangjun et al.³¹ Among them, the original article by Raghu Kalluri received the most citations, whereas the review by Luca Gianni received the largest annual average citations. The top 10 citation references are listed in Table 6. All were published in journals with JCR Q1, with half of the articles being original articles. A Ridgeline plot and timeline perspective depict the research hotspots over time in 16 clusters. Of these, “cancer stem cell” (#1) was the largest cluster and “cytomegalovirus-specific cytolytic t-cell lines and clones generated against adenovirus-pp65-infected dendritic cells” (#13) were the predominant topics in the initial studies on EMT and immunotherapy. Current hotspot research areas include “epithelial-mesenchymal transition” (#1), “therapeutic strategy” (#3), “squamous cell cancer” (#4), and “cancer therapy” (#6). Furthermore, EMT-immunotherapy research was found to receive attention in terms of squamous cell cancer as the latest research topic (Figure 7(b,c)). As shown in Figure 8, the top 25 references with the strongest citation bursts indicated that the study with the strongest burst rate (18.43) was a paper titled “Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoral immunosuppression,” published in Nature Communications by Limo Chen et al. in 2014, with the most significant citation burst values from 2016 to 2019. Notably, approximately half of the references indicated so-called burstiness from 2016, which suggests that EMT-immunotherapy research has attracted increasing scholarly attention since 2016. More importantly, the paper titled “EMT: 2016,”³² published in Cell and authored by Jean Paul Thiery et al., presented the longest citation burstiness from 2016 to the present date.

Figure 8. The top 25 references with the strongest citation bursts visualized by CiteSpace.

Figure 7. CiteSpace visualization map of the reference (a), ridgeline plot of references (b), and timeline view of references (c). Each node on the lines represent a reference, and the cluster with different color showed the time evolution.

Table 5. Top 10 co-cited references concerning the research of EMT-immunotherapy.

Title	Year	Journal	First author	Total citations	Average citation per year	Main conclusion
Depletion of Carcinoma-Associated Fibroblasts and Fibrosis Induces Immunosuppression and Accelerates Pancreas Cancer with Reduced Survival	2014	Cancer Cell	Oezdemir, Berna C	1621	158.7	Reduction of fibrosis and my fibroblasts enhanced EMT and immune surveillance. (original article)
Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease	2016	Nature Reviews Clinical Oncology	Bianchini, Giampaolo	1577	191.38	Relevant molecular findings in TNBC from the past decade and the most promising therapeutic opportunities derived from these data. (review)
Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy	2017	Nature Reviews Immunology	Nagarsheth, Nisha	1224	168.86	The potential chemokine targets and relevance cancer immunotherapy. (review)
Cancer Metastasis Is Accelerated through Immunosuppression during Snail-induced EMT of Cancer Cells	2009	Cancer Cell	Lin, Yuxin	661	129.6	Inhibition of snail-induced EMT could suppress cancer metastasis and immunosuppression. (original article)
Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications	2019	Journal of Hematology & Oncology	Kudo-Saito, Chie	654	42.4	The underly mechanism of Tumor-associated.macrophages (TAMs) in tumor metastasis and clinical therapeutic applications. (review)
Tumor Microenvironment Characterization in Gastric Cancer Identifies Prognostic and Immunotherapeutically Relevant Gene Signatures	2019	Cancer Immunology Research	Zeng, Dongqiang	528	103.4	The feature of Tumor microenvironment (TME) and new strategies for cancer treatment by using bioinformatics analysis (original articles)
Epithelial-Mesenchymal Plasticity in Cancer Progression and Metastasis	2019	Developmental Cell	Lu, Wei	501	98.2	A review of EMT in cancer progression. (review)
Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy	2020	Frontiers in Molecular Biosciences	Henke, Erik	449	112.25	A review of the role of extracellular matrix (ECM) in cancer treatment. (review)
Drug resistance and combating drug resistance in cancer	2019	Cancer Drug Resistance	Wang, Xuan	414	82.4	The mechanism of drug resistance and potential strategies for drug resistance therapy. (review)
Immunological landscape and immunotherapy of hepatocellular carcinoma	2015	Nature Reviews Gastroenterology & Hepatology	Prieto, Jesus	404	44	Description of immunotherapies in HCC and its combination therapies. (review)

Table 6. The top 10 EMT-immunotherapy related articles with the most citations.

Title	Date of publishing	Citations	Total link strength	Journal	JCR (2022)	First author
Hallmarks of Cancer: The Next Generation	2011	150	846	Cell	Q1	Douglas Hanahan
TGF beta attenuates tumor response to PD-L1 blockade by contributing to exclusion of T cells	2018	108	1016	Nature	Q1	Sanjeev Mariathasan
The epithelial-mesenchymal transition generates cells with properties of stem cells	2008	104	711	Cell	Q1	Sendurai A Mani
Inferring tumor purity and stromal and immune cell admixture from expression data	2013	102	809	Nature communication	Q1	Kosuke Yoshihara
Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoral immunosuppression	2014	88	838	Nature communication	Q1	Limo Chen
The blockade of immune checkpoints in cancer immunotherapy	2012	86	657	Nature Review Cancer	Q1	Drew M Pardoll
The basics of epithelial-mesenchymal transition	2009			The Journal of Clinical investigation	Q1	Kalluri, Raghu
Molecular mechanisms of epithelial-mesenchymal transition	2014	74	631	Nature Reviews molecular Cell Biology	Q1	Samy Lamouille
Epithelial-Mesenchymal Transition Is Associated with a Distinct Tumor Microenvironment Including Elevation of Inflammatory Signals and Multiple Immune Checkpoints in Lung Adenocarcinoma	2016	73	755	Clinical Cancer Research	Q1	Yanyan Lou
New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer	2019	72	641	Nature Reviews molecular Cell Biology	Q1	Dongre, Anushka

Keyword analysis: hotspots and emerging topics

A total of 2,695 keywords were obtained from titles and abstracts, of which 183 items satisfied the threshold. Keyword co-occurrence is indicative of interest in scientific areas and future research directions, creating a framework for studies and defining useful concepts. In this study, the top 183 keywords were grouped into four clusters of different colors (red, green, blue, and yellow). EMT, immunotherapy, tumor microenvironment, prognosis, metastasis, breast cancer, and PD-l1 were the keywords with the highest frequency of use. More importantly, time-dependent overlay visualization revealed the following emerging keywords: “immune infiltration,” “bioinformatics analysis,” “traditional Chinese medicine,” “gene signature,” “ferroptosis,” and “immune exclusion,” reflecting the interest of researchers in exploring methods to monitor obstacles in clinical immunotherapy (Figure 9(a)). Keywords denoting molecules, disease types, and cell types are indicative of more general topics and frontiers in the research field (Table 7). The most frequently occurring molecular terms were TGF-β, NFκ-B, and E-cadherin; EMT, metastasis, and cell survival were the top three physiological or pathological states, and the most common diseases addressed in these studies were breast, colorectal, and lung cancers. In terms of cell types, T cells, regulatory T cells, and stem cells were the top three most frequently occurring cell types in EMT-immunotherapy research.

Figure 9. Clustering co-occurrence map of keywords in EMT-immunotherapy study (a) and the top 18 keywords with the strongest citation bursts (b).

Table 7. Top 5 key molecules, states, disease, and cell types in studies on EMT-immunotherapy.

Rank	Molecules	Frequency	States	Frequency	Disease	Frequency	Cell types	Frequency
1	tgf-beta	121	EMT	667	Breast cancer	121	T-cells	97
2	nf-kappa-b	105	Metastasis	138	Colorectal-cancer	87	Regulatory t- cells	95
3	e-cadherin	98	Cell survival	102	Lung-cancer	68	Stem- cells	76
4	Endothelial growth-factor	46	Invasion	97	Hepatocellular-carcinoma	51	Suppressor-cells	67
5	pd-l1	38	Growth	89	Prostate-cancer	45	Dendritic cells	59

The 18 keywords with the strongest citation bursts are shown in Figure 9(b). Prostate cancer had the strongest citation burstiness (6.34), which began in 2010 and ended in 2015. The keyword “antigen” showed the longest burstiness, beginning in 2001 and ending in 2017. Moreover, terms such as “melanoma,” “prostate cancer,” “breast cancer,” and “lung cancer” are indicative of research trends in clinical applications. Our analysis suggests that NK cells and PDL blockade have gradually gained attention in recent years.

Discussion

General information on EMT-immunotherapy research

Maintaining leadership in a particular field and understanding the latest findings have become increasingly challenging in this era of information and data overload. In the present study, bibliometric analysis was used to collect bibliographic information to construct a knowledge base to define the structure of the field of EMT-immunotherapy research. A total of 1,302 articles were retrieved from the WOSCC database as of September 21, 2023, representing publications in 429 journals by 8,155 authors from 1,777 institutions in 75 countries with 2,695 co-occurrence keywords.

Global variation in publications and citation frequencies are important indicators for evaluating the developmental trends in scientific research fields.³³ From our analysis, it can be concluded that EMT-immunotherapy research first emerged in 1999 and underwent a long period of dormancy (2011–2018) before entering a stage of rapid development (2018–2022). According to the trend depicted in Figure 1, current research can be divided into three stages “germination,” “continual growth,” and “rapid increase.” The first stage (“germination”) occurred from 1999 to 2011. In this phase, studies related to EMT-immunotherapy first began to be published. The second stage, “continual growth,” occurred from 2012 to 2017—155 articles were published within six years, which implies that the role of EMT in immunotherapy has increasingly attracted the attention of more and more scholars. In the most recent stage of development, i.e., “rapid growth” (2018 to 2022), the number of publications related to EMT-immunotherapy increased tremendously; 1,128 articles were published in this period, accounting for an increase of approximately seven-fold compared to that in the second stage. Additionally, along with an increase in the number of publications, the frequency of citations also increased. The number of documents regarding EMT-immunotherapy reached a peak in 2022 (n = 331), with the publication of 211 articles, a peak in publication activity that had not decreased by September 2023. These data indicate that the “burst” period is yet to come; thus, more research on this topic is expected.

Subsequent country analysis indicated an unbalanced distribution (Figure 2(a)). According to the analysis of the strongest citation bursts by country and network visualization (Figures 2(d) and 3), although China was found to be the most productive country, the United States occupied the dominant position. Interestingly, although Chinese institutions entered this field of research at a later date, China went on to become the most productive country in recent years, which could be due to increasing investments being made in scientific research and the large number of researchers in China. The TLS value is used to evaluate the level of international cooperation among countries or research groups. The United States showed the highest TLS (and citations), indicative of its active collaboration with other countries and the strong international influence of American research on the global research community. However, nine of the top 10 most productive institutions were Chinese. This result coincides with the fact that China had the largest number of articles (in the current study period). It should be noted, however, that despite Fudan University contributing the most articles, an American institution, the University of Texas, MD Anderson Cancer Center, obtained the highest number of citations (Table 2), highlighting its significant influence on EMT-immunotherapy research.

Based on the analysis of journals and co-cited journals, as shown in Figure 6 and Table 3, the top five most popular journals were Cancers, Frontiers in Oncology, Frontiers in Immunology, International Journal of Molecular Sciences, and Frontiers in Cell and Development Biology. The cumulative occurrence of these journals shows the real-time changes in the number of articles. Among the top 10 journals, half were series of the Frontiers group of journals, which are completely open access and have gained increasing popularity recently. Furthermore, Frontiers in Immunology is considered the most influential journal based on document number and impact factors in the top 10 productive journals. Additionally, EMT-immunotherapy research has focused on basic research and clinical medicine according to the dual-map overlay map, which showed two citation paths. The thematic distribution map revealed that molecular, biological, immunological, medical, and clinical-related journals published the largest number of papers on EMT-immunotherapy. Meanwhile, journals in the areas of molecular, biological, and genetic research were the main co-cited journals. The journals of the JCR division in Q1 comprised the most productive journals (50%) and co-cited journals (80%), which indicated that these journals are critical for EMT-immunotherapy research.

Authors who are associated with a high number of published articles or the most cited articles in a particular field represent an effective resource for researchers who are starting to conduct novel research or seek stronger collaborators.³⁴ According to our results (Table 4), Claudia Palena published the most articles, and Jean Paul Thiery, who has been engaged in immunotherapy research for a long time, had the highest H-index in this research era. Thierry was not only the most influential author but also one of the top three cited authors. The map of authors and co-cited authors provides important information about which authors or their teams are more likely to publish more articles or contribute works.

Knowledge base

Knowledge base is a collection of cited references that are commonly cited in a particular field of study and can help researchers grasp the basic concepts and principles of a new direction of research.³⁵ The top-cited articles not only have a high academic value but also have a strong professional influence in the field.

The most co-cited references are listed in Table 5. In 2014, an original research article titled “Depletion of Carcinoma-Associated Fibroblasts and Fibrosis Induces Immunosuppression and Accelerates Pancreas Cancer with Reduced Survival,” published in Cancer Cell, was the most frequently cited paper. The authors of this paper reported a significant finding in EMT-immunotherapy research – fibroblasts enhance EMT and immune surveillance in pancreatic ductal adenocarcinoma (PDAC).²⁹ It is well known that the tumor microenvironment (TME) is crucial for EMT, and extracellular signals, extrinsic factors, and pathological conditions in the TME can induce EMT-TFs (Snail, Slug, Twist1, and Zeb1/2), which are essential for the execution of EMT.^20,36,37 Two years later, in 2016, investigators reviewed the challenges and opportunities related to triple-negative breast cancer (TNBC), highlighting the role of immunotherapy and novel combinatorial immune-molecular therapy in TNBC.³⁸ The review paper titled “Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease”¹ was the most frequently cited paper, based on the annual average citations. A third study revealed that chemokines in the tumor microenvironment have distinct effects on cancer therapy and progression and that the targets of chemokine networks, when combined with other immunotherapies, may represent a potential treatment for cancer.³⁹ The fourth article was an experimental study published in Cancer Cell in 2009. The authors indicated that an essential EMT-TF, Snail, can trigger EMT-induced metastasis and immune invasion.¹³ In the fifth study, investigators summarized the role of tumor-associated macrophages and their potential clinical applications.⁴⁰ The sixth most cited articles were original articles that provide a comprehensive overview of TME in gastric cancer, whose results contribute to the evaluation of responses to immunotherapies and the use of bioinformatics analysis in cancer treatment.³¹ The seventh study on our list was published in the journal Developmental Cell in 2019, which summarized the roles and underlying mechanisms of epithelial-mesenchymal plasticity in cancer therapeutics.⁴¹ The eighth study, published in 2020, was a review on the role of tumor extracellular matrix (ECM) in cancer therapy.⁴² The ninth study was an overview of the mechanism of drug resistance and potential strategies for drug resistance therapy.⁴³ The tenth article, published in Nature Reviews Gastroenterology & Hepatology, was a review article in which the research status of immunotherapy for hepatocellular carcinoma (HCC) was discussed, along with new insights into therapies for HCC based on several immune cells, vaccines, or oncolytic viruses.⁴⁴ In all of these studies, the main focus was the mechanism of tumor immunotherapy as well as strategies for improving immunotherapeutic efficacy.

The top 10 co-cited references involved three main aspects: (i) an analysis of the role of EMT in immunotherapy, with a discussion of new therapeutic strategies based on EMT suppression; (ii) the investigation of clinical therapeutic applications based on the tumor microenvironment; (iii) an analysis of potential targets, such as chemokines, extracellular matrix-related genes, and EMT-related genes, for improving immunotherapy. Taken together, these three aspects were found to represent the foundations of EMT-immunotherapy research.

Hotspots and emerging frontiers in EMT-immunotherapy research

The top 10 citation references provide an insights into the hotspots in this research field, which include immune evasion as a hallmark of cancer,⁴⁵ the role of EMT in immunotherapy,^46–49 and current opportunities and challenges for immunotherapy.^50–52 Interestingly, there were three bioinformatics-related articles in the top 10 citation references, highlighting the emergence of the use of multidimensional analysis approaches in EMT-immunotherapy research.

Reference and keyword analyses can be used to identify hotspots in research fields, which are crucial for predicting potential future directions in a particular field. The Ridgeline plot and timeline perspectives generated in the present study reflect the historic growth and continuous change in the field of EMT-immunotherapy research, from the first initial studies to recent hotspots and emerging topics (Figure 8). The references with the strongest citation bursts, including the mechanism whereby EMT induces immunosuppression and the exploration of novel therapeutic strategies based on EMT to improve immunotherapy, highlight the directions in this research field. Similarly, the results of keyword analysis reflects the research highlights. For example, the network visualization of keywords represents the popularity of topics in terms of the size of nodes, while the time-dependent visualization of keywords presents emerging topics in different colors (Figure 9). Based on the visualization outcomes, we divided the keywords into several categories (molecules, states, diseases, and cells) to understand the knowledge structure and identify general topics in EMT-immunotherapy research (Table 6).

In terms of disease type, emphasis was given to breast cancer. Breast cancer is the leading cause of death from cancer in women worldwide,⁵³ wherein triple-negative breast cancer (TNBC) has the highest rate of mortality. Breast cancer is a highly heterogeneous cancer that is accompanied by high-frequency mutations and the abnormal activation of signaling pathways, posing significant obstacles to its treatment.⁵⁴ Although, some receptor-specific targets are effective in TNBC patients under specific clinical conditions, most patients show no response; thus, more effective treatments are needed.⁵⁵ Recent research has shown that immunoconjugates, such as sacituzumab govitecan, exert therapeutic effects in patients with TNBC; additionally, the immunotherapeutic agent atezolizumab, with paclitaxel, has been approved for use in patients with PD-L1-positive expression.⁵⁶ However, due to the complexity and high malignancy of TNBC, there is a need to identify more effective treatment modalities. The malignant transformation of TNBC always involves the alteration of the EMT process.^57,58 Thus, the study of the molecular mechanisms of EMT involved in immunotherapy may provide insights into a novel and promising therapeutic target for BC.

EMT comprises various stages, including epithelial, partial EMT, and mesenchymal cell states, and plays crucial roles in tumor malignancy, immunotherapy regulation, and the shaping of the therapeutic response.⁵⁹ Cancer cells with mesenchymal features show resistance to attacks by CD8+ T or NK cells and induce immunosuppression in hypoxic TME.⁶⁰ The plasticity, inflammation, and immunosuppression in the TME induced by EMT could increase the low response to immunotherapy and therapeutic resistance, which are the main factors hindering immunotherapy. EMT is also associated with the activation of different immune checkpoint molecules, and the EMT score may represent a novel biomarker of clinical response for immunotherapy.⁶¹

TGF-β, one of the terms with the highest occurrence in the literature, is a versatile cytokine that comprises three isoforms – TGF-β1, TGF-β2, and TGF-β3. Generally, TGF-β is considered a double-edged sword in different phases of cancers, with antitumor effects in pre-malignant cancers and tumor-promoting effects in advanced cancers.⁶² Furthermore, TGF-β reportedly regulates the functions of various immune cells, including suppressing T cells (e.g. cytotoxicity T cells and helper T cells), antigen presentation of dendritic cells (DCs), and natural killer cells, thereby inducing the differentiation of regulatory T cells (Tregs).^24,63,64 Moreover, as an inducer, TGF-β plays a prominent role in EMT to promote metastasis and drug resistance in cancers.⁶⁵ TGF-β also acts as a bridge between the EMT and immunotherapy, regulating the multistep and dynamically controlled process of TGF-β-induced EMT, thereby inducing immune escape. Recently, various TGF-β inhibitors have been developed as promising strategies to explore the potential of immunotherapy for treating tumors; some of these have been used in clinical trials, including NCT05304936, NCT02452008, NCT05821595, NCT04729725, NCT05198505, and NCT05653284 (https://clinicaltrials.gov/).

In this study, among immune cells, T cells were found to be the most commonly studied cell type in the field of EMT-immunotherapy. Based on their functions, T cells are divided into two subgroups – CD4+ T helper cells (Th) and CD8+ T lymphocytes (CTL).⁶⁶ EMT is associated with the exclusion of immune cells critical in the immune response to cancer, with a significantly reduced infiltration of CD4 T cells reported in lung adenocarcinoma and CD4/CD8 T cells in squamous cell carcinoma.⁶⁷ TGF-β type II receptor induces the activation of SMAD3 in CD8+ T cells, resulting in the failure of immunotherapy.⁶⁸ Identification of the mechanism of EMT involved in T cell exhaustion and tumor immunotherapy suppression could provide a target for cancers, especially in particularly devastating tumors.

Keywords with a high burst index in recent years indicate areas of research that have great potential for development in the near future. According to the time-dependent overlay visualization and keywords citation bursts analysis, PD-1 blockage could be emerging research topic in the near future. Meanwhile, gene signature, immune infiltration, ferroptosis, traditional Chinese medicine, and bioinformatics analysis were among other emerging keywords. Traditional Chinese medicine (TCM) is believed to achieve therapeutic effects by enhancing immunity, and studies on the mechanisms by which TCM regulates immunity are likely to provide insights into how this therapeutic approach inhibits cancer progression.⁶⁹ The induction of ferroptosis, a kind of cell death dependent on lipid peroxidation, was found to enhance cancer immunotherapy.⁷⁰ Furthermore, CD8+ T cells have been reported to induce cell death through ferroptosis-specific lipid peroxidation in cells.⁷¹ Revealing the characteristics of immune cells, evaluating the response and efficacy of immunotherapy based on the immune microenvironment, and exploring novel therapeutic strategies through bioinformatics analysis were among the most popular research approaches. Establishing gene signatures with EMT based on The Cancer Genome Atlas (TCGA) or the Gene Expression Omnibus (GEO) database can provide a basis for the evaluation of prognosis and the development of novel targets for immunotherapy.^25,72 Overall, all the topics identified as hotspots in recent years were related to the mechanism of EMT in immunotherapy and the development of therapeutic strategies for improving immunotherapeutic approaches for cancer treatment.

Crosstalk between the EMT and immunotherapy

EMT-immunotherapy is intertwined with several factors, including the tumor microenvironment and drug resistance. In fact, a complex regulatory relationship exists between the EMT and immunotherapy⁶⁷: EMT-TF or EMT-related receptors at the molecular level attract immunosuppressive cells or increase the level of immunosuppressive checkpoint molecules or cytokines, such as PDL-1, IL-10, and TGF-β, resulting in the creation of a tumor immunosuppressive microenvironment.⁷³ Numerous factors play important roles in immunosuppressive function, in turn inducing EMT in cancer cells, including CTLA-4 and TIM-3.²⁰ TGF-β is produced by activated CD4+ T cells, which suppress anti-tumor immunity, highlighting its regulatory role in cancer immune evasion via autocrine TGF-β Th cells.⁷⁴ Elucidating the molecular mechanism of the bidirectional regulation between the EMT and immunotherapy is one research direction from which immunotherapy based on EMT targets could be developed.

Future research directions

The future research directions identified in this study include a focus on the mechanisms of EMT in immunotherapy. In this context, “immune infiltration,” “bioinformatics analysis,” “traditional Chinese medicine,” “gene signature,” and “ferroptosis” are expected to become popular research topics in the field of EMT-immunotherapy, including the exploration of effective targets to improve EMT-based immunotherapy or ferroptosis-related genes and its combination with immune microenvironment analysis.^72,75 In addition, in the exploration of new strategies for immunotherapy, the utilization of traditional cChinese medicine is expected to gain popularity.⁷⁶

Limitations

The study also has some inherent flaws. First, The data in our study were all retrieved from the WOSCC database. Nevertheless, WOSCC is the most commonly used bibliometric analysis database, and it can cover most of the information. Second, the data were acquired from bibliometric tools based on machine learning with natural language processing. A special data processing method may result in a biased perspective. However, compared to traditional comments,²⁶ our results are consistent and provide more objective data.

Conclusions

Currently, the field of EMT-immunotherapy research is at a stage of rapid development, with active collaboration among academic groups worldwide. Among the countries most active in this field, China contributes the largest number of publications, while the United States holds the dominant position in terms of frequency of citations and citation burstiness. Based on its high number of papers and large impact factor, Frontiers in Immunology is currently considered the most influential journal in this field. Similarly, based on citation and co-cited author analyses, Jean Paul Thiery is the most influential author. In terms of popular topics of study, the mechanisms of EMT in immunotherapy and the exploration of novel therapeutic targets for cancer are among the current top research hotspots. Additionally, bioinformatic analysis has become the most effective approach to evaluate immune status and prognosis, acting as a guide for decision-making and diagnosis in cancer immunotherapy. Finally, the combinations of distinct therapeutic strategies, such as Chinese traditional medicine and ferroptosis-related targets, are expected to become emerging topics of interest in future studies.

Author contributions

All authors contributed to the conceptualization and design of the study. Wei Du: Data curation, formal analysis, methodology, supervision, funding acquisition. Zemin Tang: Writing revision & editing, manuscript formatting. Ashuai Du: Software, formal analysis, visualization. Qinglong Yang: Investigation, visualization, conceptualization. Rong Xu: Writing original draft, writing- review & editing. All authors participated in the manuscript revision and approved the final manuscript for submission.

Disclosure statement

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

Data availability statement

All the data involving in this study can be found in online repositories. The details of repositories can be found in manuscript/supplementary material.

Additional information

Funding

The study was supported by grants from the Key Project of Scientific Research Plan of Hunan Provincial Health Commission (C202301047982, Wei Du), the Wings Scientific and Technological Foundation of The First People’s Hospital of Changde City (2022ZZ05, Wei Du) and Youth Fund project of Guizhou Provincial People’s Hospital [GZSYQN(2021)14, Qinglong Yang].