Background: The role of tumor-associated macrophages (TAMs) in patients with esophageal squamous cell carcinoma (ESCC) following surgery remains controversial. Hence, we performed the present study to systematically analyze the prognostic and clinical significance of distinct TAMs biomarkers and distributions in ESCC patients underwent surgery.

Methods: PubMed, Web of Science, and EMBASE databases were searched up to March 31, 2023. The pooled analysis was conducted to evaluate the effects of TAMs on overall survival (OS), disease-free survival (DFS), and clinicopathological characteristics using fixed-effects or random-effect model.

Results: Involving a total of 2,502 ESCC patients underwent surgery from 15 studies, the results suggested that the total count of CD68+ TAMs was inversely associated with OS and DFS in ESCC patients, which was also noticed in the relationship of CD68+ TAMs in tumor islet (TI) with OS (all P<0.05), although no association between CD68+ TAMs in tumor stroma (TS) and OS (P>0.05). Moreover, either islet or stromal CD163+ TAMs density was a prognostic factor ESCC (all P<0.05). Similarly, an elevated CD204+ TAMs density in TI predicted a poor DFS (P<0.05), although CD204+ TAMs in TI had no relationship with OS (P>0.05). Besides, a high CD68+ TAMs density was significantly associated with lymphatic vessel invasion, vascular invasion, and lymph node metastasis (all P<0.05).

Conclusion: Our results demonstrated the prognostic and clinical significance of TAMs in ESCC patients underwent surgery. TAMs should be considered a target that could improve prognostic stratification and clinical outcomes in ESCC after surgery.

Esophageal carcinoma, one of the most common malignant gastrointestinal carcinomas, is ranked seventh globally in terms of incidence, with a staggering 604,000 new cases [1]. Despite the introduction of novel therapeutic strategies such as video-assisted thoracoscopic surgery and molecularly targeted therapy, the 5-year survival rate for esophageal carcinoma remains disappointingly unchanged, particularly for individuals with advanced-stage diseases [2]. In 2020, esophageal carcinoma has the sixth-highest mortality among malignancies, which is responsible for 544,000 deaths in the world [1]. Consequently, it becomes imperative to explore biomarkers that can effectively reflect the tumor's biological behavior and prognosis in esophageal carcinoma.

The tumor microenvironment, consisting of various immune cells, plays an important role in tumor progression, invasion and metastasis of tumors [3,4]. Tumor-associated macrophages (TAMs), a class of immune cells, constitute approximately 50% of all cells and are a major component in the tumor microenvironment of solid tumors [5]. TAMs exert significant influence on numerous aspects of tumor cell biology, encompassing antigen presentation, angiogenesis, tissue repair, and tumor cell destruction [6]. Prior research suggests that TAMs drive pathological processes and possess prognostic significance in diverse cancers, including lung cancers [7], liver cancers [8], and breast cancers [9]. In general, a high infiltration of TAMs in tumor microenvironment indicates a poor prognosis, however, outcomes vary depending on macrophages biomarkers and histologic locations in patients with esophageal carcinoma [10–12]. Esophageal adenocarcinoma is the predominant subtype of esophageal carcinoma in North America and Europe, whereas esophageal squamous cell carcinoma (ESCC) is the most common subtype in China, accounting for over 90% among esophageal carcinoma patients [13]. Up to now, ESCC remains the primary subtype among esophageal carcinoma cases. Consequently, we present the first comprehensive study to assess the prognostic and clinical significance of distinct macrophage biomarkers and tissue distributions of TAMs in the tumor microenvironment of ESCC.

Literature search‘

This study was registered with PROSPERO (CRD42022324113). In line with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines [14], we conducted a comprehensive search of PubMed, Web of Science, and EMBASE databases to identify potential studies published in scholarly journals up until March 31, 2023. The Medical Subject Heading terms and/or text words entered were ‘macrophage’, ‘tumor-associated macrophage’, ‘esophageal’, ‘esophagus’, and ‘oesophagus’. We also undertaken forward and backward citation tracking to identify additional non-indexed literature. No language or country restrictions were applied to the present pooled analysis. Titles and abstracts of the articles obtained from these searches were independently screened by two reviewers to ascertain if they met the inclusion criteria.

Inclusion and exclusion criteria

We included studies reporting TAMs associated with ESCC. To be considered for inclusion, studies had to meet the following criteria: (1) they were cohort studies, such as prospective, retrospective, or case control studies. (2) TAMs must be measured at the primary tumor site using immunohistochemistry with the markers including CD68, CD163, or CD204. (3) patients were diagnosed as ESCC through pathology; and (4) the studies reported the association of TAMs with overall survival (OS), disease-free survival (DFS), and clinicopathological characteristics.

The following studies were excluded: (1) those that fell into specific types of literature, including reviews, comments, and conference abstracts; (2) TAMs were measured at metastatic or local relapse sites; (3) patients with esophageal carcinoma were diagnosed with non-ESCC, such as esophageal adenocarcinoma; and (4) studies that did not provide relevant results or had repetitive raw data.

Data extraction and quality assessment

Referring to the inclusion and exclusion criteria, relevant data were extracted independently from the original studies by two reviewers. Discrepancies would be resolved by re-evaluation and discussion with the other reviewer. The following indices were collected from each study: the last name of the first author, year of publication, demographic characteristics of participants, macrophage markers, macrophage distribution (tumor islet [TI] or tumor stroma [TS]), tumor stage, OS and DFS with hazard ratios (HRs) and 95% confidence interval (CI). In addition, we collected the prognostic information from study only reported with a Kaplan–Meier (KM) plot and a P-value derived from log-rank analysis. HRs and 95%CI were extracted from KM plot using Engauge Digitizer version 4.1 (a freely available software downloaded from http://sourceforge.net) and calculated as previously described [15]. When necessary, authors were contacted for additional unpublished data.

Two experienced reviewers independently assessed the quality of each included study using the modified Newcastle–Ottawa Scale (NOS) [16]. Studies were scored in accordance to three evaluation indexes, including patient selection, study comparability and outcome assessment. The included study was graded as high quality with an NOS score ≥ 6. Disagreements were resolved by a third reviewer.

Statistical analysis

The statistical analysis was conducted following the guidelines set forth by The Cochrane Collaboration. We employed hazard ratios (HRs) with a 95% confidence interval (CI) to assess the relationship between TAMs (tumor-associated macrophages) density and survival outcomes. To examine dichotomous data related to clinicopathological features, we utilized odds ratios (OR) along with their corresponding 95% CI. Our findings were depicted graphically using either a forest plot or a table. To evaluate variations within and between studies, we employed Cochran’s Q-statistics. Additionally, we assessed heterogeneity across studies using the I2 statistics, which range from 0 to 100%. For I2 values below 50%, indicating low heterogeneity, we conducted data analysis using a fixed-effect model. Conversely, for high heterogeneity with I2 values greater than or equal to 50%, we employed a random-effect model. Sensitivity analysis was performed by altering the statistical method and analysis model.

The statistical analysis was performed according to the recommendations from Cochrane Collaboration. The HRs with 95% CI was used to evaluate the correlation between the TAMs density and survival outcomes. The odds risk (OR) and corresponding 95% CI for the difference in clinicopathological features were used to measure the dichotomous data. Results are presented graphically using a forest plot graph or table. Cochran’s Q-statistics was used to evaluate within- and between-study variations. Heterogeneity across studies were assessed by the I2 statistics, which ranged from 0 to 100%. Data were analyzed with a fixed-effect model for I2 < 50%, which was considered as low heterogeneity. Otherwise, the random-effect model was applied for high heterogeneity with I2 ≥ 50%. Sensitivity analysis was conducted by shifting the statistical method and analysis model. Potential publication bias was assessed by funnel plots, when the number of included studies reached five or more. The pooled data were analyzed using Review Manager Version 5.3 software (The Nordic Cochrane Center, The Cochrane Collaboration, 2014, Copenhagen). P<0.05 was considered statistically significant.

Search results and study characteristics

A total of 1,317 articles were retrieved during our initial search. After electronically removing 702 duplicated articles and irrelevant studies, we excluded 562 studies based on the assessment of their titles and abstracts. Subsequently, 53 articles were evaluated in detail. Of these, 38 articles were excluded after reviewing the full text, 15 unique studies were ultimately included in the pooled analysis [10–12,17–28]. The detailed screening process was presented in Figure 1.

Flow diagram of the article selection

Figure 1
Flow diagram of the article selection
Figure 1
Flow diagram of the article selection
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The main characteristics of the included studies are summarized in Table 1. A total of 2,502 ESCC patients underwent surgery were included in the 15 studies, which were published from 2002 to 2021. As to TAMs identification, 13 articles adopted CD68 [10,11,17–25,27,28], five articles adopted CD163 [12,19,20,25,26], and three articles adopted CD204 as a macrophage marker [12,19,21,25] to detected TAMs by immunohistochemistry. Ten studies investigated the role of TAMs in both TI and TS [11,17–20,23–28], one study only investigated TAMs in TI [21], and two studies only investigated TAMs in TS [12,22]. Moreover, there were 14 articles that reported OS data [10–12,17–24,26–28], and five articles that reported DFS data [19,22,24,25,28]. The NOS scores of these studies ranged from 4 to 8 (Table 1).

Table 1
Characteristics of studies included in the pooled analysis
AuthorCountrySample sizeMaleMarkersTissue distributionTypeStageOutcome assessmentNOS
Koide (2002) Japan 56 42 CD68 Tumor islet and stroma ESCC Unavailable OS 
Guo (2007) China 137 103 CD68 Tumor islet and stroma ESCC I-IV OS 
Shigeoka (2013) Japan 70 55 CD68, CD163, CD204 Tumor islet and stroma ESCC I-IV OS, DFS 
Sugimura (2015) Japan 210 186 CD68, CD163 Tumor islet and stroma ESCC Unavailable OS 
Hatogai (2016) Japan 196 160 CD68, CD204 Tumor islet ESCC I-IV OS 
Li (2016) China 705 430 CD68 Tumor stroma ESCC I-IV OS, DFS 
Xu (2016) China 138 102 CD68 Tumor islet and stroma ESCC II-III OS 
Zhu (2016) China 220 70 CD68 Tumor islet and stroma ESCC II OS, DFS 
Hosono (2017) Japan 70 NA CD68, CD163, CD204 Tumor islet and stroma ESCC I-IV DFS 
Hu (2017) China 100 68 CD163 Tumor islet and stroma ESCC I-IV OS 
Wang (2017) China 100 117 CD68 Tumor islet and stroma ESCC I-IV OS 
Lu (2019) China 200 116 CD68, PD1 NA ESCC I-IV OS, DFS 
Yamamoto (2020) Japan 86 75 CD86, CD163, CD206 Tumor stroma ESCC I-IV OS 
Chen (2021) China 114 82 CD68, PD1 NA ESCC I-IV OS 
Jiang (2021) China 100 66 CD68, HLA-DR Tumor islet and stroma ESCC I-IV OS 
AuthorCountrySample sizeMaleMarkersTissue distributionTypeStageOutcome assessmentNOS
Koide (2002) Japan 56 42 CD68 Tumor islet and stroma ESCC Unavailable OS 
Guo (2007) China 137 103 CD68 Tumor islet and stroma ESCC I-IV OS 
Shigeoka (2013) Japan 70 55 CD68, CD163, CD204 Tumor islet and stroma ESCC I-IV OS, DFS 
Sugimura (2015) Japan 210 186 CD68, CD163 Tumor islet and stroma ESCC Unavailable OS 
Hatogai (2016) Japan 196 160 CD68, CD204 Tumor islet ESCC I-IV OS 
Li (2016) China 705 430 CD68 Tumor stroma ESCC I-IV OS, DFS 
Xu (2016) China 138 102 CD68 Tumor islet and stroma ESCC II-III OS 
Zhu (2016) China 220 70 CD68 Tumor islet and stroma ESCC II OS, DFS 
Hosono (2017) Japan 70 NA CD68, CD163, CD204 Tumor islet and stroma ESCC I-IV DFS 
Hu (2017) China 100 68 CD163 Tumor islet and stroma ESCC I-IV OS 
Wang (2017) China 100 117 CD68 Tumor islet and stroma ESCC I-IV OS 
Lu (2019) China 200 116 CD68, PD1 NA ESCC I-IV OS, DFS 
Yamamoto (2020) Japan 86 75 CD86, CD163, CD206 Tumor stroma ESCC I-IV OS 
Chen (2021) China 114 82 CD68, PD1 NA ESCC I-IV OS 
Jiang (2021) China 100 66 CD68, HLA-DR Tumor islet and stroma ESCC I-IV OS 

Abbreviations: DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; OS, overall survival; NA, not available; NOS, Newcastle–Ottawa scale.

Prognostic significance of CD68+ TAMs

A total of 13 studies were included in the analysis of the prognostic significance of CD68+ TAMs on survival outcomes in patients with ESCC. As for the total count of CD68+ TAMs, a high CD68+ TAMs density was significantly associated with poor OS (HR = 1.49, 95% CI: 1.16–1.92, P=0.002; I2 = 33%; Figure 2A) and DFS (HR = 1.85, 95% CI: 1.10–3.11, P=0.02; I2 = 60%; Figure 2B), compared with a low CD68+ TAMs density.

Forest plots of HR for survival outcomes between high and low density of total CD68+ TAMs infiltration in the tumor among ESCC patients underwent surgery.

Figure 2
Forest plots of HR for survival outcomes between high and low density of total CD68+ TAMs infiltration in the tumor among ESCC patients underwent surgery.

(A) HR of OS for total CD68+ TAMs in the tumor; (B) HR of DFS for total CD68+ TAMs in the tumor. DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophages.

Figure 2
Forest plots of HR for survival outcomes between high and low density of total CD68+ TAMs infiltration in the tumor among ESCC patients underwent surgery.

(A) HR of OS for total CD68+ TAMs in the tumor; (B) HR of DFS for total CD68+ TAMs in the tumor. DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophages.

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In addition, higher CD68+ TAMs density predicted worse OS than lower CD68+ TAMs density in TI, with a pooled HR of 1.30 (95% CI: 1.02–1.65, P=0.03; I2 = 17%; Figure 3A). However, the pooled HR showed that CD68+ TAMs infiltration in TS was not associated with OS (HR = 0.97, 95% CI: 0.52–1.81, P=0.93; I2 = 91%; Figure 3B).

Forest plots of HR for OS between high and low density of CD68+ TAMs in ESCC patients underwent surgery.

Figure 3
Forest plots of HR for OS between high and low density of CD68+ TAMs in ESCC patients underwent surgery.

(A) HR of OS for CD68+ TAMs in TI; (B) HR of OS for CD68+ TAMs in TS. ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

Figure 3
Forest plots of HR for OS between high and low density of CD68+ TAMs in ESCC patients underwent surgery.

(A) HR of OS for CD68+ TAMs in TI; (B) HR of OS for CD68+ TAMs in TS. ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

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Prognostic significance of CD163+ TAMs

To assess the role of CD163+ TAMs on survival outcomes in patients with ESCC, a total of five studies were included in the analysis. This pooled analysis was performed in fixed-effect model for the low heterogeneity in the followed results (I2 < 50%). Relative to a high density of CD163+ TAMs, a low density of CD163+ TAMs indicated better OS in TI (HR = 2.45, 95% CI: 1.19–5.05, P=0.02; I2 = 0; Figure 4A), which was similar to CD163+ TAMs in TS (HR = 2.25, 95% CI: 1.59–3.17, P<0.00001; I2 = 42%; Figure 4B).

Forest plots of HR for OS between high and low density of CD163+ TAMs in ESCC patients underwent surgery.

Figure 4
Forest plots of HR for OS between high and low density of CD163+ TAMs in ESCC patients underwent surgery.

(A) HR of OS for CD163+ TAMs in TI; (B) HR of OS for CD163+ TAMs in TS. ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

Figure 4
Forest plots of HR for OS between high and low density of CD163+ TAMs in ESCC patients underwent surgery.

(A) HR of OS for CD163+ TAMs in TI; (B) HR of OS for CD163+ TAMs in TS. ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

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Prognostic significance of CD204+ TAMs

Given the absent of heterogeneity, the fixed-effect model was used in assessing the association between CD204+ TAMs and survival outcomes (I2 = 0), despite only three studies included. The pooled analysis evaluating the role of CD204+ TAMs on OS showed no statistically significant differences in TI (HR = 1.35, 95% CI: 0.91–2.00, P=0.14; I2 = 0; Figure 5A), but higher 204+ TAMs density indicated worse DFS compared with lower 204+ TAMs density in TI (HR = 3.42, 95% CI: 1.52–7.68, P=0.003; I2 = 0; Figure 5B)

Forest plots of HR for survival outcomes between high and low density of CD204+ TAMs in TI among ESCC patients underwent surgery.

Figure 5
Forest plots of HR for survival outcomes between high and low density of CD204+ TAMs in TI among ESCC patients underwent surgery.

(A) HR of OS for CD204+ TAMs in TI; (B) HR of DFS for CD204+ TAMs in TI. DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet.

Figure 5
Forest plots of HR for survival outcomes between high and low density of CD204+ TAMs in TI among ESCC patients underwent surgery.

(A) HR of OS for CD204+ TAMs in TI; (B) HR of DFS for CD204+ TAMs in TI. DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; HR, hazard risk; OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet.

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Association between TAMs and clinicopathological characteristics

We also analyzed the association between TAMs (CD68+ or CD163+) and clinicopathological characteristics in ESCC patients who underwent surgery. Unlike sex, histologic grade, and T grade, the pooled results suggested that a high density of total CD68+ TAMs was significantly associated lymphatic vessel invasion (OR = 2.55, 95% CI: 1.43–4.54, P=0.001; I2 = 0), vascular invasion (OR = 3.25, 95% CI: 1.94–5.47, P<0.00001; I2 = 29%), and lymph node metastasis (OR = 1.85, 95% CI: 1.05–3.28, P=0.03; I2 = 20%). Especially, a high CD68+ TAMs density had a significant association with lymph node metastasis in TI (OR = 2.10, 95% CI: 1.19–3.71, P=0.01; I2 = 0) or TS (OR = 1.58, 95% CI: 1.00–2.51, P=0.05; I2 = 0). However, the results in TI did not reveal any significant association between CD163+ TAMs and any clinicopathological characteristics, including histologic grade and lymph node metastasis (Table 2).

Table 2
Association between TAMs and clinicopathological characteristics of ESCC patients underwent surgery
Clinicopathological characteristicsReferencesNo. of studiesModelPooled OR (95% CI)P-valueHeterogeneity
I2 (%)P-value
CD68+ TAMs (TI+TS) 
Sex (males vs. females) Males Fixed 0.89 (0.60–1.33) 0.58 0.39 
Histologic grade (poor vs. well-moderate) Poor Fixed 1.21 (0.60–2.45) 0.60 30 0.24 
Lymphatic vessel invasion (positive vs. negative) Positive Fixed 2.55 (1.43–4.54) 0.001 0.72 
Vascular invasion (positive vs. negative) Positive Fixed 3.25 (1.94–5.47) < 0.00001 29 0.24 
T grade (T2-4 vs. T1) T2-4 Random 1.23 (0.15–10.10) 0.85 93 < 0.001 
Lymph node metastasis (positive vs. negative) Positive Fixed 1.85 (1.05–3.28) 0.03 20 0.26 
CD68+ TAMs (TI) 
Lymphatic vessel invasion (positive vs. negative) Positive Fixed 2.10 (1.19–3.71) 0.01 0.38 
CD68+ TAMs (TS) 
Age (≥60 y vs. <60 y) ≥60 years Fixed 1.13 (0.71–1.80) 0.60 0.98 
Sex (males vs. females) Males Fixed 0.79 (0.51–1.23) 0.30 0.92 
Location of the primary tumor (upper vs. middle + lower) Upper Fixed 1.20 (0.63–2.31) 0.58 0.90 
Histologic grade (poor vs. well-moderate) Poor Random 1.40 (0.54–3.59) 0.49 74 0.05 
T grade (T3-4 vs. T1-2) T3-4 Random 1.94 (0.47–8.01) 0.36 89 0.003 
Lymph node metastasis (positive vs. negative) Positive Fixed 1.58 (1.00–2.51) 0.05 0.86 
p-stage (III-IV vs. I-II) III-IV Random 1.46 (0.72–2.95) 0.30 50 0.16 
CD163+ TAMs (TI) 
Histologic grade (poor vs. well-moderate) Poor Fixed 1.23 (0.58–2.59) 0.59 0.41 
Lymph node metastasis (positive vs. negative) Positive Fixed 1.73 (0.95–3.15) 0.07 0.43 
Clinicopathological characteristicsReferencesNo. of studiesModelPooled OR (95% CI)P-valueHeterogeneity
I2 (%)P-value
CD68+ TAMs (TI+TS) 
Sex (males vs. females) Males Fixed 0.89 (0.60–1.33) 0.58 0.39 
Histologic grade (poor vs. well-moderate) Poor Fixed 1.21 (0.60–2.45) 0.60 30 0.24 
Lymphatic vessel invasion (positive vs. negative) Positive Fixed 2.55 (1.43–4.54) 0.001 0.72 
Vascular invasion (positive vs. negative) Positive Fixed 3.25 (1.94–5.47) < 0.00001 29 0.24 
T grade (T2-4 vs. T1) T2-4 Random 1.23 (0.15–10.10) 0.85 93 < 0.001 
Lymph node metastasis (positive vs. negative) Positive Fixed 1.85 (1.05–3.28) 0.03 20 0.26 
CD68+ TAMs (TI) 
Lymphatic vessel invasion (positive vs. negative) Positive Fixed 2.10 (1.19–3.71) 0.01 0.38 
CD68+ TAMs (TS) 
Age (≥60 y vs. <60 y) ≥60 years Fixed 1.13 (0.71–1.80) 0.60 0.98 
Sex (males vs. females) Males Fixed 0.79 (0.51–1.23) 0.30 0.92 
Location of the primary tumor (upper vs. middle + lower) Upper Fixed 1.20 (0.63–2.31) 0.58 0.90 
Histologic grade (poor vs. well-moderate) Poor Random 1.40 (0.54–3.59) 0.49 74 0.05 
T grade (T3-4 vs. T1-2) T3-4 Random 1.94 (0.47–8.01) 0.36 89 0.003 
Lymph node metastasis (positive vs. negative) Positive Fixed 1.58 (1.00–2.51) 0.05 0.86 
p-stage (III-IV vs. I-II) III-IV Random 1.46 (0.72–2.95) 0.30 50 0.16 
CD163+ TAMs (TI) 
Histologic grade (poor vs. well-moderate) Poor Fixed 1.23 (0.58–2.59) 0.59 0.41 
Lymph node metastasis (positive vs. negative) Positive Fixed 1.73 (0.95–3.15) 0.07 0.43 

Abbreviations: CI, confidence interval; ESCC, esophageal squamous cell carcinoma; OR, odds ratio; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

Sensitivity analysis and publication bias

Our analyses were robust in terms of choice of the models and statistical methods. The relationship between total CD68+TAMs and OS did not alter by shifting a fixed-effect model to a random-effect model (HR = 1.47, 95% CI: 1.05–2.04, P=0.02). According to the funnel plot of the standard error by log OR, there was no significant publication bias in the present pooled analysis (Figure 6).

Funnel plot of studies with TAM density for potential publication bias assessment.

Figure 6
Funnel plot of studies with TAM density for potential publication bias assessment.

(A) OS and CD68+ TAMs in the tumor; (B) OS and CD68+ TAMs in TI; (C) OS and CD68+ TAMs in TS. OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

Figure 6
Funnel plot of studies with TAM density for potential publication bias assessment.

(A) OS and CD68+ TAMs in the tumor; (B) OS and CD68+ TAMs in TI; (C) OS and CD68+ TAMs in TS. OS, overall survival; TAM, tumor-associated macrophage; TI, tumor islet; TS, tumor stroma.

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Because of the insidious onset, the diagnosis of ESCC is usually delayed, resulting in rapid invasion and challenging treatment [29]. Currently, therapeutic approaches for ESCC consist mainly of surgery, chemotherapy and radiotherapy, with high recurrence rates and fatality rates [2]. TAMs, an important component in tumor microenvironment, might become a promising direction for ESCC therapy. The recent ongoing experimental and pre-clinical TAM-targeted studies have shown that TAMs are connected with prognosis in ESCC patients, yielding different conclusions. Therefore, we conducted a comprehensive analysis to investigate the association between TAMS (CD68+, CD163+ or CD204+) and survival outcomes (OS or DFS) in ESCC through pooling data from 2,502 patients who underwent surgery.

In this pooled analysis, a total of 15 studies were included to analyze the prognostic and clinical significance of TAMs in ESCC patients who underwent surgery. Among these studies, 13 studies used CD68 as a biomarker for TAM identification in tumor tissue, while five and three studies used CD163 and CD204, respectively. Our results suggested that a high CD68+ TAMs density in the tumor microenvironment was significantly associated with poor prognosis (OS and DFS) than a low CD68+ TAMs density. Similarly, greater CD68+ TAMs density in TI predicted worse OS, although no significant association was observed between CD68+ TAMs in TS and OS. Moreover, higher CD163+ TAMs density indicated worse OS in TI and TS. Unlike no statistical significance between CD204+ TAMs and OS, a high density of CD204+ TAMs predicted poor DFS in TI. In addition, we also analyzed the association between TAMs and clinicopathological characteristics in ESCC patients who underwent surgery, which demonstrated that a high density of CD68+ TAMs was significantly associated with lymphatic vessel invasion, vascular invasion, and lymph node metastasis. However, it is important to note that there is substantial heterogeneity, necessitating further studies with larger sample size to validate these conclusions.

From an oncological viewpoint, TAMs have been commonly polarized into two distinct macrophage phenotypes: pro-inflammatory M1 (classically activated macrophages) with tumor suppressive capabilities, and anti-inflammatory M2 (alternatively activated macrophages) with tumor supportive capabilities [12]. M2 macrophages, which contribute to the progression of ESCC by promoting tumor cell growth, invasion and metastasis, as well as restraining anti-tumor immune response cells [30], are characterized by the specific receptors known as CD163 (hemoglobin scavenger receptor) and CD204 (macrophage scavenger receptor I) [31,32]. This may explain that greater CD163+ and CD204+ TAMs density suggested worse prognosis. However, because of the limited studies included in our analysis, we did not find a significant association between CD204+ TAM density in TI and OS. As a biomarker used to identify TAMs, CD68 has been widely accepted by scholars. In our pooled analysis, 13 out of 15 included studies used CD68 for TAM identification. Although the total CD68+ TAMs infiltration in the tumor was significantly associated with poor OS, the survival outcomes of CD68+ TAMs in TI and TS were inconsistent. It has been reported that TAMs infiltrating in TI might be the M2 type predominantly, while TAMs infiltrating in TS might display characteristics of the M1 type [23,27]. This could be the reason why greater CD68+ TAMs density in TI predicted worse OS. However, no significant association was observed between CD68+ TAMs in TS and OS, owing to the attainable limited data.

For avoiding the confounding factors as possible as we can, this pooled analysis exclusively enrolled ESCC patients who underwent surgery. Moreover, we have extensively incorporated studies examining the prognostic and clinical significance of TAMs in ESCC, to make our findings more reliable. Besides, the sensitivity analysis was robust, and no publication bias was detected, which ensured the validity of the present results. Finally, the subgroup analyses were conducted according to different TAMs markers (CD68, CD163 and CD204) and histologic locations (TI+TS, TI, and TS) to assess the effect of TAMs on the prognosis of ESCC, including OS and DFS, which made our pooled analysis more informative and persuasive.

In addition to these strengths, several limitations are existing in our study. On the one hand, the pooled analysis was performed in meticulous detail to minimize heterogeneity, including distinct TAMs markers, different histologic locations, as well as OS or DFS; therefore, there was not enough of such information available from the included studies to perform a pooled analysis, such as HLA-DR+ TAMs, M1 and M2 TAMs. On the other hand, all included studies were retrospective study, which might result in the selection bias in our results. Furthermore, the heterogeneity was significant in some pooled analyses, we still should use caution when drawing conclusions, although a random effects model was used on account of this heterogeneity.

This study represents the first comprehensive analysis of the prognostic and clinical significance of TAMs in ESCC, using existing literature. The findings of our pooled analysis suggested that the higher density of total CD68+ TAMs infiltration in the tumor indicated poor OS and DFS, while greater CD163+ and CD204+ TAMs density suggested worse prognosis in ESCC patients underwent surgery, Moreover, a high CD68+ TAMs density was closely link to lymphatic vessel invasion, vascular invasion, and lymph node metastasis. Given the limitations, further large-scale studies are still required to confirm the conclusion in our study.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

The authors declare that there are no competing interests associated with the manuscript.

This research was supported by grants from the National Natural Science Foundation of China [grant numbers 81974367, 81572281, 81702278, 82003065, and 82172655]; the National Natural Science Foundation of Hunan Province [grant number 2022JJ30925, 2020JJ4132]; the Project Program of National Clinical Research Center for Geriatric Disorders (Xiangya Hospital) [grant number 2021LNJJ17]; and National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases (Lung Cancer) [grant number z027002].

Bin Yi: Software, Formal analysis, Visualization, Methodology, Writing—original draft. Jun Zeng: Software, Formal analysis, Visualization, Methodology, Writing—original draft. Linfeng Li: Software, Formal analysis, Visualization, Methodology, Writing—original draft. Junjie Zhang: Data curation. Yufan Chen: Data curation. Yang Gao: Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review & editing.

We are grateful to the works by the authors of included 15 studies, and all reviewers and editors for reviewing this work.

CI

confidence interval

DFS

disease-free survival

ESCC

esophageal squamous cell carcinoma

HRs

hazard ratios

KM

Kaplan–Meier

NOS

Newcastle–Ottawa scale

OS

overall survival

PRISMA

Preferred Reporting Items for Systematic Reviews and Meta‐Analyses

TAM

tumor-associated macrophage

TI

tumor islet

TS

tumor stroma

1.
Sung
H.
,
Ferlay
J.
,
Siegel
R.L.
,
Laversanne
M.
,
Soerjomataram
I.
,
Jemal
A.
et al.
(
2021
)
Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries
.
CA Cancer J. Clin.
71
,
209
249
[PubMed]
2.
Wald
O.
,
Smaglo
B.
,
Mok
H.
and
Groth
S.S.
(
2017
)
Future directions in esophageal cancer therapy
.
Ann. Cardiothorac. Surg.
6
,
159
166
[PubMed]
3.
Vitale
I.
,
Manic
G.
,
Coussens
L.M.
,
Kroemer
G.
and
Galluzzi
L.
(
2019
)
Macrophages and metabolism in the tumor microenvironment
.
Cell Metab.
30
,
36
50
[PubMed]
4.
Kaymak
I.
,
Williams
K.S.
,
Cantor
J.R.
and
Jones
R.G.
(
2021
)
Immunometabolic interplay in the tumor microenvironment
.
Cancer Cell
39
,
28
37
[PubMed]
5.
Mantovani
A.
,
Marchesi
F.
,
Malesci
A.
,
Laghi
L.
and
Allavena
P.
(
2017
)
Tumour-associated macrophages as treatment targets in oncology
.
Nat. Rev. Clin. Oncol.
14
,
399
416
[PubMed]
6.
Wang
Y.
,
Lin
Y.X.
,
Qiao
S.L.
,
Wang
J.
and
Wang
H.
(
2019
)
Progress in tumor-associated macrophages: from bench to bedside
.
Adv. Biosyst.
3
,
e1800232
[PubMed]
7.
Thielmann
C.M.
,
Costa
D.S.M.
,
Muley
T.
,
Meister
M.
,
Herpel
E.
and
Muckenthaler
M.U.
(
2019
)
Iron accumulation in tumor-associated macrophages marks an improved overall survival in patients with lung adenocarcinoma
.
Sci. Rep.
9
,
11326
[PubMed]
8.
Song
X.
,
Li
N.
,
Liu
Y.
,
Wang
Z.
,
Wang
T.
,
Tan
S.
et al.
(
2022
)
CD169-positive macrophages enhance abscopal effect of radiofrequency ablation therapy in liver cancer
.
Transl. Oncol.
15
,
101306
[PubMed]
9.
Huang
X.
,
Cao
J.
and
Zu
X.
(
2022
)
Tumor-associated macrophages: An important player in breast cancer progression
.
Thorac. Cancer
13
,
269
276
[PubMed]
10.
Chen
Y.
,
Feng
R.
,
He
B.
,
Wang
J.
,
Xian
N.
,
Huang
G.
et al.
(
2021
)
PD-1H expression associated with CD68 macrophage marker confers an immune-activated microenvironment and favorable overall survival in human esophageal squamous cell carcinoma
.
Front Mol. Biosci.
8
,
777370
[PubMed]
11.
Jiang
C.H.
,
Liang
W.H.
,
Li
F.P.
,
Xie
Y.F.
,
Yuan
X.
,
Zhang
H.J.
et al.
(
2021
)
Distribution and prognostic impact of M1 macrophage on esophageal squamous cell carcinoma
.
Carcinogenesis
42
,
537
545
[PubMed]
12.
Yamamoto
K.
,
Makino
T.
,
Sato
E.
,
Noma
T.
,
Urakawa
S.
,
Takeoka
T.
et al.
(
2020
)
Tumor-infiltrating M2 macrophage in pretreatment biopsy sample predicts response to chemotherapy and survival in esophageal cancer
.
Cancer Sci.
111
,
1103
1112
[PubMed]
13.
Zeng
H.
,
Zheng
R.
,
Zhang
S.
,
Zuo
T.
,
Xia
C.
,
Zou
X.
et al.
(
2016
)
Esophageal cancer statistics in China, 2011: Estimates based on 177 cancer registries
.
Thorac. Cancer
7
,
232
237
[PubMed]
14.
Moher
D.
,
Liberati
A.
,
Tetzlaff
J.
and
Altman
D.G.
(
2009
)
Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement
.
J. Clin. Epidemiol.
62
,
1006
1012
[PubMed]
15.
Altman
D.G.
and
Bland
J.M.
(
2011
)
How to obtain the confidence interval from a P value
.
BMJ
343
,
d2090
[PubMed]
16.
Stang
A.
(
2010
)
Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses
.
Eur. J. Epidemiol.
25
,
603
605
[PubMed]
17.
Koide
N.
,
Nishio
A.
,
Hiraguri
M.
,
Kishimoto
K.
,
Nakamura
T.
,
Adachi
W.
et al.
(
2002
)
Differences and relationships of thymidine phosphorylase expression in tumor-associated macrophages and cancer cells in squamous cell carcinoma of the esophagus
.
Dis. Esophagus
15
,
67
73
[PubMed]
18.
Guo
S.J.
,
Lin
D.M.
,
Li
J.
,
Liu
R.Z.
,
Zhou
C.X.
,
Wang
D.M.
et al.
(
2007
)
Tumor-associated macrophages and CD3-zeta expression of tumor-infiltrating lymphocytes in human esophageal squamous-cell carcinoma
.
Dis. Esophagus
20
,
107
116
[PubMed]
19.
Shigeoka
M.
,
Urakawa
N.
,
Nakamura
T.
,
Nishio
M.
,
Watajima
T.
,
Kuroda
D.
et al.
(
2013
)
Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma
.
Cancer Sci.
104
,
1112
1119
[PubMed]
20.
Sugimura
K.
,
Miyata
H.
,
Tanaka
K.
,
Takahashi
T.
,
Kurokawa
Y.
,
Yamasaki
M.
et al.
(
2015
)
High infiltration of tumor-associated macrophages is associated with a poor response to chemotherapy and poor prognosis of patients undergoing neoadjuvant chemotherapy for esophageal cancer
.
J. Surg. Oncol.
111
,
752
759
[PubMed]
21.
Hatogai
K.
,
Kitano
S.
,
Fujii
S.
,
Kojima
T.
,
Daiko
H.
,
Nomura
S.
et al.
(
2016
)
Comprehensive immunohistochemical analysis of tumor microenvironment immune status in esophageal squamous cell carcinoma
.
Oncotarget
7
,
47252
47264
[PubMed]
22.
Li
J.
,
Zhang
B.Z.
,
Qin
Y.R.
,
Bi
J.
,
Liu
H.B.
,
Li
Y.
et al.
(
2016
)
CD68 and interleukin 13, prospective immune markers for esophageal squamous cell carcinoma prognosis prediction
.
Oncotarget
7
,
15525
15538
[PubMed]
23.
Xu
B.
,
Chen
L.
,
Li
J.
,
Zheng
X.
,
Shi
L.
,
Wu
C.
et al.
(
2016
)
Prognostic value of tumor infiltrating NK cells and macrophages in stage II+III esophageal cancer patients
.
Oncotarget
7
,
74904
74916
[PubMed]
24.
Zhu
Y.
,
Li
M.
,
Bo
C.
,
Liu
X.
,
Zhang
J.
,
Li
Z.
et al.
(
2017
)
Prognostic significance of the lymphocyte-to-monocyte ratio and the tumor-infiltrating lymphocyte to tumor-associated macrophage ratio in patients with stage T3N0M0 esophageal squamous cell carcinoma
.
Cancer Immunol. Immunother.
66
,
343
354
[PubMed]
25.
Hosono
M.
,
Koma
Y.I.
,
Takase
N.
,
Urakawa
N.
,
Higashino
N.
,
Suemune
K.
et al.
(
2017
)
CXCL8 derived from tumor-associated macrophages and esophageal squamous cell carcinomas contributes to tumor progression by promoting migration and invasion of cancer cells
.
Oncotarget
8
,
106071
106088
[PubMed]
26.
Hu
J.M.
,
Liu
K.
,
Liu
J.H.
,
Jiang
X.L.
,
Wang
X.L.
,
Chen
Y.Z.
et al.
(
2017
)
CD163 as a marker of M2 macrophage, contribute to predicte aggressiveness and prognosis of Kazakh esophageal squamous cell carcinoma
.
Oncotarget
8
,
21526
21538
[PubMed]
27.
Wang
X.L.
,
Liu
K.
,
Liu
J.H.
,
Jiang
X.L.
,
Qi
L.W.
,
Xie
Y.F.
et al.
(
2017
)
High infiltration of CD68-tumor associated macrophages, predict poor prognosis in Kazakh esophageal cancer patients
.
Int. J. Clin. Exp. Pathol.
10
,
10282
10292
[PubMed]
28.
Lu
Y.
,
Guo
L.
and
Ding
G.
(
2019
)
PD1(+) tumor associated macrophages predict poor prognosis of locally advanced esophageal squamous cell carcinoma
.
Future Oncol.
15
,
4019
4030
[PubMed]
29.
Napier
K.J.
,
Scheerer
M.
and
Misra
S.
(
2014
)
Esophageal cancer: a review of epidemiology, pathogenesis, staging workup and treatment modalities
.
World J. Gastrointest Oncol.
6
,
112
120
[PubMed]
30.
Komohara
Y.
,
Fujiwara
Y.
,
Ohnishi
K.
and
Takeya
M.
(
2016
)
Tumor-associated macrophages: potential therapeutic targets for anti-cancer therapy
.
Adv. Drug. Deliv. Rev.
99
,
180
185
[PubMed]
31.
Binnemars-Postma
K.
,
Storm
G.
and
Prakash
J.
(
2017
)
Nanomedicine Strategies to target tumor-associated macrophages
.
Int. J. Mol. Sci.
18
,
1
27
[PubMed]
32.
Zheng
X.
,
Turkowski
K.
,
Mora
J.
,
Brune
B.
,
Seeger
W.
,
Weigert
A.
et al.
(
2017
)
Redirecting tumor-associated macrophages to become tumoricidal effectors as a novel strategy for cancer therapy
.
Oncotarget
8
,
48436
48452
[PubMed]
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