Wang JL, Niu YX, Li HX, Cui FC, Chen X. Clinical significance of mesenchymal stem cells exosomal microRNAs in predicting prognosis and inflammatory response of esophageal cancer. World J Stem Cells 2026; 18(7): 119714 [DOI: 10.4252/wjsc.119714]
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Wang JL, Niu YX, Li HX, Cui FC, Chen X. Clinical significance of mesenchymal stem cells exosomal microRNAs in predicting prognosis and inflammatory response of esophageal cancer. World J Stem Cells 2026; 18(7): 119714 [DOI: 10.4252/wjsc.119714]
Author contributions: Wang JL conducted the majority of experiments and wrote the manuscript; Niu YX designed the study and served as a scientific advisor and guarantor; Li HX corrected the manuscript; Cui FC was involved in applying the analytical tools; Chen X participated in the collection of human material.
AI contribution statement: The authors confirm that no generative AI or AI-assisted technologies were used in the preparation of this manuscript.
Institutional review board statement: This study was approved by Henan Provincial People’s Hospital (Approval No. 2022-08), and the study followed the ethical guidelines of the Declaration of Helsinki.
Informed consent statement: Informed consent was obtained from all study participants.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Received: February 27, 2026 Revised: March 19, 2026 Accepted: May 19, 2026 Published online: July 26, 2026 Processing time: 147 Days and 2.1 Hours
Abstract
BACKGROUND
Esophageal squamous cell carcinoma (ESCC) is the predominant histological type of esophageal cancer, accounting for over 90% of cases in Asia. Although the traditional tumor-node-metastases staging can to some extent assess prognosis, the biological heterogeneity leads to significant differences in survival among patients with the same stage.
AIM
To elucidate the relationship between two mesenchymal stem cells (MSCs) exosomal microRNAs (miRNAs) and ESCC.
METHODS
A total of 312 patients who underwent radical resection for ESCC were recruited. Preoperative blood samples were collected, and stem cells were isolated using the automatic blood cell separator. MSCs were further sorted by flow cytometry, and exosome was isolated using kit. The morphologies and particle sizes of exosomes were evaluated. The relative expression of miR-375/miR-655 in peripheral blood MSCs (PBMSCs) exosomes were assessed by quantitative real-time polymerase chain reaction. Associations with clinicopathological variables were assessed using the χ2 test or Fisher’s exact test, and Spearman’s rank correlation was employed to analyze the relationship between miR-375/miR-655 and inflammatory factors. Kaplan-Meier curves and log-rank tests assessed overall survival, and Cox models performed univariate/multivariate analyses on overall survival impact. Receiver operator characteristic curves evaluated the predictive performance of miR-375.
RESULTS
miR-375 and miR-655 showed elevated expression in PBMSC-derived exosomes from patients, in contrast to PBMSCs, and were associated with a lower incidence of poorly differentiated tumors, advanced tumor-node-metastases stages, and lymph node metastasis. The miR-375/miR-655 low expression group exhibited significantly elevated levels of interleukin (IL)-6, IL-1β, CC-chemokine ligand 2 (CCL2), and CCL5 compared to the high expression group. Spearman correlation analysis indicated a negative correlation between miR-375/miR-655 and IL-6, IL-1β, CCL2, and CCL5. Patients exhibiting high miR-375/miR-655 expression alongside low IL-6/IL-1β/CCL2/CCL5 expression demonstrated significantly longer overall survival compared to those with the opposite expression profiles. Multivariate Cox analysis determined miR-375 as an independent predictor of overall survival with notable accuracy.
CONCLUSION
Overexpression of miR-375 and miR-655 in PBMSCs exosomes derived from patients is associated with inflammatory factors and unfavorable prognosis in ESCC. Their combined evaluation provides improved prognostic stratification and may serve as a valuable adjunct for individualized clinical management.
Core Tip: Overexpression of miR-375 and miR-655 in peripheral blood mesenchymal stem cells exosomes derived from patients is associated with inflammatory factors and unfavorable prognosis in esophageal squamous cell carcinoma. Their combined evaluation provides improved prognostic stratification and may serve as a valuable adjunct for individualized clinical management. The findings aim to provide new evidence for the biological and clinical relevance of these biomarkers and offer a reference for personalized prognosis assessment and clinical decision-making in esophageal squamous cell carcinoma management.
Citation: Wang JL, Niu YX, Li HX, Cui FC, Chen X. Clinical significance of mesenchymal stem cells exosomal microRNAs in predicting prognosis and inflammatory response of esophageal cancer. World J Stem Cells 2026; 18(7): 119714
Esophageal cancer is recognized as one of the primary contributors to cancer-related mortality on a global scale, standing as the sixth leading cause of cancer fatalities. This is attributed to its aggressive nature and unfavorable prognosis[1]. GLOBOCAN 2020 data indicates around 600000 new cases[2]. Esophageal squamous cell carcinoma (ESCC) is the predominant pathological type, accounting for more than 90% of cases in Asian countries, particularly China[3]. Although multimodal treatments like surgery, radio-chemotherapy, and immunotherapy have improved local disease management, the prognosis for ESCC remains poor. The five-year survival rate for these patients rarely exceeds 30%, and the three-year survival rate for those in advanced disease stages often drops below 50%[4,5]. Consequently, discovering novel molecular markers that can refine risk assessment, optimize treatment planning, and predict prognosis is of great clinical importance.
Mesenchymal stem cells (MSCs) have been identified in various adult and fetal/perinatal tissues, including peripheral blood[6,7]. MSCs exhibit plastic adherence when cultured in growth media. MSCs require specific cell surface antigen expression. These cells should be capable of differentiating into diverse mesodermal or non-mesodermal cell types under specific culture conditions[8]. MSCs possess a homing ability that allows them to migrate to sites of inflammation and contribute to tissue repair[9]. This tropism toward inflamed and malignant tissues enables MSCs to be recruited into the tumor microenvironment, where they interact closely with cancer cells and are influenced by tumor-derived signals. Consequently, the exosomal cargo-including microRNAs (miRNAs)-packaged by these tumor-associated MSCs may reflect the pathological status of the microenvironment and, in turn, modulate tumor progression through paracrine signaling[10]. Furthermore, MSCs play multiple biological roles, including multilineage differentiation, immunosuppression, and tissue repair development[11]. Due to their advantages, MSCs are widely utilized in clinical research[12].
Paracrine actions of MSCs are considered a significant therapeutic approach. Tumor necrosis factor-alpha-stimulated gene/protein-6 from MSCs can enhance liver fibrosis recovery by regulating M2 macrophages and upregulating matrix metalloproteinase 12 expression[13]. Recent studies have highlighted that, beyond soluble factors, extracellular vesicles, particularly exosomes, are crucial paracrine elements of MSCs[14]. Exosomes are membrane particles sized 40 nm to 150 nm, essential for intercellular and inter-organ communication[15]. Exosomes carry bioactive molecules such as proteins, mRNAs, and miRNA, facilitating functional delivery between different cell types and even across species[16]. A recent study demonstrated that exosomes derived from MSCs, enriched with miRNA-182-5p and miR-23a-3p, ameliorated lipopolysaccharide-induced acute lung injury by inhibiting epithelial-to-mesenchymal transition[17]. miR-221 expression is often elevated in exosomes from GC-MSCs and certain cancer tissues, correlating significantly with lymph node metastasis, venous invasion, and tumor-node-metastases (TNM) stage[18]. Exosomal miRNAs play a crucial regulatory role in cancer progression, drug resistance, metastasis, and immune response[19].
Extensive research has focused on cytokines and chemokines such as interleukin (IL)-6-IL-1β-CC-chemokine ligand 2 (CCL2)-CCL5 in the study of ESCC. These inflammatory mediators are crucial for disease onset and progression and are strongly linked to patient prognosis. Evidence suggests that IL-6 promotes cancer cell proliferation and invasiveness by activating the signal transducer and activator of transcription-3 signaling pathway[20]. Furthermore, serum IL-6 levels are significantly linked to overall survival and disease recurrence in ESCC patients[21]. IL-6 promotes tumor progression by enhancing the recruitment of tumor-associated macrophages via the upregulation of CCL2 and CCL5 expression[22]. IL-1β plays a crucial role in ESCC. Research indicates that IL-1β enhances ESCC cell proliferation, migration, and invasion through phosphatidylinositol 3-kinase-protein kinase B pathway activation[23]. CCL2 and CCL5 are crucial in modulating the tumor microenvironment. CCL2 promotes function of ESCC cells through the activation of the Toll-like receptor 4 signaling pathway[24]. Additionally, CCL2 forms an immunosuppressive microenvironment by interacting with CCR2, promoting tumor immune escape[25]. CCL5 facilitates the recruitment of cancer-associated fibroblasts through its interaction with CCR5, consequently augmenting the proliferation and invasive capabilities of tumor cells[26]. Studies have shown that high CCL5 expression is associated with poor prognosis in ESCC-patients[27].
Recent studies have proposed that MSCs exosomal miR-375 and miR-655 were involved in regulating the malignant progression of ESCC. MSCs exosomal miR-375 was proved to retard the progression of ESCC via downregulating enabled homolog[28]. Similarly, it has been discovered that exosomal miR-655 from MSCs can inhibit the development of ESCC[29]. However, systematic studies examining the link between these two miRNAs and inflammatory factors in ESCC are limited and yield inconsistent results. This retrospective study analyzed 312 surgically treated ESCC cases to evaluate miR-375 and miR-655 expression, investigate their associations with clinicopathological features and survival outcomes, and determine their prognostic significance. The findings aim to provide new evidence for the biological and clinical relevance of these biomarkers and offer a reference for personalized prognosis assessment and clinical decision-making in ESCC management.
MATERIALS AND METHODS
Ethical statement
The Medical Ethics Committee of Henan Provincial People’s Hospital approved the study protocol (Approval No. 2022-08). All procedures adhered to the Declaration of Helsinki principles. To ensure patient confidentiality, all data were anonymized and de-identified before analysis.
Study subjects
This retrospective study was conducted 312 patients diagnosed with ESCC between (January 2019 to December 2021) were enrolled. Preoperative blood samples were collected obtained 12 hours prior from all patients at initial diagnosis for ESCC. All cases were histopathologically confirmed as ESCC through preoperative biopsy and postoperative pathological examination. All patients underwent surgery without prior administration of neoadjuvant chemotherapy, radiotherapy, or immunotherapy. Patients presenting with distant metastases at diagnosis, other synchronous malignancies, or incomplete clinical data were excluded.
Inclusion criteria included: (1) Individuals aged 18 years to 80 years, regardless of sex; (2) Histologically confirmed ESCC diagnosis; (3) Completion of curative (R0) resection; (4) Availability of comprehensive clinicopathological and follow-up data; and (5) Well-preserved blood samples appropriate for experimental analysis[30].
Exclusion criteria include: (1) A history of other malignancies; (2) Prior neoadjuvant therapy; (3) Perioperative death from non-cancer causes; (4) Incomplete clinical or survival data; and (5) Insufficient blood quality for experimental analysis.
Clinical data collection and follow-up
Data pertaining to patients were retrieved from the institution. Factors considered included age, sex, body mass index, smoking and alcohol history, familial cancer history, tumor characteristics (location, size, histological grading), TNM staging (8th edition American Joint Committee on Cancer/Union for International Cancer Control), lymph node metastasis, preoperative carcinoembryonic antigen and carbohydrate antigen 19-9 levels (Roche Diagnostics, Switzerland), and inflammatory markers (IL-6, IL-1β, CCL2, CCL5) (Thermo Fisher Scientific, MA, United States). Additionally, the dataset comprised information on follow-up duration and survival outcomes. Overall survival refers to the duration from initial diagnosis to either disease-related death or the last follow-up.
Isolation and culture of peripheral blood MSCs
Stem cells were isolated from peripheral blood using the Rvy kit through an automatic blood cell separator (FRESENIUS KABI, Baden-Hoewerk, Germany). MSCs were further sorted by flow cytometry after cells were labeled with CD73 and CD105 (Abcam, Cambridge, United Kingdom). Cells were cultured in DMEM medium.
Exosome isolation
Exosome isolation was conducted following the QIAGEN exosome isolation kit instructions (Duesseldorf, Germany). Initially, peripheral blood MSCs (PBMSCs) underwent centrifugation to eliminate large cell fragments. The supernatants were then subjected to a 12000 × g centrifugation for 45 minutes at 4 °C to remove smaller debris. Finally, the supernatants were ultracentrifuged at 120000 × g for 120 minutes at 4 °C to pellet small vesicles. The pellets were resuspended in phosphate buffered saline (PBS, Thermo Fisher Scientific, MA, United States) and filtered through a 0.22 μm filter to eliminate impurities. The pellets obtained were resuspended in PBS after a second ultracentrifugation cycle at 120000 × g and approximately 4 °C, as previously described. Finally, a BCA test kit (MeilunBio, Dalian, Liaoning Province, China) was used to measure the protein quantities found in these separated exosomes.
Transmission electron microscope
Exosomes were placed on a formvar-coated copper grid for ten minutes following separation. The exosomes were then embedded in a solution containing 0.4% uranyl acetate (Sigma-Aldrich, MA, United States) and 0.13% methylcellulose (Sigma-Aldrich, MA, United States). The grid was analyzed using an 80V electron microscope (HITACHI, Tokyo, Japan).
Nanoparticle tracking analysis
The size distribution of the exosomes was measured using a Nanoparticle Tracking Analyzer. Exosome samples, ranging from 50 to 400, were diluted with 1 × PBS to achieve the desired concentration. The size of the exosomes was measured in accordance with the instrument parameters.
Total RNA was extracted from exosomes or PBMSCs using TRIzol reagent (Thermo Fisher Scientific, MA, United States). A HiCapacity RT kit (Thermo Fisher Scientific, MA, United States) was used to reverse transcribe 0.5 μg of total RNA into cDNA. The SYBR Green Master Mix (Takara, Kyoto, Japan) was used for quantitative real-time polymerase chain reaction analysis. RNA expression levels were quantified using the 2-ΔΔCt method and normalized against U6[31,32]. The primer sequences were: miR-375 (forward: 5’-CGGGTTTGTTCGTTCGGCT-3’, reverse: 5’-GTGCAGGGTCCGAGGTATT-3’), miR-655 (forward: 5’-AATAGTGCCTAAAGTGCTGC-3’, reverse: 5’-GTGCAGGGTCCGAGGTAT-3’).
Statistical analysis
Quantitative data are expressed as mean ± SD and analyzed using the independent-sample t-test. Categorical data are presented as n (%) and analyzed using the χ2 test or Fisher’s exact test, depending on suitability. Spearman’s rank correlation was employed to analyze the relationship between miR-375/miR-655 and inflammatory factors. Kaplan-Meier survival curves were created. The Cox proportional hazards model was employed for univariate and multivariate analyses. Receiver operator characteristic curves were generated to assess the predictive ability of miR-375, miR-655, and their combination for poor prognosis, with AUC, sensitivity, and specificity calculated. A P-value below 0.05 in a two-tailed test was considered statistically significant.
RESULTS
The high expression of miR-375 and miR-655 in PBMSCs exosomes of ESCC patients
High-throughput sequencing showed that miR-375-3p was down-regulated in exosomes isolated from esophageal epithelial cells and ESCC cell lines[33]. Functional studies have shown that miR-655/miR-375 derived from extracellular vesicles or exosomes of human umbilical cord MSCs can inhibit the progression of ESCC[28,29]. However, these findings are derived from tumor cell-derived exosomes or exogenous MSC models. It is unclear whether miR-375/miR-655 is endogenously expressed or dysregulated in MSCs of ESCC patients. Based on this, we selected miR-375 and miR-655 as candidate miRNAs for verification. We extracted PBMSCs exosomes from patients to verify the expression of these two miRNAs. Transmission electron microscope and nanoparticle tracking analysis characterization results demonstrated the morphologies and particle sizes of exosomes derived from patients (Figure 1A and B). Quantitative real-time polymerase chain reaction analysis revealed significantly elevated expression levels of miR-375 and miR-655 in exosomes derived from patient PBMSCs compared to the PBMSCs themselves (Figure 1C and D, P < 0.001).
Figure 1 The expression levels of miR-375 and miR-655 in peripheral blood mesenchymal stem cells exosomes and peripheral blood mesenchymal stem cells from esophageal squamous cell carcinoma patients.
A: Transmission electron microscopy characterization of exosomes isolated from peripheral blood mesenchymal stem cells (PBMSCs) of esophageal squamous cell carcinoma (ESCC) patients (scale bar: Left, 200 nm; right, 100 nm); B: Nanoparticle tracking analysis characterization of exosomes isolated from PBMSCs of ESCC patients; C and D: Quantitative real-time polymerase chain reaction was used to measure the miR-375 (C), and miR-655 (D) levels in PBMSCs exosomes and PBMSCs of ESCC patients. cP < 0.001. PBMSCs: Peripheral blood mesenchymal stem cells.
Comparison of clinical data of ESCC patients with different miR-375 and miR-655
Patients exhibiting low miR-375 expression demonstrated significantly higher rates of poorly differentiated tumors, advanced TNM stages, and lymph node metastasis compared to those with high miR-375 expression (all P < 0.05, Table 1). No statistically significant differences were observed in age, body mass index, tumor location, or serum markers (carcinoembryonic antigen and carbohydrate antigen 19-9). Patients exhibiting low miR-655 expression demonstrated significantly higher rates of poor differentiation, advanced stage-disease, and lymph-node-metastasis compared to those with high miR-655 expression (all P < 0.05, Table 1).
Table 1 Comparison of clinical data of esophageal squamous cell carcinoma patients with different miR-375 and miR-655 expression levels, mean ± SD/n (%).
Correlation analysis of miR-375 and miR-655 expression with inflammatory factors
In the group with low miR-375 expression, IL-6, IL-1β, CCL2, and CCL5 levels were significantly elevated compared to the high expression group (all P < 0.001, Figure 2A). A significant negative correlation between miR-375 and IL-6 (r: -0.581,P < 0.001), IL-1β (r: -0.413, P < 0.001), CCL2 (r: -0.505, P < 0.001), and CCL5 (r: -0.507, P < 0.001) (Figure 2B). The low miR-655 expression group exhibited significantly elevated levels of IL-6, IL-1β, CCL2, and CCL5 compared to the high-expression group (all P < 0.001, Figure 3A). miR-655 was negatively correlated with IL-6 (r: -0.568,P < 0.001), IL-1β (r: -0.448,P < 0.001), CCL2 (r: -0.511, P < 0.001), and CCL5 (r: -0.518, P < 0.001) (Figure 3B).
Figure 4 Kaplan-Meier analysis of overall survival according to two microRNAs and inflammatory factors.
A and B: Kaplan-Meier curves depict overall survival probabilities for patients stratified by miR-375 (A), and miR-655 (B); C-F: Kaplan-Meier curves depict overall survival probabilities for interleukin-6 levels using a cutoff of 21.99 (C), interleukin-1β levels using a cutoff of 7.99 (D), CC-chemokine ligand 2 using a cutoff of 245.52 (E), CC-chemokine ligand 5 using a cutoff of 50.42 (F). IL: Interleukin; CCL: CC-chemokine ligand.
Cox regression analysis
Cox regression analyses were conducted. Figure 5A presents a univariate analysis identifying several factors significantly linked to overall survival. miR-375 [hazard ratio (HR) = 0.399, P < 0.001], miR-655 (HR = 0.737, P = 0.01), IL-6 (HR = 1.029, P < 0.001), IL-1β (HR = 1.059, P = 0.005), and CCL5 (HR = 1.015, P = 0.01) were identified as risk predictive factors. In the multivariate analysis adjusting for key covariates, miR-375 remained as independent predictors of overall survival (HR = 0.447, P = 0.001, Figure 5B).
Figure 5 Univariate and multivariate Cox regression analysis for overall survival.
A: Forest plot of univariate Cox regression analysis for overall survival; B: Forest plot of multivariate Cox regression analysis for overall survival. TNM: Tumor node metastasis; CI: Confidence interval; IL: Interleukin; CCL: CC-chemokine ligand.
Cox regression model
Cox regression demonstrated that miR-375 could serve as an independent predictor for overall survival. To further assess its predictive efficacy. We constructed a nomogram for this regression model (Figure 6A). The model’s predictive efficacy was assessed using receiver operator characteristic analysis (Figure 6B, Table 2). The findings indicate that the model accurately predicts patient overall survival at various time points.
Figure 6 Multivariate Cox regression model for overall survival.
A: Nomogram of multivariate Cox regression analysis for progression-free survival; B: Receiver operating characteristic analysis of multivariate Cox regression analysis for progression-free survival with different time. AUC: Area under the curve.
Table 2 Predictive efficiency of multivariate Cox regression model for progression-free survival among different time.
This study found a notable rise in miR-375 and miR-655 expression levels in PBMSCs exosomes influenced by ESCC compared to PBMSCs. This suggests a strong association between these molecules and the processes of malignant transformation as well as the functional significance of these miRNAs in ESCC pathogenesis.
In our study examining the expression levels of PBMSCs exosomal miR-375-miR-655 in ESCC, we found a significant association between their decreased expression and aggressive tumor features, including advanced TNM staging. In previous studies, miR-375 has generally been regarded as a factor negatively associated with the malignant progression of ESCC. Wu et al[34] reported that high levels of miR-375 were associated with a decrease in the degree of ESCC metastasis. Serum miR-375 levels are significantly lower in ESCC patients compared to healthy individuals[35]. The expression of miR-655 was significantly reduced compared to adjacent cancer tissues, similar to miR-375[36]. High levels of miR-655 are positively correlated with the inhibitory state of epithelial-to-mesenchymal transition in ESCC patients[37]. The study found that ESCC patients with low PBMSCs exosomal miR-375 and miR-655 expression exhibited significantly higher rates of poorly differentiated tumors compared to those with high expression levels. Previous reports and our results have confirmed that PBMSCs exosomal miR-375 and miR-655 are negatively correlated with the progression of ESCC.
Additionally, our findings revealed a notable negative association between elevated expression levels of these miRNAs and increased concentrations of inflammatory cytokines. This observation aligns with previous research that emphasizes the influence of inflammatory mediators on the regulation of miR-375 and miR-655 expression. Research indicates that miR-375 can reduce inflammatory factor levels in gastric carcinogenesis induced by Helicobacter pylori[38]. In conditions like osteoarthritis, elevated miR-655 levels can suppress inflammatory factors, including IL-17[39]. The interplay between inflammation and PBMSCs exosomal miRNAs suggests that the inflammatory milieu not only facilitates tumor progression but may also provide therapeutic targets, as targeting these inflammatory pathways could attenuate the invasive characteristics of ESCC cells.
In addition to their clinical significance and association with inflammatory factors, we also evaluated the prognostic value of these two miRNAs for patients with ESCC. During follow-up period, patients exhibiting low expression of either PBMSCs exosomal miR-375 or miR-655 experienced significantly shorter overall survival. Kaplan-Meier analysis revealed significantly longer median survival times for high-expression groups compared to low-expression groups, as indicated by the Log-rank. These data indicate that PBMSCs exosomal miR-375 and miR-655 are not merely markers of tumor aggressiveness but also independent predictors of prognosis. Prior evidence from ESCC similarly revealed that lower levels of miR-375 and miR-655 were linked to unfavorable survival, and our findings extend this pattern to ESCC[40,41]. This study demonstrates that miR-375 independently predicts prognosis in ESCC, as confirmed by univariate and multivariate Cox regression analyses.
From a translational standpoint, the simultaneous assessment of PBMSCs exosomal miR-375 and miR-655 may serve as a valuable adjunct to conventional postoperative risk stratification in ESCC. In particular, for intermediate-risk patients whose prognosis cannot be accurately defined by imaging or histopathological criteria alone, integrating molecular markers could refine outcome predictions. Furthermore, combining these biomarkers with TNM stage, radiologic, and genomic parameters could contribute to the construction of comprehensive nomograms or prognostic models to guide individualized therapy and adjuvant treatment decisions.
It is worth noting that the PBMSCs analyzed in this study may represent a heterogeneous cell population. MSCs derived from peripheral blood are relatively rare under physiological conditions. It is generally believed that there are multiple sources, including bone marrow MSCs mobilized to peripheral blood and tissue resident progenitor cell niches[42,43]. In addition, there is growing evidence that bone marrow MSCs can be mobilized or recruited into the circulation to respond to inflammatory stimuli or tissue damage. Previous studies have shown that circulating MSC-like cells can be recruited or mobilized to respond to tumor-related inflammation, tissue damage, or system signaling prompts[42,43]. In malignant tumors, including ESCC, the tumor microenvironment may further affect the phenotype and functional characteristics of circulating MSCs, which may lead to changes in exosomes such as miRNAs. In addition, these circulating MSC may represent an activated or specific subset different from tissue-resident MSC, and their phenotype and function may be regulated by the circulating environment or tumor microenvironment, thus reflecting the difference from tissue-derived MSC[44]. However, due to the lack of specific lineage tracking markers, the exact source and biological characteristics of PBMSCs are not fully understood, and their heterogeneity should be considered when explaining the existing research results. Therefore, although our results show that PBMSCs-derived exosomes miR-375 and miR-655 are associated with tumor progression and inflammatory status, it is necessary to further study the source, subgroup composition, functional heterogeneity of PBMSCs in ESCC patients and their differences with tissue-resident MSC.
In addition to the association between PBMSC-derived exosomes miR-375 and miR-655 and tumor progression and inflammation, an important question is whether the exosome miRNA profile represents the stable intrinsic characteristics of MSCs or is dynamically regulated by the tumor microenvironment. New evidence shows that MSC-derived exosomal miRNAs exhibit great plasticity in response to inflammatory cytokines, hypoxia, or tumor-derived factors[45,46]. For example, treatment of MSCs with synthetic peptide analogs induced ≥ 2-fold regulation of 182 miRNAs in extracellular vesicles, including a 10-fold reduction in tumor-associated miR-1246, indicating that external stimuli can quantitatively reprogram extracellular vesicle miRNA content[45]. It is suggested that the miRNA expression profile of PBMSCs in patients with ESCC not only reflects the composition characteristics of the tumor, but also may be affected by the continuous interference of the tumor microenvironment. Studies have shown that MSCs exposed to different inflammatory environments obtain different immunomodulatory phenotypes, and their exosome miRNAs mediate context-dependent effects that promote or inhibit tumor progression[46,47]. More and more attention has been paid to the dual role of MSC-derived exosomes in cancer, and their functional results may be determined by specific miRNA signals obtained by microenvironment signals[48]. In our study, the negative correlation between PBMSCs exosomal miR-375/miR-655 and inflammatory cytokine levels was consistent with this dynamic regulation model, increasing the possibility that the inflammatory tumor environment may help regulate miRNA packaging to PBMSC-derived exosomes. In the future, further study of the static and dynamic regulation of MSC exosome miRNAs in tumors is crucial for understanding their functional role as biomarkers and translation utility.
In addition, from a more macroscopic point of view, the interaction between MSCs and tumor cells is bidirectional[49]. This two-way interaction not only includes the effects of MSCs and their exosomes on the biological behavior of tumor cells, but also covers the reverse regulation of tumor microenvironment on the phenotype, function and secretory activity of MSCs[49,50]. MSCs can be recruited to the tumor site and reprogrammed under the action of tumor-derived signals (such as inflammatory factors, hypoxic conditions, or tumor-derived exosomes) to obtain a tumor-associated fibroblast phenotype, which in turn promotes tumor progression, angiogenesis, immune escape, and therapeutic resistance by secreting specific factors or exosome miRNAs[49,51,52]. However, MSCs and their exosomes may also exert tumor suppression by delivering tumor suppressor miRNAs (such as miR-375 and miR-655 found in this study) or regulating immune cell function[46]. In this study, we observed that down-regulation of miR-375 and miR-655 in PBMSCs exosomes of ESCC patients was associated with tumor progression and poor prognosis, which may be the result of reprogramming of PBMSCs function by tumor microenvironment; at the same time, the expression of these miRNAs is negatively correlated with the level of inflammatory factors, suggesting that PBMSCs may participate in the regulation of tumor-related inflammatory responses through their exosome miRNAs, thereby reversely affecting the tumor microenvironment. Therefore, the interaction between PBMSCs and ESCC cells may form a dynamic feedback loop: The tumor microenvironment shapes the miRNA expression profile of PBMSCs, and PBMSCs exosome miRNAs in turn regulate the malignant behavior of tumor cells[49]. Future research should further explore the specific molecular mechanism of this two-way interaction and how to use or intervene in this interaction process to achieve more effective treatment strategies.
This research is subject to certain limitations. First, the retrospective design could inherently introduce selection bias. Second, although the sample size was expanded to 312 cases, subgroup analyses - such as stratification by differentiation grade or adjuvant therapy - were not conducted. Third, the present research was confined to immunohistochemical characterization and did not delve into molecular signaling pathways underlying the miR-375/miR-655 axis. Future work incorporating western blotting, quantitative real-time polymerase chain reaction, and functional assays in cellular and animal models is warranted to elucidate their mechanistic interplay. Future studies should implement stricter controls for confounding variables such as comorbidities and lifestyle factors. Fourth, whether the changes in the expression of exosomal miR-375 and miR-655 observed in this study are specific to PBMSCs or reflect the systemic MSC response that can be detected in other MSC populations, we cannot confirm. In the future, we can compare the miRNA profiles of exosomes from multiple MSC sources (such as paired bone marrow-derived MSCs, adipose-derived MSCs and PBMSCs) from the same ESCC patient; different MSC populations were exposed to ESCC-derived conditioned medium to explore whether miRNA changes are unique to PBMSC.
CONCLUSION
This study concludes that miR-375 and miR-655 are significantly expressed in PBMSCs exosomes of ESCC and are strongly associated with its clinicopathological progression and poor prognosis. Importantly, exosomal miR-375 and miR-655 from PBMSCs showed negative correlations with inflammatory markers (IL-6, IL-1β, CCL2, and CCL5), highlighting their significant role in indicating the inflammatory burden of ESCC. Low levels of PBMSCs exosomal miR-375 and miR-655 showed significantly decreased overall survival, with miR-375 identified as an independent prognostic indicator in ESCC. In conclusion, this research substantiates the prognostic relevance and inflammatory response association of PBMSCs exosomal miR-375 and miR-655 in ESCC. The detection of these biomarkers not only enhances prognostic precision but also provides a theoretical and practical foundation for integrating molecular indicators into postoperative management and personalized treatment strategies.
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