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Abstract

Background

CD137L plays a substantial role in immune regulation and has been associated with tumor progression. However, its expression pattern and clinical significance in colorectal cancer remain unclear. The current study was planned to evaluate the expression levels of CD137L in colorectal cancer tissues and investigate its association with clinicopathological characteristics and patient survival.

Methodology

36 tissue samples were collected from colorectal cancer patients followed by RNA extracted. Following the cDNA synthesis, qRT-PCR was conducted to evaluate the CD137L expression, normalized against GAPDH and the comparative expression was determined using the ΔΔCt method. Chi-square test was applied to evaluate the relation of CD137L expression with clinical parameters. Meanwhile, the TCGA database was explored to find the relationship between the prognosis of colorectal cancer patients and different levels of CD137L expression and to analyze the distribution characteristics of differentially expressed genes.

Results

CD137L expression was significantly correlated with patient survival (P < 0.05), with higher expression observed in patients who were alive at the time of analysis. Clinical parameters such as age and gender were insignificantly associated (P > 0.05) with CD137L expression. However, a significant correlation (P = 0.03) was noted between CD137L expression and tumor staging, suggesting its potential involvement in CRC progression. Furthermore, Analysis of TCGA data showed that patients with elevated CD137L expression exhibited improved overall survival compared to those with lower expression levels. Enrichment analysis revealed that CD137 was primarily enriched with immune cell proliferation, T cell activation and Th1/Th2 balance-related signaling pathways.

Conclusion

CD137L may serve as a reliable indicator for forecasting the outcome of colorectal cancer patients, providing guidance for colorectal cancer prognosis.

Keywords

CD137L colorectal cancer cancer prognosis survival analysis

Introduction

Colorectal Cancer (CRC) is among the most prevalent malignant tumors globally, with high prevalence and mortality rates. Though, traditional treatments such as surgical resection and chemotherapy have increased the survival rate in patients to a certain extent, a considerable number of patients still have poor prognosis due to tumor recurrence and metastasis. Therefore, identifying the new biomarkers and therapeutic targets is of significant importance for enhancing the preclinical diagnosis and treatment efficacy of CRC.^1,2

CD137L (4-1BBL) is a co-stimulatory molecule of the tumor necrosis factor superfamily (TNFSF), primarily localized on the surface of antigen-presenting cells (APCs) and certain tumor cells. The interaction between CD137L and its receptor CD137 plays a vital role in immune responses. CD137 is a co-stimulatory receptor present on activated T cells and natural killer (NK) cells. By binding with CD137L, it can enhance the proliferation, survival and effector functions of T cells, thereby promoting immune responses. CD137L play a critical role in tumor immunity. By activating CD137, CD137L enhances the oncolytic activity of T cells, supports the proliferation and survival of tumor-specific T-cells, and reduce the function of immunosuppressive cells including regulatory T cells (Tregs). CD137L also enhances the activity of NK cells, strengthening their killing ability against tumor cells. These effects together significantly improve the body's immune response to tumors, thereby inhibiting tumor growth and spread.^3–5 Several studies have shown that by activating the CD137L signaling pathway, the effectiveness of immunotherapy for CRC can be significantly enhanced. This effect is especially pronounced when combined with other immune-related checkpoint inhibitors, such as PD-1/PD-L1, further strengthens anti-tumor immune response. These results offer new strategies for CRC immunotherapy.^6,7

while CD137L has been confirmed as a therapeutic target for CRC in many studies, its prognostic value remains unclear. Therefore, current study was planned to investigate the potential of CD137L as a prognostic indicator for CRC. Doing so, we analyzed CD137L expression associated with the prognosis of CRC using the TCGA database. Meanwhile, clinical tumor specimens were isolated from CRC patients to study the relationship between CD137L expression and clinical factors, providing insights into its potential clinical application.

Materials and methods

Collection of samples and clinical data

A total of 36 tissue samples were obtained from CRC patients undergoing surgical resection at Beijing Shijitan Hospital, Beijing during June 2022 to June 2024. Tumor staging was pre-classified by expert pathologists following the pTNM staging system guidelines of the American Joint Committee on Cancer (AJCC). Patient clinical data including age, gender and tumor stage were retrieved from medical records. Furthermore, patient overall survival rates were assessed through follow-up.

RNA extraction and quantitative reverse transcription PCR PCR analysis

To analyze CD137L expression, RNA was isolated from tumor tissues using Qiagen RNeasy FFPE Kit following the guidelines provided by the kit manufacturer. The concentration and purity of RNA was assessed via a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). After that, the cDNA was synthesized from 1 µg of extracted RNA through reverse transcription using the PrimeScript RT Reagent Kit (TaKaRa Biotechnology Dalian, China). The obtained cDNA was aliquoted and stored at −80⁰C until further use. CD137L mRNA expression was noticed using a Real-Time PCR System (Bio-Rad) in conjunction with the FastStart Universal SYBR Green Master Kit (Roche Diagnostics, Mannheim, Germany). Specimens were tested in triplicate and the relative expression of CD137L was measured after normalization with the internal Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene. The primers used for CD137L were: Fwd primer, 5′- GAGCTTTCGCCCGACGAT-3′; Rev primer, 5′-CCTCTTTGTAGCTCAGGCCC-3′, And those for GAPDH were: Fwd primer, 5′-ATCCTGGGCTACACTGAGCACC-3′; Rev primer, 5′-AAGTGGTCGTTGAGGGCAATGC-3′.⁸

TCGA data acquisition and bioinformatics analysis

Gene expression and clinical data were retrieved from The Cancer Genome Atlas (TCGA) through the UCSC Xena browser (https://xenabrowser.net/), specifically using the codes such as TCGA-COAD (Colon Adenocarcinoma) and TCGA-READ (Rectum Adenocarcinoma) datasets. A total of 620 colorectal cancer patient samples with available RNA-seq expression data (HTSeq-FPKM format) and complete overall survival (OS) information were included in the analysis. Participants were stratified into high and low CD137L expression groups on the basis of median expression value. Differential expression analysis was performed with a log₂|fold change| cutoff of 1 and a q-value threshold of 0.01. Kaplan–Meier survival curves were constructed to evaluate the relationship between CD137L expression and overall survival.

Protein-protein interaction (PPI) network data were obtained from STRING database v11.5 using a confidence score threshold of >0.4 (top 5%). Cytoscape software (version 3.9.0) was used to compute the Maximal Clique Centrality (MCC) values, and the top 10 genes having highest MCC scores were selected as hub genes. These hub genes were then analyzed to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the WebGestalt platform, with a false discovery rate (FDR) < 0.05 considered statistically significant.

Statistical analysis

Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 6.0 software. The relationship among the expression level of CD137L and pathological features of CRC was evaluated using the χ2 test. For survival analysis, Kaplan–Meier survival curves were generated to compare overall survival between patients with high versus low CD137L expression levels. The log-rank test was applied to assess differences in survival distributions and the hazard ratio (HR) was calculated to estimate the relative risk of death between groups.

Results

Relationship between expression levels of CD137L and clinicopathological characteristics

Among the 36 CRC patients, 21 with high CD137L expression were alive, whereas only 1 patient with high expression had died. In contrast, among those with low CD137L expression, 10 were alive, while 4 had died, indicating a statistically significant (P < 0.05) association between CD137L expression and survival status. Clinical parameters such as age, gender, metastasis, and recurrence status showed no significant differences in the high and low CD137L expression groups, however, they were closely associated with patient prognosis and TNM tumor staging (Table 1).

Table 1.

The relationship between CD137L expression and clinicopathological characteristics in colorectal cancer patients.

	Characteristics	High expression	Low expression	P-value
Status	Alive	21	10	0.005
	Dead	1	4	0.005
Age*	Mean (SD)	66.6 (13.2)	66 (12.3)	0.566
	Median [MIN, MAX]	68 [31,90]	67 [31,90]	0.566
Gender	FEMALE	10	6	0.076
	MALE	12	8	0.076
pTNM_stage	I	5	3	0.03
	II	7	3
	IIA	3	2
	IIB	1
	III	1
	IIIA	1	1
	IIIB	2	2
	IIIC		2
	IV	1	1
	IVA	1

*Age is reported both as mean ± standard deviation and median [min, max] to provide a complete summary due to non-normal distribution.

Kaplan-Meier survival analysis of CD137L in CRC patients

Gene expression data of CRC patients was statistically analyzed through Kaplan-Meier method and survival probabilities of two groups with high and low expression of CD137L were compared. The results showed that H group (with higher expression of CD137L) has a significant survival probability as compared to L group (with lower expression of CD137L) of CRC patients (Figure 1). The log-rank test supports a statistically significant difference (p = 0.028) in survival of these two groups. Additionally, the hazard ratio (HR) of 0.67 indicated a 33% reduction in the death risk in G1 group (high-expression group) as compared to G2 (low-expression group) as shown in Figure 1. The relationship between expression level and improved survival outcomes highlighted CD137L expression as a good prognostic biomarker in CRC patients.

Figure 1.

Relationship between prognosis and CD137L expression in 620 colorectal patients in TCGA-CESC database.

Go enrichment analysis of CD137L

GO enrichment analysis demonstrated a significant role of CD137L in immune system regulation. The biological processes of CD137L were notably elevated in immune response, immune system process, activation of T cell, and regulation of leukocyte proliferation, thus, shaping the immune microenvironment. Similarly, the enrichment of molecular functions such as MHC class II receptor function, antigen binding and cytokine receptor activity highlighted the contribution of CD137L in antigen presentation and immune signaling which are essential mechanisms in maintaining anti-tumor immunity. Additionally, cellular components like external surface of the plasma membrane and clathrin-coated vesicle membrane underscores the localization of CD137L on immune cell surfaces which play a significant role in receptor trafficking and facilitating the immune interactions as shown in Figure 2.

Figure 2.

GO results of CD137L enrichment.

KEGG enrichment analysis of CD137L

KEGG enrichment analysis of CD137L disclosed that it is significantly involved in immune related pathways. The analysis revealed that CD137L is enriched with antigen processing and presentation, T-helper cells types1 (Th1) and Type2 (Th2) cell differentiation and cytokine–cytokine receptor interaction as shown in Figure 3. These pathways help in shaping the adaptive immune responses and promoting effective communication in immune cells. Moreover, enrichment in pathways like Staphylococcus aureus infection” and “Leishmaniasis indicated its involvement in host defense mechanisms, potentially contributing to anti-tumor immunity. Furthermore, the association of CD137L with some autoimmune pathways like systemic lupus erythematosus and rheumatoid arthritis highlighted its role in immune regulation and dysregulation which are crucial in cancer progression.

Figure 3.

KEGG results of CD137L enrichment.

Discussion

This study explained the potential role of CD137L expression in the prognosis of CRC patients by integrating analysis of TCGA database and clinical samples. The results showed a statistically significant relationship between high CD137L expression and improved survival in CRC patients, which is of significant importance for the clinical management of CRC.

In current study, high expression of CD137L in CRC patients showed significantly better survival rate compared to the patients with low expression of CD137L. Our results aligned with the previous studies highlighting the protective role of CD137L in breast and lung cancer where high expression was associated with better prognosis.^9,10 Further analysis of the TCGA database exposed that CD137L is mainly involved in immune cell proliferation, activation of T cells and signaling pathways related to Th1/Th2 balance. These processes are important in shaping the effective anti-tumor immune responses and may help explain the observed survival benefit. Additionally, CD137L has been found to improve the anti-tumor immune response in the body by regulating the immune microenvironment by promoting Th1-type immune response while damping the Th2-type immune response.^11–14

When explaining these findings, it is important to consider the complex role of CD137L in immune regulation. CD137L acts as a co-stimulatory ligand for CD137 (4-1BB), a receptor presented on activated T-cells and NK cells. Engagement of this pathway enhances the cytotoxic function, proliferation, and survival of effector immune cells, potentially inhibiting tumor growth and metastasis.^15,16 In CRC, high CD137L expression may therefore reflect a robust anti-tumor immune response, explaining the favorable outcomes observed in patients with higher expression. Several immunotherapeutic agents targeting the CD137/CD137L interaction have shown promise in preclinical and early clinical studies. Urelumab (BMS-663513), i.e., a fully human agonistic monoclonal antibody targeting CD137, demonstrated enhanced T cell activation and antitumor activity in multiple tumor models including colorectal cancer. However, dose-limiting hepatotoxicity was observed in early clinical trials suggesting further dose optimization strategies.^17,18 Similarly, utomilumab (PF-05082566) also targets CD137 with a more favorable safety profile and has been tested in combination with PD-1/PD-L1 inhibitors in several Phase I/II trials.¹⁹ These agents aim to stimulate co-stimulatory signaling to enhance T-cell mediated immune responses. The therapeutic potential of combining CD137 agonists with immune checkpoint inhibitors or cancer vaccines is an active area of investigation, indicating that the CD137/CD137L axis holds considerable promise in the design of next-generation immunotherapies for colorectal and other cancers.

GO enrichment analysis of CD137L further demonstrated its biological relevance. CD137L was shown to be significantly enriched in immune-related biological processes including the activation of T cell, immune response, and regulation of leukocyte proliferation. Similarly, molecular function terms such as MHC class II receptor activity, cytokine receptor binding, and antigen binding suggest a critical role in immune signaling and antigen presentation. The involvement of cellular components like the plasma membrane and clathrin-coated vesicle membrane is consistent with CD137L's localization on the surface of immune cells, emphasizing its role in receptor-ligand interactions. These enriched GO terms collectively support CD137L's participation in regulating tumor immunity through enhanced immune cell interaction and signaling.

KEGG pathway analysis also highlighted the association of CD137L with antigen processing and presentation, cytokine–receptor signaling pathways and differentiation of Th1 and Th2 cells. These are central pathways in anti-tumor immune regulation. Notably, CD137L's enrichment in Th1/Th2 differentiation supports its role in promoting a Th1-dominant immune profile, which has greater anti-tumor potential. Enrichment in infection-related pathways such as leishmaniasis and Staphylococcus aureus infection indicates broader immune activation, suggesting that CD137L-mediated immune stimulation may contribute to tumor control in CRC. These findings align with the observed survival benefit in patients expressing high levels of CD137L, further reinforcing its role as a beneficial immunomodulatory factor in colorectal cancer progression.

The findings of this study have lightened up a new path for immunotherapy of CRC. As a potentiating molecule, CD137L can significantly enhance the T cells activation and NK cells, thereby promoting cancer immunosurveillance.²⁰ Therefore, the high expression of CD137L may be a potential immunotherapeutic target for immunotherapy. By activating the CD137L signaling pathway, the immune response of patients can be further enhanced and the growth and spread of tumors can be inhibited.²¹ In addition, CD137 L also plays a vital role in regulating the immune microenvironment, which can promote Th1 immune response and inhibit Th2 immune response, thereby improving the tumor microenvironment and the effect of immunotherapy.²² Future research can explore immunotherapy based on CD137L, such as the development of monoclonal antibodies or agonists against CD137L, and combined with existing immune checkpoint inhibitors to form a multimodal treatment plan to further enhance the patient survival and quality of life. These findings offer a novel approach to personalized CRC treatment while also serving as a valuable reference for advancing immunotherapy strategies in other cancers.

Despite the promising findings, several limitations should be acknowledged. The clinical sample size of current study was quite small, which may disrupt the stability and reproducibility of the results. Moreover, while this study identified correlations between CD137L expression and clinical outcomes, mechanistic insights at the cellular and molecular levels remain to be explored. Future research employing gene editing, functional assays, and animal models are warranted to clarify the mechanistic underpinnings of CD137L in colorectal cancer biology.

Conclusion

In conclusion, CD137L serves as a promising prognostic biomarker in CRC and holds potential as a therapeutic target in immuno-oncology. These findings provide a foundation for the development of novel CD137L-based immunotherapies and underscore the need for further preclinical and clinical research to translate these insights into clinical practice.

Supplemental Material

sj-docx-1-cbm-10.1177_18758592251375149 - Supplemental material for CD137L and colorectal cancer prognosis: Insights from clinical and TCGA data analysis

Supplemental material, sj-docx-1-cbm-10.1177_18758592251375149 for CD137L and colorectal cancer prognosis: Insights from clinical and TCGA data analysis by Kaiyu Xu in Cancer Biomarkers

Footnotes

Acknowledgements

No.

Ethical approval and informed consent

The present study received approval from the Ethics Committee of Beijing Shijitan Hospital (250327,V1.0). Written informed consent was obtained from all participants before the collection of cancer tissues and use of associated clinical data,in accordance with institutional regulations and the declaration of Helsiki.

Funding

The author received no financial support for the research,authorship,and/or publication of this article.

Declaration of conflicting interests

The author declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Supplemental material

Supplemental material for this article is available online.

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