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CD44 on cancer stem cell is a potential immunological and prognostic pan-cancer biomarker

Cancer Cell International volume 25, Article number: 134 (2025) Cite this article

Abstract

Background

CD44, a widely recognized cancer stem cell marker, displayed a vital participation in the cancer immune invasion and may related with the response to the immunotherapy. However, the role of CD44 in cancer immunology is not well defined. Therefore, we intended to explore its prognostic value and potential immunological functions across 33 human cancer types.

Methods

Based on the data of patients from The Cancer Genome Atlas (TCGA), Sangerbox was used to analyze the correlations between CD44 expression and tumor-infiltrated immune cells, immune checkpoints, neoantigens, microsatellite instability (MSI), and tumor mutational burden (TMB) in human cancers. A mouse model xenografted with shRNA-CD44 MC38 was established.

Results

The elevated CD44 was associated with tumor stage and prognosis in several different cancers. GSEA results showed that upregulated CD44 involved in cancer stem cell associated process, antigen processing and presentation, and immune cells proliferation and activation. CD44 plays an essential role in the tumor immune regulation and immune checkpoints inhibitor response. The correlation of CD44 gene expression and infiltration levels of immune cells varied across different cancer types. Notably, the upregulation of CD44 expression is positively correlated with regulatory CD4 T cells, macrophages M1 and M2 in several analyzed cancers. Furthermore, we verified the effect of CD44 on tumor growth and immune microenvironment in mouse xenografted with shRNA-CD44 MC38. Moreover, DNA methylation existed in CD44 expression and associated with dysfunctional T-cell phenotypes via different mechanisms, thus resulting in tissue-dependent prognoses.

Conclusion

CD44 is both a cancer stem cell marker and a potential prognostic and immunological biomarker in various malignant tumors. Moreover, CD44 could be a novel target for immune-based therapy.

Introduction

Immunotherapy, including immune checkpoints inhibitors (ICIs), therapeutic vaccines and engineered T cells emerged as important strategies of cancer treatment [1]. Especially ICIs, such as programmed death-1/programmed cell death ligands (PD1/PD-L1), cytotoxic T lymphocyte-associated antigen-4 (CTLA4), and anti-lymphocyte activation gene 3 (LAG3) have shown promising clinical benefits in different malignancies and brought encouraging perspectives to patients with cancer. However, not all patients can benefit from ICIs because of the primary or acquired resistance [2]. In addition, there are still no effective prognostic biomarkers for the ICIs treatment. Therefore, more effective targets or biomarkers is urgently required to be identified. The Cancer Genome Atlas (TCGA), a public database, provided a chance to perform pan-cancer analyses and evaluate essential genes in the immune infiltration and cancer prognosis [3].

CD44, a cartilage link protein family member, is a receptor for hyaluronic acid (HA) and interact with various ligands, such as versican, osteopontin, fibronectin, and matrix metalloproteinases (MMPs). This transmembrane glycoprotein participates in a wide variety of cellular functions, including lymphocyte activation, recirculation, homing, and tumor metastasis [4, 5]. CD44 is overexpressed as standard isoform (CD44s) or alternatively spliced variant isoforms (CD44v) in cancer stem cell (CSCs) and frequently underwent alternative splicing to support cancer progression and related with poor survival [5]. CD44 has been recognized as a CSCs marker and therapeutic targets in various cancers. For therapeutic intervention targeting CD44, including CD44 neutralizing antibodies, pharmacological inhibitors, peptide mimetics, HA oligomers and aptamers are developed in preclinical and clinical trials [6].

CD44 participated in several processes of immune responses.CD44 expression is upregulated on naive T cells after activation via the T cell receptor (TCR).CD44 was essential for the generation of memory T helper 1 (Th1) cells in many diseases [7]. As for cancer, CD44 is highly expressed in gastric cancer and associated with gastric immune invasion. CD44 can be used as a prognostic biomarker of gastric cancer [8]. In triple-negative breast cancer (TNBC) and non–small cell lung cancer (NSCLC), CD44 positively regulated the PD-L1 expression through binding to the regulatory region of PD-L1 locus [9]. CD44 displayed a vital participation in the cancer immune invasion and may related with the response to the immunotherapy. However, the role of CD44 in caner immune microenvironment and cancer therapy remains poorly understood.

In our pan-cancer analysis study, we comprehensively described the cellular location and mRNA expression of CD44 in various cancer. Then, we investigated prognostic values of CD44 in TCGA cancer types. To explore the role of CD44 in the possible mechanism of immune invasion, we analyzed the relationship between CD44 and infiltrated immune cells in tumor, immune checkpoint genes, neoantigen, tumor mutation burden (TMB) level, and microsatellite instability (MSI) event. The relationship between CD44 methylation with T-cell dysfunctions and the effectiveness of ICI therapies were also evaluated.

Materials and methods

Data and software availability

The uniformly standardized pan-cancer dataset was downloaded from The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/). Gene expression data for CD44 (ENSG00000026508) were extracted from the public databases using Sangerbox (https://doiorg.publicaciones.saludcastillayleon.es/10.1002/imt2.36 Version 3.0). Cancer types with fewer than 3 samples were excluded. Then, the expression of CD44 were Log2(x + 0.001) transformed and visualized by R software (Version 4.0.2; https://www.Rproject.org) and “ggplot2” package. The UALCAN interactive web resource (https://ualcan.path.uab.edu/) was used to analyze the CD44 methylation. Promoter methylation levels are displayed by beta values ranging from 0 (unmethylated) to 1 (fully methylated) for each tumor. The correlation of CD44 methylation with prognosis and dysfunctional T-cell phenotypes were conducted using the TIDE server. In addition, the representative immunofluorescence staining of CD44 were retrieved from the Human Protein Atlas (HPA) database (http://www.proteinatlas.org).

Multivariate Cox regression analysis and survival analysis

To assess whether the high expression level of CD44 was the independent predictor overall survival (OS) and cancer-specific survival (CSS) of patients with different cancer. After excluding samples with follow-up time shorter than 30 days, the univariate Cox regression analysis and log-rank test were conducted. The “survival” package was utilized to plot the survival curves. The relationship between CD44 expression and survival outcomes in pan-cancer was manifested as forest plots using the R packages “forestplot”.

The biological significance of CD44 expression in tumors

Gene Set Enrichment Analysis (GSEA) was performed to explore the biological functions of CD44 in tumors. Samples were classified into high-CD44 and low-CD44 groups. Then, GSEA software was used to enrich Gene Ontology terms (GO) and ImmuneSigDB gene sets from the Molecular Signatures Database (MSigDB, https://www.gsea-msigdb.org/gsea/msigdb).

Relationship between CD44 expression and immunity

We estimated whether the mRNA expression of CD44 was associated with the immune infiltration landscapes in different tumor types. We extracted gene expression profiles from each tumor and mapped them to GeneSymbols. Using the IOBR R package (version 0.99.9) [10], we applied the QUANTISEQ method to assess the immune cell infiltration scores (including B cells, macrophages, monocytes, neutrophils, NK cells, T cells, Tregs, dendritic cells, and others) in each tumor [11]. Finally, we used the psych R package (version 2.1.6) to calculate Pearson’s correlation coefficients between genes and immune cell infiltration scores. The correlation between CD44 and immune checkpoints, including PD-1, PD-L1, CTLA-4, and LAG-3 was also evaluated. Moreover, we explored the relationship between CD44 and neoantigens, MSI, TMB in human TCGA cancers.

Immunofluorescence staining of CD44 in colon cancer cell and tissue

The paraffin-embedded sections of tumor tissues were heated at 50 °C for 1.5 h. The deparaffinized slides were processed for antigen retrieval by microwave heating in EDTA Tris–HCl buffer (pH8.0) for 15 min. To reduce background staining, the sections of tumor tissues or cells were treated for 1 h with 1% normal goat serum (Boster, Wuhan). The samples were incubated with the rabbit antibody against CD44 (Proteintech, Wuhan) overnight at 4 °C. Next, the secondary rabbit antibody with FITC (Boster, Wuhan) were incubated for 1 h. The immunohistochemistry images were collected using a confocal microscope (Nikon, Japan). Colon cancer and adjacent tissues from patients who had surgical resection at the West China Hospital between 2016 and 2017 were collected for this study. None of the patients received radiotherapy, chemotherapy, or immunotherapy before the operation.

Cell culture and in vitro testing

The mouse colon cell line MC38 was purchased from the American Type Culture Collection (MD, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin (Gibco, USA) in a humidified chamber with 5% CO2 at 37 °C. Stable MC38 cell line expressing shRNA-CD44 was established using lentiviral delivery system (STable1). Then, the antibody CD44 (clone IM7) was used to detect the expression of CD44 in MC38 and MC38-shRNA CD44 via flow cytometry. The CCK8 kit (Dojindo, Japan) was used to evaluate the cell proliferation. Colony formation assay was employed for testing the ability of two cell lines to form colonies. The migrated cells were detected via Transwell chamber assay.

Mouse model

MC38 and MC38-shRNA CD44 cells in the logarithmic growth phase (1 × 106 cells/200 μl) were subcutaneously inoculated into the right flanks of female C57BL/6 mice (6 weeks old). After 15 days, the 4 mice each group were sacrificed to obtain the tumor issues. The survival time of the remaining mice were observed until 35 days after the inoculation.

Immune cell markers analysis

Cells disserted from tumor tissue were stained with antibodies and analyzed by flow cytometry. The following antibodies and dye were obtained from BioLegend: CD45 (Alexa 700 30-F11), CD3e (BV510 145-2c11), CD4 (PerCP-Cy5.5 RM4-5), CD25 (PE 3C7), Foxp3 (Alexa 647 MF23), F4/80 (BV510 T45-2342), CD11c (BV605 HL3), MH-II (V500 M5/114.15.2), Ly-6G/Ly-6C (APC RB6-8C5), CD11b (PERCP-CY5.5 M1/70), CD86 (PE BU63), CD206 (PE-CY7 C068C2) and fixable viability stain 620. Stained cells were analyzed on BDLSR Fortessa ™ (BD Biosciences) and FlowJo software.

Statistical analysis

Two groups and multiple groups were analyzed using a Student’s t-test and one-way analysis of variance (ANOVA), respectively. Survival analysis was conducted using Kaplan–Meier method. Pearson or Spearman correlation analysis was utilized to calculate correlation coefficients. Multiple testing corrections to control the false discovery rate (FDR) were applied to avoid false positives. Those mentioned analyses above is conducted by R software (Version 4.0.2). A two-tailed p-value < 0.05 was defined as the threshold of significance.

Results

CD44 localized in cytoplasm and membrane of tumor cell and significantly associated with cancer

To evaluate the distribution and expression of CD44 in tumor cell, we retrieved the immunofluorescence results of A-431, U-2OS, and U251-MG from the HPA database. The endoplasmic reticulum (ER) and microtubules were marked with yellow and red, respectively. It was observed that CD44 overlapped with ER and microtubules but displayed no staining in the nuclei, suggesting that CD44 colocalized with these markers in the cytoplasm of tumor cell (Fig. 1A). In addition, our immunofluorescence staining results also demonstrated the location of CD44 in the colon cancer cell lines (HT29 and HCT116) (Fig. 1B). Furthermore, compared with the adjacent tissue, the number of cancer cells expressing CD44 were increased in cancer tissue of colon cancer patients (Fig. 1C). These results suggested that CD44 exhibits similar expression patterns and levels across various cancer cell lines and colon cancer patients had increased tumor cells expressing CD44 in tumor tissue.

Fig. 1
figure 1

Localization and expression of CD44 in cancer cell lines and tissue. A The immunofluorescence staining of the subcellular distribution of CD44 within the nucleus, endoplasmic reticulum (ER), and microtubules of A-431, U-2OS, and U251-MG from the HPA database. B The immunofluorescence staining of CD44 in the HT29 and HCT116 colon cells. C The number of CD44 positive cells per field of view in adjacent tissue and tumor tissue from colon cancer patient. ****p < 0.0001

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CD44 was aberrantly expressed in various tumor tissues and associated with tumor stages

To further clarify the association of CD44 and cancer, the CD44 expression was evaluated in several cancer types from TCGA database. Compared with the normal tissues, CD44 was significantly overexpressed in 25 tumor types. The top 5 were glioma (GBMLGG, p = 1.6e-204), brain lower grade glioma (LGG, p = 6.0e-160), glioblastoma multiforme (GBM, p = 1.5e-83), colon adenocarcinoma/rectum adenocarcinoma esophageal carcinoma (COADREAD, p = 1.6e-82) and colon adenocarcinoma (COAD, p = 7.0e-72). In contrast, we observed significant downregulation in six tumors such as uterine corpus endometrial carcinoma (UCEC, p = 2.4e-3), lung adenocarcinoma (LUAD, p = 2.2e-28), and prostate adenocarcinoma (PRAD, p = 1.6e-6) (Fig. 2A and STable2-3). Additionally, compared with stage I, we found that CD44 expression was significantly increased in stage IV of stomach adenocarcinoma (STAD, p = 0.01), stomach and esophageal carcinoma (STES, p = 0.02) and pan-kidney cohort (KIPAN, p = 0.01). Conversely, breast invasive carcinoma (BRCA, p = 1.4e-3) exhibited a relative decrease of CD44 expression at stage IV (Fig. 2B and STable4). Then, we analyzed the CD44 expression in different grades of pan-cancer. There was a continuous upregulation of CD44 expression from G1 to G4 in Pan-kidney cohort (KIPAN, p = 5.2e-5), kidney renal clear cell carcinoma (KIRC, p = 5.2e-5). GBMLGG (p = 1.8e-5), STAD (p = 0.01), and LGG p = 1.8e-5) had increased CD44 expression from G2 to G3. While head and neck squamous cell carcinoma (HNSC, p = 2.1e-3) and ESCA, p = 8.8e-3), STES (p = 0.04) showed decrease of CD44 expression from G2 to G3 (Fig. 2C and STable5). These above results suggested that CD44 upregulated in the advanced stage of most types of tumors. CD44 may evolve with the progress and development of cancer and may affect the survival outcome of patients with cancer.

Fig. 2
figure 2

CD44 expression in pan-cancer. A Differential CD44 mRNA expression in tumor tissues from TCGA database. B CD44 expression of tumor tissues in different stages (I–IV) of STAD, KIPAN, STES, and BRCA. C CD44 mRNA expression in different grades (G1–4) of KIPAN, KIRC, GBMLGG, STAD, LGG, HNSC, ESCA, and STES. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001; ns, not significant

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CD44 is a potential prognostic marker in pan-cancer

Based on TCGA mRNA-seq data and clinical information, a Cox proportional hazards regression model was used to determine the association of CD44 expression levels with the prognosis of patients with various cancer. TGCT (testicular germ cell tumor), GBMLGG, LGG, KIRP (kidney renal papillary cell carcinoma), KIPAN, HNSC, and PAAD with higher CD44 expression had poorer overall survival and cancer specific survival (Fig. 3A, B). Consistently, Kaplan–Meier survival curves of OS and CSS of GBMLGG, LGG and PAAD showed significant difference in high and low CD44 expression groups. On the contrary, increased CD44 mRNA level uniquely related with considerable outcome in UVM (Fig. 3C, D), suggesting the distinct role of CD44 in the development of UVM.

Fig. 3
figure 3

Survival analysis comparing the high and low CD44 expression on CSS and OS of different cancers in the TCGA database. A Forest plot of univariate Cox regression analysis of OS. B Forest plot of univariate Cox regression analysis of CSS. C Kaplan–Meier OS diagram of GBMLGG, LGG, PAAD, and UVM. D Kaplan–Meier CSS diagram of GBMLGG, LGG, PAAD, and UVM

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Gene set enrichment analysis

GO and ImmuneSigDB enrichment analyses were performed in high-CD44 and low-CD44 group of pan-cancer (Fig. 4A, B). In the high-CD44 group, we found that many CSCs related gene sets and immune associated sets were enriched. GO enrichment analyses suggested that these genes were mainly concentrated in cell adhesion and migration, epithelial-mesenchymal transition (EMT), Notching signaling pathway, phosphatidylinositol phosphate biosynthesis process (Fig. 4A). GO and ImmuneSigDB enrichment analysis commonly displayed that upregulated CD44 involved in MHC II biosynthesis, cytokine, chemokine, and interleukin production, T cell and B cell proliferation, and regulations on T cell activation, suggesting CD44 involves in the several process of immune response.

Fig. 4
figure 4

Go and ImmuneSigDB enrichment analysis of CD44 in indicated tumor types. A Go enrichment results of PAAD, GBM, BRCA, and PRAD. B ImmuneSigDB enrichment results of PAAD, GBM, BRCA, and PRAD. Values of p < 0.05 and results higher than 5 were considered and displayed

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CD44 expression is related with immune cell infiltration in human cancers

The subtypes and amounts of infiltrating lymphocytes in tumor were important predictors of the survival of patients with cancer [12]. Hence, we obtained the content of 11 specific immune cells in each sample of total 44 cancer types. This analysis covered 11,180 tumor samples across 44 cancer types. Our results indicated that CD44 expression significantly correlated with tumor purity. The heatmap described the results at p < 0.005. CD44 expression was correlated with the infiltration levels of CD4+T cells in 17 cancer types, B cells in 17 cancer types, CD8+T cells in 22 cancer types, neutrophils in 25 cancer types, and dendritic cells in 20 cancer types. KIPAN, GBMLGG, LGG, and TCGT displayed a strong correlation between CD44 expression and quantities of immune infiltrated cells, including Treg, CD8+T cell, macrophages M1 and M2 (Fig. 5A). While CD44 expression showed a negative relationship with amounts of infiltrated monocytes in several cancer types.

Fig. 5
figure 5

Integrative analysis of relationship between CD44 expression and immune microenvironment. A The correlations between infiltrated immune cells and CD44 expression levels in different cancers. B Correlation between CD44 expression and 150 immunomodulators (chemokines, receptors, MHC, and immunostimulators). (C) Correlation between CD44 and four immune checkpoints, PD-L1, CTLA-4, PD-1, and LAG-3. The dots represent cancer types. The Y-axis represents the Pearson correlation, while the X-axis represents -log10P. *p < 0.05, **p < 0.01, ***p < 0. 001

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Correlations between CD44 expression and immune marker sets, TMB, and MSI in pan-cancer

We found that chemokines (41 genes), immune receptors (18 genes), MHC (21 genes), immunoinhibitors (24 genes), immunostimulators (46 genes) were obviously activated in the group with overexpressed CD44 group (Fig. 5B). Among them, CD44 expression is positively correlated with immune chemokines, such as (chemokine C–C motif) CCL2, CCL5, CCL15, CCL20, CCL21, CXCL13, and their receptors, such as CCR1, CCR2, CCR5, CCR7, and CXCR3. These chemokines and receptors can improve the infiltration of CD8+ T cells, TH17 cells, and antigen-presenting cells. MHC associated genes that reveal the capacity of antigen presentation and processing also had a positive relationship with CD44 expression. Furthermore, it was observed that CD44 expression had a positively correlation with immune checkpoints, such as PD-1, PD-L1, CTLA-4, and LAG-3 in many types of cancer. In GBMLGG, LGG, and OV (Ovarian serous cystadenocarcinoma), CD44 expression showed a strong positively correlation with PD-1 and PD-L1. OV, KIPAN, and KIRP displayed an obvious relationship between CD44 expression and CTLA-4, and LAG-3 (Fig. 5C). Moreover, TMB, MSI, and neoantigen load both effected the therapeutic efficacy of ICIs. Our results showed that there was a strongly positive association between CD44 expression neoantigen, MSI, and TMB in COAD, READ (rectum adenocarcinoma), SARC (sarcoma), and UCEC (uterine corpus endometrial carcinoma) (SFig.1A). In the contrast, CD44 expression had a strongly negative association with MSI in GBMLGG (SFig.1B). CHOL (cholangiocarcinoma) showed a significantly negative relationship between CD44 expression and neoantigen load and TMB (SFig.1A and SFig.1C). These findings further revealed that CD44 is a potential predictor for the sensitivity of immunotherapy based on the immune checkpoints.

CD44 methylation related with T-cell dysfunctions and poor prognoses of cancer cohorts

We calculated the promoter methylation level of CD44 in pan-cancer. Different beta-values were hypermethylation (0.7–0.5) or hypomethylation (0.3–0.25). Many TCGA cancer types showed the hypermethylation of CD44. In LUSC, LUAD, ESCA, BRCA, the promoter methylation levels of CD44 were significantly higher than those in normal groups. While PAAD, COAD, LIHC, TGCT had decreased CD44 methylation levels in (Fig. 6A). Hypomethylation of CD44 was positively associated with dysfunctional T cell phenotypes and survival outcomes in 20 cancer types (Fig. 6B). Then, Kaplan–Meier survival analyses were performed to explore the relationship between CD44 promoter methylation and patient prognosis. Brain cancer, PAAD, and HNSC cohort with higher methylation levels of CD44 had better survival prognosis. In contrast, the hypomethylation level of CD44 was positively associated with T-cell dysfunctions but was a protective factor in patients in LUAD (Fig. 6C). Together, these results indicated that epigenetic methylation of CD44 is associated with dysfunctional T-cell phenotypes via different mechanisms that ultimately result in poor prognoses of PAAD, COAD, LIHC, TGCT cohorts while prolonging the survival of LUAD cohort.

Fig. 6
figure 6

Epigenetic modification of CD44 mediates dysfunctional T-cell phenotypes and poor prognoses of cancer cohorts. A The CD44 methylation levels (beta values) of tumor and normal tissues from TCGA database. B The correlation between CD44 methylation and cytotoxic T-cell levels (CTLs), dysfunctional T-cell phenotypes, and risk factors of TCGA cancer cohorts. C Overall survival curves of cancer groups with high methylation levels and those with low methylation levels of CD44

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CD44 promoted the tumor growth and remodeled the immune microenvironment of mouse colon cancer

To further verify the function of CD44 in cancer, we constructed stable MC38 cell line expressing shRNA -CD44 using lentiviral delivery approach. Then, the flow cytometry detected that the expression of CD44 was significantly decreased in MC38 shRNA-CD44 cell line (Fig. 7A). We found that CD44 knockdown reduced the proliferation, colony number and migration cells in MC38 (Fig. 7B–D). Furthermore, CD44 knockdown also inhibited subcutaneous xenograft tumor growth in mice (Fig. 7E). The mouse xenografted with shRNA-CD44 MC38 had a prolonged survival outcome (Fig. 7F). Importantly, the immune environment of mouse colon cancer was detected by multicolor flow cytometry. Total CD45+cells were higher in the group xenografted with shRNA-CD44 MC38.Of these, the group xenografted with shRNA-CD44 MC38 had obviously increased CD4+, CD8+ T cells and MDSC (myeloid derived suppressor cell) in CD45 cells. In contrast, Treg cell in CD45 cells of the mouse xenografted with shRNA-CD44 MC38 was significantly decreased. Dendritic cells, macrophages M2 and M1 in in CD45 cells showed no significant difference among two groups (Fig. 7G).

Fig. 7
figure 7

The effect of CD44 on tumor growth and the immune microenvironment of mouse xenografted with colon cancer. A The flow cytometry to detect the expression of CD44 in MC38 and MC38 shRNA-CD44 cell line. B The CCK8 assay of two cell lines. (C) The colony assay and statistical bar chart of two cell lines. (D) The migration assay and statistical bar chart of two cell lines. (E) The subcutaneous xenograft tumors in mice were generated using two cell lines (F) The survival curve of two groups xenografted with different cell lines. (G) The tumor immune environment detected by multicolor flow cytometry

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Discussion

A subpopulation of patients with cancer had limited response rates to ICIs [13]. The tumor genomic and microenvironment characteristics were explored to find the biomarkers of responses to ICIs [14]. The biomarkers with good predictive value were required to evaluate the immune response and select beneficial patients with cancer. CD44, a CSCs marker, is upregulated and had association with the immune response in many tumors. CD44 may be a promising biomarker for ICIs treatment and therapeutic targets. Therefore, we comprehensively investigated the expression of CD44 and its predictive values in 33 TCGA cancer.

CD44, including some of its alternatively spliced variants, is one of most common cell surface biomarkers for CSCs and maintains stem cell phenotype and innates the metastasis [15]. It has been reported that the CD44-HA binding can induce EMT, promoting the invasiveness and metastasis of cancers. The CD44-HA binding level varies in different malignancies. Besides, different alternatively spliced variants of CD44 have shown conflicting effects on the development of various cancers. CD44v3 is overexpressed in HNSC tissue and was associated with cell migration [16]. CD44v3, CD44v6, and CD44v7/8 were correlated with advanced stages of breast cancer [17]. However, CD44v3 expression correlated with better outcomes in neuroblastomas and cutaneous melanoma [18]. In our results, compared to normal tissue, CD44 is obviously overexpressed in a variety of tumors. The increased CD44 expression was observed in stage IV and G3 or G4 of several cancer cohorts, implying that the CD44 expression associated with tumor metastasis. On the contrary, CD44 expression in HNSC, ESCA, and STES from G2 to G3 was decreased. These contradictory results may be a result of the different expression of CD44 isoforms in different cancer types. Despite of the not consistent results of CD44 in the caner metastasis, it suggested that CD44 was potentially a therapeutic target treating metastatic cancers. It has been reported that the invasion of colon carcinoma cells was decreased when HA binding was suppressed by a CD44 blocking antibody [19]. Moreover, we found the number of CD44 positive cells was significantly upregulated in the tumor tissue of colon cancer patients compared with adjacent tissue. In vitro, CD44 promoted the proliferation and migration of colon cancer line, indicating CD44 exerts a promotive effect on tumor progression of colon cancer.

Given the significance of CD44 in the tumor recurrence, the prognosis analysis of CD44 in pan-cancer was conducted in our study. CD44 was a risk prognostic factor for most cancer types, including GBMLGG, LGG, KIRP, KIPAN, HNSC, PAAD, and TGCT. Consistently, a previous study also revealed that CD44 overexpression was significantly associated with pancreatic cancer, colorectal and breast cancer [20,21,22]. Our result also showed that the low expression of CD44 was positively with promising survival outcome in mouse colon cancer. Mechanically, CD44s regulated TGF-β signaling mediated mesenchymal phenotype and increased expression of vimentin, and decreased E-cadherin expression, thus resulting in the poor prognosis of hepatocellular carcinoma [23]. On the contrary, patients with high CD44 expression uniquely had a better survival in UVM. A previous study demonstrated that the loss of CD44 promoted lung metastasis during breast cancer progression [24]. The CD44 expression and its prognostic values varied in different types of cancers. The specific role of CD44 or CD44v in each cancer needs to be investigated.

We also explored biological signaling pathways CD44 involved in cancer. It was known that the dysregulation of several signaling pathways, such as Notching and Hippo pathways contributes to cancer, especially the development of CSCs. Our results of Go analysis displayed that Notching signaling pathway and phosphatidylinositol phosphate biosynthesis process were enhanced in the high-CD44 group. It has been proved that phosphatidylinositol phosphate biosynthesis process regulated the activity of Hippo signaling pathway as the upstream modulator [25]. The Go analysis displayed that cell adhesion and migration, EMT processes were enriched in the group with high CD44 expression. Increased expression of EMT transcription factors, such as OCT4 and SOX2, contributes to the phenotype and functions of CSCs [26]. The dendritic cell (DC)-based cancer vaccines targeting CSCs reduced the lung metastasis and prolonged the survival via conferring the specific cytotoxic T lymphocytes in the adjuvant setting [27]. Similarly, as a CSCs marker in various cancers, we found that CD44 had an important role in the immune response via regulating MHC II biosynthesis, cytokines, chemokines, and interleukin production, T cell and B cell proliferation, and regulations on T cell activation. Especially, CD44 expression was correlated with the infiltration levels of macrophages M1, M2 and Tregs in most analyzed cancer types, suggesting that CD44 may have an essential role in the emergency of immunosuppressive microenvironment.

We further verified the effect of CD44 on tumor microenvironment in mouse colon cancer. CD4+, CD8+T cells in CD45+ cells were significantly increased in the mouse xenografted with shRNA-CD44 MC38. In addition, Tregs in CD45+ cells of were significantly decreased. CD44 expression is positively associated with transcription factor FoxP3 levels and the suppressive function of Tregs [28]. It has been reported that Tregs was functionally impaired in CD44-knockout mice [29]. Consistently, in TCGA database, READ had positive association between CD44 expression and infiltrated Tregs. However, there was no significant difference between CD44 expression and Tregs infiltrations in COAD or COADREAD. The different anatomical locations of colon cancer may influence the effect of CD44 on Tregs. In ovarian cancer, CSCs with high CD44 expression can activate STAT3 in macrophages and drive M2 polarization [30]. Notably, macrophage M2 and M1 in CD45+ cells showed no significant relationship between CD44 expression in colon mouse model. On the contrary, macrophage M2 had a positive association with CD44 expression in COAD, COADREAD and READ of TCGA database. Differences in the immune microenvironments may contribute to the heterogeneity observed between human and mouse tumor models.

Immune checkpoints, such as PD-1 and CTLA-4, involves the tumor invasion from the immune attack and determines the immunotherapy results [31]. In our study, immune checkpoints, such as PD-1, PD-L1, CTLA-4, LAG-3 had a positively strong correlation with CD44 expression in many types of cancer, especially in GBMLGG and OV. It suggested that CD44 might coordinate the activities of these immune checkpoint genes via different signal pathways and mediate immune invasion. TMB reflects the total number of somatic mutations per coding area in the genome of tumor cells [32]. TMB level is commonly regarded as a notable biomarker to predict the effectiveness of immunotherapy in many tumors [33]. MSI is also an important biomarker of ICI s and high-frequency MSI is an independent risk factor for the prognosis of in colorectal cancer [34]. Our results demonstrated that there was a strongly positive association between CD44 expression and TMB, MSI, and neoantigen load in COAD, READ, SARC, UCEC, and READ. In the contrast, LUAD, LUSC, BLCA, CHOL showed a negative relationship. CD44 was both a tumor stem cell marker and an immunosuppressive molecule. The level of CD44 expression may alter the TMB, MSI, and neoantigen load in cancer, thus exerting an effect on the patient response to ICIs. These findings further revealed that CD44 is a potential pan-cancer immunotherapy target and biomarker to predict the therapeutic efficacy of immunotherapy targeting immune checkpoints.

Furthermore, we found that CD44 was hypomethylated in various cancer types. Meanwhile, mRNA levels of CD44 were overexpressed in those cancers. Epigenetic methylation of CD44 may affect its transcriptome in cancer. The epigenetic marker 6 mA DNA methylation affects the gene expression. 6 mA DNA methylation can promote mRNA metabolism and translation, leading to the gene activation. Besides, m6A can alter RNA folding and structure to promote translation and affects splicing [35]. The switching of CD44s and CD44v generated by alternative splicing of middle exons was associated with EMT and tumor metastases [5]. It may explain that methylation of CD44 was associated with cancer survival. Interestingly, our results showed that hypomethylation of CD44 mediated in dysfunctional T cell phenotypes and survivals in 20 cancer types. Brain cancer, PAAD, and HNSC cohort with higher methylation levels of CD44 had better survival prognosis. Inversely, the hypomethylation level of CD44 was positively mediated T-cell dysfunctions but was a protective factor of patients with LUAD via a different mechanism. The methylation level of CD44 in different cancer further supports our conclusion that CD44 displayed a tissue-dependent regulation on cancer immunity and survival prognosis.

Conclusion

Our pan-cancer analysis described the CD44 expression in normal and tumor tissues and revealed the correlation between CD44 expression and clinical stage. The high expression of CD44 was associated with unfavorable survival outcome. CD44 can be served as a prognostic factor for in several different tumors. Moreover, in various cancer types, CD44 expression was related with immune cell infiltration, TMB, MSI, and neoantigens across various cancer types. CD44 was hypomethylated and associated with T-cell dysfunctions and poor prognoses of patients with cancer. The effect of CD44 expression on prognosis and cancer immunity varied with tumor types. Our findings elucidated the role of CD44 as onco-immunological marker and provided a reference for the personalized immune-based therapy in the future.

Data availability

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

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Acknowledgements

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Funding

This work was financially supported by National Key R&D·Program of China (2023YFC3403200) and the National Natural Science Foundation of China (Grant No. 82103460).

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Author notes

  1. Ya-Juan Zhu, Si-Ying Li and Shan-Shan Yang equally contributed to this work.

Authors and Affiliations

  1. Department of Biotherapy, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Ya-Juan Zhu, Yang Du & Ji-Yan Liu

  2. Department of Liver Surgery, Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Si-Ying Li

  3. Molecular Medicine Research Center, West China Hospital, Sichuan University, Chengdu, China

    Shan-Shan Yang

  4. Department of Head and Neck Cancer Surgery, West China School of Stomatology, Sichuan University, Chengdu, China

    Zhuo-Yuan Zhang

  5. State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China

    Zhuo-Yuan Zhang

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Contributions

YJZ and SYL: Writing—drafting. ZYZ and JYL: Conceptualization, Methodology, Supervision. SSY and YD: Writing—review & editing. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Zhuo-Yuan Zhang or Ji-Yan Liu.

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All animal procedures were performed according to the protocol approved by the West China Hospital Animal Care and Use Committee (No. 20240904001) and complied with the principles of the Declaration of Helsinki. Human specimens were obtained from the West China Hospital after the West China Hospital’s Medical Ethics Committee approval (No. 2024664) and patients’ informed consent.

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Zhu, YJ., Li, SY., Yang, SS. et al. CD44 on cancer stem cell is a potential immunological and prognostic pan-cancer biomarker. Cancer Cell Int 25, 134 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03748-4

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Keywords

Cancer Cell International

ISSN: 1475-2867

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