Correlation analysis of CD44 and gastric cancer progression.

To clarify the function of CD44 in gastric cancer, we analyzed the TCGA-STAD dataset and observed that the expression levels of CD44 in gastric cancer tissues were significantly elevated compared to those in normal tissues (p < 0.001, Fig. 1a). Based on the expression levels of CD44, the tumor samples were divided into two groups: one group exhibited high CD44 expression, while the other exhibited low CD44 expression. Our analysis revealed that high CD44 expression was linked to a poorer prognosis compared to the group with low CD44 expression (p = 0.039, Fig. 1b). Similarly, CD44 expression level was markedly higher in patients diagnosed with M1 (metastatic disease) compared to those diagnosed with M0 (non-metastatic disease) (p < 0.01, Fig. 1c). The results of pathway enrichment analysis of differentially expressed genes showed that individuals with high CD44 expression levels are mainly linked to the JAK-STAT and cell adhesion pathways (green, NES > 1), whereas those with low CD44 expression are primarily enriched in the DNA repair pathway (orange, NES < 1) (Fig. 1d).

Fig. 1

Correlation analysis of CD44 and gastric cancer progression. a Through the analysis of TCGA-STAD (Gastric cancer) dataset, it was found that the expression of CD44 in tumor tissues was higher than that in normal tissues. b Survival analysis showed that the prognosis of the group with high CD44 expression was worse than that of the group with low CD44 expression. c The expression of CD44 in patients with metastasis was higher than that in patients without metastasis. d Enrichment analysis of differentially expressed genes in CD44 high expression and CD44 low expression groups

Clinically, the above results suggest a potential link between high CD44 expression and the advancement of gastric cancer. Given the significant role of CD44 expression in gastric cancer progression, it is crucial to further explore its relationship with other oncogenic properties, such as tumor stemness, to understand its broader impact on tumor biology.

CD44v6 expression positively correlates with CSC marker levels in gastric cancer tissues

Based on bioinformatics data analysis, CD44 overexpression was correlated with poor prognosis and metastatic disease in gastric cancer. To further explore CD44 expression and the specific roles of its isoforms in gastric cancer, we examined clinical gastric cancer samples.

Hematoxylin–eosin (HE) staining revealed significant differences in tissue architecture between normal and tumor tissues. Furthermore, IHC analysis indicated a notable increase in CD44 expression in gastric cancer tissues relative to adjacent normal tissues, underscoring the potential role of CD44 in promoting tumor progression (Fig. 2a).

Fig. 2

CD44v6 expression positively correlates with CSC marker levels in gastric cancer tissues. a Compared with normal tissues, the expression of CD44 was increased in gastric cancer, and the expression of its isomer CD44 was more significant. b Expression of CD44 isomers in gastric cancer. c CD133 and CD44v6 were co-expressed in gastric cancer. d CD24 and CD44v6 were co-expressed in gastric cancer

The overall expression of CD44 may mask the distinct functions of its isoforms. To further investigate the roles of CD44 isoforms in gastric cancer development, we performed additional IHC analysis. Among CD44 isoforms, CD44v6 exhibited the highest expression in gastric cancer tissues, significantly surpassing other isoforms, including CD44s, CD44v3-v5, and CD44v7-v9. CD44v6 expression was predominantly localized to the cell membrane, consistent with its proposed function as a CSC surface marker (Fig. 2b).

Building on the reported association between CD44v6 and tumor stemness in other cancers [23], we sought to further investigate its specific role in gastric CSCs. CD133 and CD24, recognized as hallmarks of CSCs, were examined using immunofluorescence staining. Regions with elevated CD44v6 expression colocalized with increased levels of CD133 (Fig. 2c) and CD24 (Fig. 2d). Collectively, these results show that CD44v6 is crucial for maintaining stem cell-like properties in gastric cancer cells and in driving gastric cancer development, particularly through its association with CSC traits.

CD44v6 modulates CSC marker expression and maintains gastric CSC characteristics

Following our findings of significantly increased CD44v6 expression in gastric cancer tissues and its correlation with CSC markers, we next investigated its functional role by examining how CD44v6 expression levels affect gastric CSC characteristics and related mechanisms.

Specifically, two gastric cancer cell lines, MKN-45 and NCI-N87, were utilized as experimental models to investigate the effects of CD44v6 knockdown and overexpression. Western blot analyses confirmed effective CD44v6 knockdown in both cell lines, showing significantly reduced relative intensity compared to the control group (p < 0.05, Fig. 3a). The reduction in CD44v6 was accompanied by decreased levels of CD44, CD24, and CD133, which are widely recognized as key CSC markers involved in regulating self-renewal, differentiation potential, and tumor aggressiveness (p < 0.001). Additionally, EpCAM expression, a molecule critical for cell adhesion and CSC maintenance, showed a moderate to weak decrease (p < 0.05, Fig. 3b).

Fig. 3

CD44v6 modulates CSC marker expression and maintains gastric CSC characteristics. a, b CD44v6− in MKN-45 and NCI-N87 cells was controlled by Western blot to evaluate the CSC markers CD44, CD24, CD133, and EpCAM. c, d Both CD44v6− and CD44v6+ in MKN-45 and NCI-N87 cells were performed by real-time PCR to evaluate the CSC-related genes Oct4, Nanog, and Sox2, respectively. Significant differences to control cells: * p < 0.05, **p < 0.01, and ***p < 0.001

Furthermore, the knockdown of CD44v6 resulted in a significant decrease in the mRNA levels of Oct-4 and Nanog, two vital transcription factors for sustaining self-renewal and pluripotency (p < 0.05). However, Sox-2, another transcription factor, remained unaffected by CD44v6 modulation (Fig. 3c). Conversely, overexpression of CD44v6 led to increased levels of Oct-4 and Nanog, with Sox-2 expression remaining unchanged (Fig. 3d).

These observations demonstrate that CD44v6 plays a pivotal role in regulating key CSC markers and maintaining the stem cell-like properties of gastric cancer cells.

CD44v6 enhances self-renewal, proliferation, and migration in gastric cancer cells

Building on the role of CD44v6 in regulating CSC marker expression, we further assessed its impact on the self-renewal, colony formation, migration, and proliferation abilities of gastric cancer cells.

The colony formation assay showed that MKN-45 CD44v6+ and NCI-N87 CD44v6+ cells exhibited significantly enhanced colony formation (p < 0.05), while MKN-45 CD44v6− and NCI-N87 CD44v6− cells showed markedly reduced colony formation than the control cells (p < 0.05), indicating a pivotal role of CD44v6 in supporting self-renewal characteristics (Fig. 4a, b). Microscopic examination of the soft agar colony formation assay showed that CD44v6− cells formed fewer and smaller colonies in comparison with the control group (Fig. S1a). Quantitative analysis confirmed a marked decrease in the number of cell clusters formed by CD44v6− cells (p < 0.001, Fig. S1b). Additionally, the colony diameter of CD44v6− cells was significantly smaller than that of the control group (p < 0.001, Fig. S1c, S1d). This evidence underscores the critical function of CD44v6 in enhancing the self-renewal and tumorigenic capabilities of gastric cancer cells, thereby supporting its potential as a therapeutic intervention point in gastric cancer.

Fig. 4

CD44v6 enhances self-renewal, proliferation, and migration in gastric cancer cells. a Anchorage-independent growth capacity of both CD44v6− and CD44v6+ in MKN-45 and NCI-N87 cells were characterized by soft agar colony formation assays. b Quantitative analysis of colony numbers and size in soft agar assays. c Subconfluent monolayers of cells as in were scratched with a pipette tip. Wound healing was recorded for 48 h. d Quantitative analysis of scratch test. e Both CD44v6− and CD44v6+ in MKN-45 and NCI-N87 cells were controlled for cell proliferation by crystal violet and CCK-8. f Quantitative analysis of cell proliferation. A representative example of CD44v6−, CD44v6+ and control cells and mean ± SD of the wound area in three independent assays are shown. Mean ± SD of three independent assays are shown, significant differences to control cells: * p < 0.05, **p < 0.01, and ***p < 0.001

The migration of CD44v6−, CD44v6+, and control cells was assessed using the wound healing assay. Notably, the wound healing rate was significantly enhanced in MKN-45CD44v6+ and NCI-N87CD44v6+ cells, while CD44v6− cells exhibited weaker migration abilities than the control group. (p < 0.001, Fig. 4c, d).

Additionally, the viability of CD44v6−, CD44v6+, and control cells was assessed using crystal violet and CCK-8 assays. Cell viability, assessed using crystal violet staining, showed that CD44v6 + cells had a significantly increased survival rate compared to both control and CD44v6− cells in both gastric cancer cell lines (Fig. 4e). The proliferation of CD44v6− cells was notably diminished at 36 h, 48 h, and 72 h (p < 0.05). In contrast, CD44v6+ cells exhibited heightened proliferation compared to control cells, particularly relative to CD44v6− cells (p < 0.05, Fig. 4f).

Overall, these results establish CD44v6 as a key enhancer of multiple malignancy-associated behaviors in gastric cancer cells, underscoring its importance as a promising therapeutic intervention to inhibit gastric cancer progression.

CD44v6 enhances chemotherapy resistance in gastric cancer cells

After demonstrating the notable effect of CD44v6 on self-renewal, colony formation, proliferation, and migration of gastric cancer cells, we next explored its potential impact on chemotherapy resistance, specifically its effect on apoptosis triggered by cisplatin treatment. Cells were treated with 50 μg/mL cisplatin for a duration of 48 h, and apoptosis was assessed via Annexin V/PI staining. The results confirmed that the cisplatin administration effectively decreased the apoptosis resistance of CD44v6− cells compared to control cells (p < 0.001). By contrast, CD44v6+ cells exhibited significantly enhanced apoptosis resistance in both MKN-45 and NCI-N87 cell lines when compared to both CD44v6− and control cells (p < 0.001), as seen in Fig. 5a, b.

Fig. 5

CD44v6 enhances chemotherapy resistance in gastric cancer cells. a, b Apoptosis was evaluated by Annexin V/PI staining after 48-h culture in identical amounts of cisplatin (50 μg/ml). Mean ± SD of three independent assays are shown, significant differences to control cells: * p < 0.05, **p < 0.01, and ***p < 0.001

CD44v6 not only influences the biological behaviors of gastric cancer cells but also significantly impacts their response to the chemotherapeutic cisplatin. Our data underscore the importance of CD44v6 as a prospective treatment target, especially in designing strategies to combat drug resistance.

CD44v6 knockdown suppresses subcutaneous tumor growth and stem cell marker expression in gastric cancer models

To verify the regulatory impact of CD44v6 on gastric cancer, tumor growth was monitored in both the control and CD44v6 knockdown groups using in vivo imaging techniques. The experimental process is shown in Fig. 6a. Results indicated that tumor growth was significantly suppressed in the knockdown group than the control group, with a notable reduction in fluorescence intensity (p < 0.001, Fig. 6b, c). Macroscopically, both the appearance and size of tumors were significantly reduced in the CD44v6 knockdown group, further verifying the suppressive impact of gene knockdown on tumor growth (Fig. 6d). Detailed measurement data showed that, when compared to the control group, CD44v6 knockdown significantly reduced tumor volume (p < 0.01, Fig. 6e) and weight (p < 0.001, Fig. 6f).

Fig. 6

CD44v6 knockdown suppresses subcutaneous tumor growth and stem cell marker expression in gastric cancer models. a Animal subcutaneous tumor modeling diagram. b Control group and CD44v6 knockdown group were imaged in vivo. c Fluorescence quantitative analysis in vivo imaging of animals. d, e Gross and quantitative analysis of tumor volume in experimental group and control group. f Quantitative analysis of tumor weight in experimental group and control group. g HE staining and expression of CD44, CD44v6, CD24, CD133 and Ki67 in the control group. h HE staining and expression of CD44, CD44v6, CD24, CD133, and Ki67 in the CD44v6 knockdown group. Mean ± SD of three independent assays are shown, significant differences to control cells: * p < 0.05, **p < 0.01, and ***p < 0.001

Histological examination revealed that in the control group, HE staining and the expression of CD44, CD44v6, CD24, CD133, and Ki67 were markedly highly expressed (Fig. 6g). In contrast, in the CD44v6 knockdown group, both HE staining and the expression of these tumor markers were significantly reduced, closely correlating with the slower tumor growth (Fig. 6h).

Collectively, these in vivo findings highlight the crucial regulatory role of CD44v6 in gastric cancer progression, emphasizing its promise as a target for precision therapy and drug development (Fig. 7).

Fig. 7

Schematic diagram of targeting CD44v6 to inhibit gastric cancer progression as a potential therapy