CD44 plays a key role in the communication of CSCs with the microenvironment and the regulation of stem cell properties. UALCAN was used to analyze the expression of CD44 in bladder cancer (BLCA) and normal tissue. The UALCAN was utilized to analyze the prognostic value of CD44 in BLCA. The TIMER database was used to explore the relationship between CD44 and PD-L1; CD44 and tumor-infiltrating immune cells. The regulatory effect of CD44 on PD-L1 was verified by cell experiments in vitro. IHC confirmed the results of the bioinformatics analysis. GeneMania and Metascape were used to analyze protein–protein interaction (PPI) investigations and functional enrichment analysis. We found that BLCA patients with high CD44 expression had worse survival than those with low CD44 expression (P < 0.05). IHC and the TIMER database results showed that CD44 expression was positively correlated with PD-L1 expression (P < 0.05). At the cellular level, the expression of PD-L1 was significantly inhibited after CD44 expression was inhibited by siRNA. Immune infiltration analysis showed that CD44 expression levels in BLCA were significantly correlated with immune infiltration levels of different immune cells. IHC staining results further confirmed that the expression of CD44 in tumor cells was positively associated with the number of CD68⁺ macrophages and CD163⁺ macrophages (P < 0.05). Our results suggest that CD44 is a positive regulator of PD-L1 in BLCA and may be a key regulator of tumor macrophages infiltration and may be involved in M2 macrophage polarization. Our study provided new insights into the prognosis and immunotherapy of BLCA patients through macrophage infiltration and immune checkpoints.

Background CD44 is the most common surface marker of CSCs (cancer stem cells) and plays a key role in the communication of CSCs with the microenvironment and the regulation of stem cell properties. Methods UALCAN was used to analyze the expression of CD44 in BLCA and normal tissue. Kaplan–Meier plotter and UALCAN were utilized to analyze the prognostic value of CD44 in BLCA. The TIMER database was used to explore the relationship between CD44 and tumor-infiltrating immune cells. The expression of CD44, the macrophage marker (CD68⁺), and the M2-type marker (CD163⁺) was evaluated using immunohistochemistry in 15 BLCA samples. GeneMania and Metascape were used to analyze protein-protein interaction investigations and functional enrichment analysis Results We found that patients with bladder cancer with high CD44 expression had worse survival than those with low CD44 expression (P < 0.05). Furthermore, CD44 expression is significantly associated with PD-L1 expression. Immune infiltration analysis showed that CD44 expression levels in BLCA were significantly correlated with immune infiltration levels of different immune cells. And the analysis showed that infiltration of macrophages means a worse prognosis for patients. Immunohistochemical (IHC) staining results further confirmed that the expression of CD44 was positively associated with the expression of CD68⁺ and CD163⁺ (P < 0.05). Conclusion Our results suggest that CD44 may be a potential immunotherapeutic target for bladder cancer, and macrophage infiltration in bladder cancer has prognostic value for bladder cancer patients. CD44 may be a key regulator of tumor macrophage infiltration and may be involved in M2 macrophage polarization. Our study sheds new light on prognosis and immunotherapy in bladder cancer patients by targeting macrophage infiltration and immune checkpoints.