Panoply™ Human CD40 Knockdown Stable Cell Line

CD40 expression in esophageal cancer (EC) is associated with poor prognosis, although its molecular role remains unclear. Here, researchers explored the role of CD40 in EC progression and metastasis, focusing on its interaction with CD154 and upregulation of MMP-9. CD40 expression in EC cell lines was confirmed using quantitative PCR, western blotting, flow cytometry, and immunocytochemistry. Functional assays demonstrated that stimulation with recombinant soluble CD154 enhanced the migration and invasion of CD40-overexpressing EC cells without affecting their viability. Co-culture experiments with platelets demonstrated that platelet-derived CD154 acted on CD40-overexpressing esophageal cancer cells, resulting in upregulated MMP-9 secretion, which may drive tumor invasion. Analysis of serum from patients undergoing esophagectomy revealed that lower MMP-9 levels were associated with longer survival in patients with pathological stage I, while the opposite trend was observed in patients with stages II-IV. These results suggest that activation of CD40 enhances tumor cell invasiveness through upregulation of MMP-9. This dual role of CD40, enhancing antitumor immunity through expression on antigen-presenting cells while promoting tumor invasion through secretion of MMP-9 when expressed on esophageal cancer cells, may complicate immunotherapeutic strategies targeting CD40, as such interventions may inadvertently promote malignancy in the tumor microenvironment.

To clarify the role of the CD40 gene in TE cells, the researchers constructed CD40 knockdown TE-10 cells. TE-10 cells were transfected with si-CD40 or si-control to generate CD40 knockdown TE-10 cells and control TE-10 cells (Mock), respectively. Then, the knockdown efficiency was evaluated by measuring CD40 mRNA levels by qPCR (Figure 1A). In addition, the researchers also evaluated the response of these cells to sCD154 stimulation by measuring MMP-9 mRNA levels. Regardless of sCD154 stimulation or not, CD40 knockdown TE-10 cells always maintained low MMP-9 mRNA levels (Figure 1B). In addition, migration and invasion experiments were performed using CD40 knockdown TE-10 cells. The results showed that CD40 knockdown TE-10 cells exhibited reduced migration and invasion abilities compared with control cells (Figure 1C and D).

Figure 1. Experiments using CD40-knockdown TE-10 cells using siRNA. (Umemoto K, et al., 2025)