Fig 2: Identification of CD44 was a critical protein mediating exosome- transmission of LNM phenotype (A) A flow chart shows the identification of differential protein profile between HGC-27-exosomes and AGS-exosomes by label-free quantitation; (B) CD44 protein detection in different GC cell-exosomes; (C) Comparison of CD44 protein in GC cells; (D) Change of CD44 protein levels in AGS after treatment with exosomes; (E) Effect of CHX pretreatment on CD44 protein in AGS incubation with HGC-27-exosomes; (F) Screening for the most efficient si-CD44 in HGC-27 and HGC-27-L; (G–J) Effect of CD44 knockdown on the migration and invasion capacity (G, H) and FAO (I, J) of HGC-27 and HGC-27-L; (K) CD44 protein detection in exosomes derived from HGC-27 and HGC-27-L with CD44 knockdown; (L–O) Effect of CD44-less exosomes on migration and invasion capacity (L, M) and FAO (N, O) of AGS; (P–R) Effect of CD44-less exosomes on LNM capacity of AGS in vivo. Ruler unit, mm. CHX, cycloheximide; ex, exosomes. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Exosomal CD44 increased FAO possibly via regulating the RhoA/YAP/Prox1/CPT1A signaling axis (A–C) Western blotting of CPT1A, RhoA, YAP, phosphorylated-YAP (p-YAP) and Prox1 in GC cells (A), exosome-treated AGS (B) and si-CD44-transfected HGC-27(C); (D) Protein analysis in Verteporfin (VP) treated HGC-27; (E–H) Effect of VP treatment on FAO (E, F), migration and invasion capacity (G, H) of HGC-27 and HGC-27-L cells; (I–L) Effect of VP pretreatment on lymphatic GC cell-exosomes increasing FAO (I, J), migration and invasion capacity (K, L) of AGS; (M–O) Effect of VP pretreatment on HGC-27-exosome enhancing LNM capacity of AGS in vivo. Ruler unit, mm. ex, exosomes. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Schematic overview showing lymphatic metastatic GC cell-exosomal CD44 increases FAO to confer LNM phenotype on primary GC cells via the RhoA/YAP/CPT1A signaling axis. Metastatic GC cells in LNs secrete CD44-rich exosomes, which are taken up by GC cells in primary GC tissues and upregulate its intracellular CD44 protein. CD44 then increases RhoA expression, stimulates YAP expression, de-phosphorylation and nuclear translocation. YAP and TAZ function as transcriptional co-factors to promote Prox1 transcription and protein generation. Prox1 further translocates into nuclei and increases CPT1A transcription. CPT1A protein upregulation promotes FAO reprogramming and increases cell migration and invasion, leading to acquiring LNM capacity by primary GC cells. CD44, YAP and CPT1A might be potential therapeutic targets to suppress exosome-mediated LNM phenotype transfer from lymphatic metastatic GC cells to primary GC cells. LNs, lymph nodes.