Characterization and proteomic analysis of plasma-derived small extracellular vesicles in locally advanced rectal cancer patients.

Background: Neoadjuvant chemoradiotherapy (nCRT) stands as a pivotal therapeutic approach for locally advanced rectal cancer (LARC), yet the absence of a reliable biomarker to forecast its efficacy remains a challenge. Thus, this study aimed to assess whether the proteomic compositions of small extracellular vesicles (sEVs) might offer predictive insights into nCRT response among patients with LARC, while also delving into the proteomic alterations within sEVs post nCRT.

Methods: Plasma samples were obtained from LARC patients both pre- and post-nCRT. Plasma-derived sEVs were isolated utilizing the TIO₂-based method, followed by LC-MS/MS-based proteomic analysis. Subsequently, pathway enrichment analysis was performed to the Differentially Expressed Proteins (DEPs). Additionally, ROC curves were generated to evaluate the predictive potential of sEV proteins in determining nCRT response. Public databases were interrogated to identify sEV protein-associated genes that are correlated with the response to nCRT in LARC.

Results: A total of 16 patients were enrolled. Among them, 8 patients achieved a pathological complete response (good responders, GR), while the remaining 8 did not achieve a complete response (poor responders, PR). Our analysis of pretreatment plasma-derived sEVs revealed 67 significantly up-regulated DEPs and 9 significantly down-regulated DEPs. Notably, PROC (AUC: 0.922), F7 (AUC: 0.953) and AZU1 (AUC: 0.906) demonstrated high AUC values and significant differences (P value < 0.05) in discriminating between GR and PR patients. Furthermore, a signature consisting of 5 sEV protein-associated genes (S100A6, ENO1, MIF, PRDX6 and MYL6) was capable of predicting the response to nCRT, yielding an AUC of 0.621(95% CI: 0.454-0.788). Besides, this 5-sEV protein-associated gene signature enabled stratification of patients into low- and high-risk group, with the low-risk group demonstrating a longer overall survival in the testing set (P = 0.048). Moreover, our investigation identified 11 significantly up-regulated DEPs and 31 significantly down-regulated DEPs when comparing pre- and post-nCRT proteomic profiles. GO analysis unveiled enrichment in the regulation of phospholipase A2 activity.

Conclusions: Differential expression of sEV proteins distinguishes between GR and PR patients and holds promise as predictive markers for nCRT response and prognosis in patients with LARC. Furthermore, our findings highlight substantial alterations in sEV protein composition following nCRT.