Abstract
Regulatory T cells (Tregs) are often enriched in tumors, where their immunosuppressive function has a key role in tumor persistence and progression. In colorectal cancer (CRC), however, Tregs are frequently associated with an improved clinical outcome. Tumor-infiltrating Tregs have been shown to exhibit a distinct signature comprising the co-stimulatory molecules (OX40, 4-1BB), cytokine receptors (IL1R2, IL21R, CCR8, CD30), and co-inhibitory molecules (PD-L1, TIGIT). Here, we showed by flow cytometry that circulating CD45RO+ Tregs from patients with CRC (n = 25) have elevated CD30 and OX40 expression compared to healthy subjects (n = 14). We identified co-expression of CD30 and OX40 on circulating CD45RO+ Tregs using single-cell images captured by the DEPArray™ system. The frequency of CD30+OX40+CD45RO+ Tregs was significantly higher in CRC patients than in healthy subjects (P < 0.001). Importantly, receiver operating characteristic analysis confirmed that this CD30+OX40+ Treg subset could strongly discriminate between CRC patients and healthy subjects with the highest accuracy of 92.3%, an AUC of 0.92, a sensitivity of 88%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 82.35%, and a trade-off value of 3.44%, compared to other Treg subsets. Consistently, multiplex-IHC/IF of tumor-infiltrating Tregs revealed a significant association between high densities of CD30+OX40+ Tregs and improved overall survival; no such association was found for other subsets. These data suggest a potential role for CD30+OX40+ Tregs as a diagnostic or prognostic biomarker in CRC.
Highlights
Colorectal cancer (CRC) is a major health and socioeconomic problem worldwide
De Simone et al [21] reported that specific cytokine receptors and co-signaling molecules are differentially expressed by tumor-infiltrating Tregs in CRC patients recruited in Italy., Based on these findings, we set out to ascertain whether CRC patients from our South-East Asian cohort expressed these markers
Gating on CD45RA−CD45RO+ circulating Tregs, we determined that PD-L1, IL-1R2, IL-21R, and CCR8 were generally absent (Supplementary Fig. 1a) and that the proportions of expressing cells were similar between CRC patients and healthy subjects
Summary
Two cohorts of CRC patients were studied: (1) a prospective cohort of 30 patients for FCM analysis, and (2) a retrospective cohort of 217 patients for tissue microarray and multiplex-IHC/IF studies. MIHC/IF was performed using an Opal Multiplex fIHC kit (Akoya Bioscience, Menlo Park, California, USA), as previously described [23,24,25,26,27,28,29,30,31,32,33,34,35]. FFPE tissue sections were cut onto Bond Plus slides (Leica Biosystems, Richmond, Illinois, USA) and heated at 60 °C for 20 min [36]. Following TSA deposition, slides were again subjected to HIER to strip the tissue-bound primary/secondary antibody complexes and ready for labeling of the marker. These steps were repeated until all markers were labeled. Survival outcomes were estimated with the Kaplan–Meier analysis and compared between groups by Cox regression adjusted for age at diagnosis, and the grade and stage of cancer. *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001; ns: P > 0.05
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
