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Page 2 of 15 Chen et al. Cancer Drug Resist 2024;7:9 https://dx.doi.org/10.20517/cdr.2023.151

Conclusion: Our findings indicate that circNCOA3 might be useful as a potential biomarker to predict the efficacy
and prognosis of CRC patients treated with anti-PD-1 therapy.

Keywords: circRNA, circNCOA3, colorectal cancer, anti-PD-1 therapy, immune evasion

INTRODUCTION
Colorectal cancer (CRC) is one of the most commonly diagnosed tumors and a leading cause of cancer-
[1]
related deaths in the world . Thanks to the development of novel treatment strategies in recent years, the
mortality rate of CRC is declining. However, the prognosis of CRC is still unsatisfactory, especially in
[2]
patients with advanced stages or old age . Currently, the main treatment option for advanced CRC patients
is chemotherapy combined with targeted therapy . However, drug resistance is common in CRC and
[3]
affects the therapeutic efficacy . A growing body of literature indicates that escape from immune
[4,5]
surveillance is critical for cancer growth and progression . Clinically, it has been confirmed that immune
[6]
checkpoint inhibitors, especially programmed cell death 1 (PD-1) antibody therapy, are effective in various
cancers . For CRC, anti-PD-1 therapy is efficacious in metastatic microsatellite-instability-high (MSI-H)
[7,8]
or mismatch-repair-deficient (dMMR) tumors [9-11] . The KEYNOTE-177 phase III clinical trial proved the
effectiveness of PD-1 antibody therapy in first-line treatment of CRC. Nevertheless, about 30% of MSI-H/
dMMR CRC patients display primary resistance to PD-1 antibody treatment . Among patients with
[11]
microsatellite stable (MSS) CRC, the objective response rate (ORR) to PD-1 antibody treatment is nearly
0% , and it could be increased with the combination of Regorafenib. Previous studies explored the
[12]
association of some factors with primary resistance, including a relatively low tumor mutational burden,
elevated systemic inflammation, heterogeneity of MSI-H/dMMR, and tumor intrinsic metabolic
reprogramming [13-16] . However, the underlying molecular mechanism of anti-PD-1 resistance in CRC
remains virtually unknown.

Circular RNAs (circRNAs) were identified as a novel type of non-coding RNAs or protein-coding RNAs
with covalently closed loops and lack 5’ to 3’ polarity or polyadenylation tail . circRNAs generally arise
[17]
from alternative back-splicing of pre-mRNA transcripts and are stable, conserved, and expressed in multiple
[18]
cancer cells and tissues . circRNAs have been found to be involved in various aspects of tumor formation,
such as proliferation and metastasis [19,20] . Recent studies found that circRNAs participate in tumor immune
microenvironment regulation and mediate PD-1 antibody responsiveness . However, the role of circRNAs
[21]
in CRC immune regulation and PD-1 antibody responsiveness has not been established.

METHOD
Cell lines and tissues
For RNA microarray and real-time PCR analysis, paraffin-embedded or fresh-frozen tissues, or serum were
obtained from metastatic CRC patients who underwent colonoscopy with biopsies or surgery and PD-1
inhibitor treatment at Sun Yat-sen University Cancer Center (SYSUCC) between July 2018 and May 2022.
Response rates were determined based on the RECIST 1.1 guideline. The study was approved by the ethics
committee of the SYSUCC, and informed consent was obtained from each patient. Progression-free survival
(PFS) and overall survival (OS) were assessed from the date of PD-1 antibody therapy to the date of
progression (PFS) or death from any cause or last contact (OS).

CRC cell lines (HT29, SW620, HCT116, SW480, DLD-1, LoVo), normal colon epithelial cell NCM460, and
mouse MC38 colon cancer cells and HEK 293T cells were obtained from the cell bank of the Shanghai
Institute of Cell Biology (Shanghai, China). The cell lines were maintained and cultured following the
supplier’s protocols.