Colorectal cancer (CRC) is a common malignancy and the fourth leading cause of cancer-related deaths globally (1). If diagnosed in the early stage, the 5-year survival rate of CRC patients is as high as 70%–90% (2). Nevertheless, CRC patients with tumor metastases present a worse prognosis, with a 5-year survival rate of only approximately 20% (3). Furthermore, due to changes in people’s dietary and lifestyle habits, a growing number of patients with CRC are diagnosed at an advanced stage, which leads to challenging therapeutic resection of primary tumors and metastases (4).

Consequently, improving the prognosis of CRC patients largely depends on early and accurate diagnosis. At present, colonoscopy and tissue biopsy are the most efficient methods for CRC screening (5). Nonetheless, colonoscopy is an invasive procedure that can be traumatic for subjects, and the whole operation is occasionally hard to complete due to poor compliance of patients with CRC (2). Additionally, considering the invasiveness and cost of these operations, it is impractical to perform comprehensive screening as part of a general physical examination. Therefore, there is an urgent demand for more noninvasive and efficacious biomarkers for clinical diagnosis. Over recent years, the identification of blood biomarkers has become an important issue because of the pain-free operation of blood biomarkers testing (6). Blood biomarkers such as carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA) are broadly applied for CRC detection (7, 8). Yet, these three biomarkers, alone or in combination, are not sufficient for diagnosing CRC due to their poor specificity and sensitivity (8, 9). Hence, there is an urgent need to optimize the diagnosis of CRC by other efficient blood biomarkers.

N6-methyladenosine (m⁶A) modification, which was encoded by the methyltransferase complex consisting of “writers”, “erasers”, and “readers”, has emerged as a critical regulator in a multitude of diseases (10, 11). The modification of m⁶A is enriched close to the 3′ untranslated terminal region (UTR) and the stop codon, thus influencing RNA transcription, processing, and translation (12, 13). Over recent years, activation of m⁶A modification has been reported in CRC tumor cells (10, 13). Upregulated m⁶A modification contributes to tumor progression by maintaining SOX2 expression in CRC cells through IGF2 mRNA binding proteins 2 (IGF2BP2)-dependent mechanisms (14, 15). Moreover, activating the glycolytic pathway by m⁶A methylation promotes CRC tumorigenesis, indicating that m⁶A modification of CRC tumor cells might become a therapeutic target (16, 17). Besides, the m⁶A-modified status of peripheral blood has been recently reported as a new promising hallmark in diabetes and gastric cancer (18, 19). Nevertheless, whether the m⁶A modification of peripheral blood RNA may act as a new diagnostic biomarker or therapeutic target for CRC remains unclear.

In this study, we examined the levels of m⁶A in peripheral blood RNA of CRC patients and NCs to assess its value as a diagnostic biomarker. We also used bioinformatics, which revealed that elevated m⁶A levels were mainly associated with monocytes and suppressed their immune response, indicating that m⁶A modifications of peripheral blood immune cells might become a therapeutic target for CRC.

Most patients are already at an advanced stage by the time they are diagnosed with CRC, which substantially contributes to the poor prognosis (4). Hence, improving the prognosis of CRC patients depends on an early and accurate diagnosis. However, the currently used clinical tumor biomarkers for CRC such as CEA, CA125, and CA19-9 are not specific or sensitive enough to detect CRC patients (7, 9). Therefore, optimizing the diagnosis of CRC with other validated biomarkers is of urgent importance. The present study identified the m⁶A status of peripheral blood immune cells as a novel marker for CRC screening. In addition, it might also serve as a new target for CRC treatment.

Despite a growing body of reports that have linked m⁶A dysregulation to various cancers, the role of m⁶A modifications in CRC tumor tissues remained controversial (10, 20). Stimulating m⁶A modification promotes β-catenin translation to drive the epithelial–mesenchymal transition of CRC cells, while some studies found that m6A regulation suppresses proliferation and metastasis (15, 21, 22). Our research revealed for the first time that the m⁶A RNA levels of peripheral blood immune cells were dramatically higher in patients with CRC than in healthy subjects ( Figure 1A ). Our results demonstrated that m⁶A RNA was more strongly modified in peripheral blood immune cells of CRC, yet m⁶A modification in CRC tumor tissue needs to be further explored. Additionally, the m⁶A status of peripheral blood immune cells was substantially elevated in CRC patients with distant metastases compared to those without metastases, implying that it could also discriminate if the tumor had metastasized ( Figures 1B, C ). Although the m⁶A levels were reduced in treated CRC patients, more clinical samples were requested to determine whether they could be used as an indicator of oncologic efficacy, such as relapse and drug resistance ( Figures 1D, E ). It has been discussed that the m⁶A levels might be applied as a biomarker for gastric cancer, but the regulation of m⁶A modification in different tumors varied significantly (18, 23). Therefore, it is worthwhile to investigate further whether the m⁶A levels had diagnostic value in other tumors.

CEA, CA125, and CA19-9 are widely used in physical screening for CRC (9). Nevertheless, due to their poor specificity and sensitivity, these three biomarkers alone or in combination are not sufficient to diagnose CRC (7). As shown in Figure 2 , the AUC for m⁶A to differentiate CRC patients from healthy subjects was 0.946 (95% CI, 0.914–0.977), which was significantly higher than the AUC for CEA (0.817; 95% CI, 0.754–0.881), CA125 (0.732; 95% CI, 0.659–0.806), and CA19-9 (0.771; 95% CI, 0.700–0.842). The combination of CEA, CA125, and CA19-9 with m⁶A further increased the AUC to 0.977 (95% CI, 0.961–0.994). Besides, forest plots from multiple logistic regression analysis showed that the m⁶A levels were an independent risk factor associated with the diagnosis of CRC compared to these common tumor biomarkers ( Figure 2D ). Our study presented a considerable challenge to the value of these tumor biomarkers.

“Writers”, “erasers”, and “readers” together formed the methyltransferase complex responsible for m⁶A modification. Wilms tumor 1-associated protein (WTAP), Methyltransferase-like 3 (METTL3), and METTL14 were classified as “writers” catalyzing the formation of m⁶A (24–26). AlkB homolog 5 (ALKBH5) and Fat mass and obesity-associated protein (FTO) represented “erasers”, meaning they could induce selective removal of methylation code from the target mRNA (27, 28). “Readers” were able to decode m⁶A modification, comprising YT521-B homology domain-containing protein (YTHDF) as well as IGF2BP families (16, 29). m⁶A modifications altered the expression of target genes and changed the consequent biological features (30). To further understand the role of the elevated m⁶A levels in CRC tumor progression, we screened for the most variable “writers”, “erasers”, and “readers” in CRC peripheral blood immune cells by limma differential analysis. Members of the IGF2BP family (IGF2BP1, IGF2BP2, and IGF2BP3), which belonged to “readers”, were the most markedly changed molecules in the methyltransferase complex ( Figure 3 ). Simultaneously, IGF2BP2 revealed the greatest increase, thus suggesting a potentially crucial role in peripheral blood immune cell m⁶A modification ( Figure 3 ). Unlike other readers, IGF2BPs acted as a unique family of m⁶A readers that target a multitude of mRNA transcripts and enhance the conservation and stability of their candidate mRNAs in an m⁶A-dependent way (14, 15, 31). Our study further demonstrated that elevated IGF2BP2 might interact with several essential genes to negatively regulate immunity, such as cytokine production and chemotaxis ( Figure 5 and Supplementary Figure 3 ). Although we found that increased IGF2BPs expression combined with elevated m⁶A levels affected cancer immunity in CRC, we have not yet clarified the mechanism of increased IGF2BPs, which is also the biggest limitation of the current study. Taken together, m⁶A modification and IGF2BPs expression were likely to be novel targets for CRC treatment, but further in vivo experimental studies are required.

Previous studies reported that elevated m⁶A levels of peripheral blood in patients with gastric cancer might be due to downregulation of FTO and ALKBH5, which belonged to “erasers” (18). Our qRT-PCR results also revealed a slight downregulation of FTO and ALKBH5 in peripheral blood cells of CRC patients, partially explaining the increased m⁶A levels ( Supplementary Figure 4 ). Other unknown methylases and demethylases may also be involved in the changes of m⁶A levels that deserved further exploration (32). Additionally, monocytes were identified as the immune cells most strongly associated with the increased regulation of upregulated m⁶A levels in peripheral blood of CRC patients ( Figure 4 ). It has been noted that the presence of a large number of m⁶A-modified infiltrating immune cells in the tumor tissue microenvironment promotes tumor progression (33, 34). Furthermore, imbalanced m⁶A regulation strongly conferred immune disruption and tumor evasion, primarily by affecting immune cell migration, rather than apoptosis or survival (35). These observations were generally consistent with our findings in peripheral blood immune cells. Moreover, the number of monocytes in the CD14⁺CD16⁺HLA-DR^hi subpopulation of patient’s peripheral blood was found to be the most accurate predictor of progression-free survival and overall survival after receiving PD-1 inhibitor therapy (36). Whether the subset of monocytes with elevated m⁶A levels had a similar role in tumor immunotherapy to the CD14⁺CD16⁺HLA-DR^hi subset deserves further investigation.

In conclusion, the highlights of our research were the first identification of m⁶A RNA levels in peripheral blood immune cells as a novel biomarker for the diagnosis of CRC and the provision of a new strategy for the treatment of CRC by targeting m⁶A levels or IGF2BPs expression in peripheral blood immune cells.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Zhongshan People’s Hospital. The patients/participants provided their written informed consent to participate in this study.

HY, HH, and CL conceived and designed this study. JX, ZH, and PJ performed the experiments and analyzed the data. RW and HJ contributed to the data analysis and discussion. All authors contributed to the article and approved the submitted version.

This study was supported by the fund from the National Nature Science Foundation of China (81900775; 81902693); Educational Commission of Guangdong Province (2017KTSCX155); Guangdong Basic and Applied Basic Research Foundation (2019A1515011318); Natural Science Foundation of Guangdong Province (2018A030310298); the Science Foundation of Guangzhou First People’s Hospital (Q2019004; KYQD0046); China Postdoctoral Science Foundation (2019M662991); Key Medical and Health Projects of Zhongshan City (2020K0012); Guangzhou Science and Technology Planning Project (202102020142).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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