Bai BJ, Xu DY, Chen L, Ding LY, Song ZF. Clinical value of carbon nanoparticles in identifying metastatic lymph nodes in colorectal cancer: A single-center observational study. World J Gastrointest Oncol 2026; 18(8): 122077 [DOI: 10.4251/wjgo.122077]
Corresponding Author of This Article
Zhang-Fa Song, MD, Doctor, Department of Colorectal Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, No. 3 Qinchun East Road, Hangzhou 310016, Zhejiang Province, China. songzhangfa@zju.edu.cn
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Oncology
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Bai BJ, Xu DY, Chen L, Ding LY, Song ZF. Clinical value of carbon nanoparticles in identifying metastatic lymph nodes in colorectal cancer: A single-center observational study. World J Gastrointest Oncol 2026; 18(8): 122077 [DOI: 10.4251/wjgo.122077]
Bing-Jun Bai, Deng-Yong Xu, Li Chen, Zhang-Fa Song, Department of Colorectal Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, Zhejiang Province, China
Bing-Jun Bai, Zhang-Fa Song, Zhejiang Key Laboratory of Biological Treatment, Hangzhou 310016, Zhejiang Province, China
Bing-Jun Bai, Zhang-Fa Song, Key Laboratory of Integrated Traditional Chinese and Western Medicine Research on Anorectal Diseases of Zhejiang Province, Hangzhou 310016, Zhejiang Province, China
Li-Ya Ding, Department of Pathology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, Zhejiang Province, China
Author contributions: Bai BJ and Xu DY were responsible for the study conception and design, as they are co-first authors; Chen L analyzed the data and wrote the manuscript; Ding LY and Song ZF critically revised the article for important intellectual content; all the authors reviewed and approved the final version to be published.
AI contribution statement: No AI tool was involved in the generation of research data, interpretation of results, or formulation of conclusions.
Institutional review board statement: The study was approved by the Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (No. 2024-0110).
Informed consent statement: Written informed consent was obtained from all participants.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
STROBE statement: The authors have read the STROBE Statement—a checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-a checklist of items.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at songzhangfa@zju.edu.cn. Consent for data sharing was not obtained but the presented data are anonymized and risk of identification is low.
Corresponding author: Zhang-Fa Song, MD, Doctor, Department of Colorectal Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, No. 3 Qinchun East Road, Hangzhou 310016, Zhejiang Province, China. songzhangfa@zju.edu.cn
Received: April 13, 2026 Revised: June 7, 2026 Accepted: June 17, 2026 Published online: August 15, 2026 Processing time: 118 Days and 6.1 Hours
Abstract
BACKGROUND
Carbon nanoparticles (CNPs) are increasingly utilized for tracing lymph nodes (LNs) in colorectal cancer (CRC) surgeries. However, the relationship between CNP-stained LNs and metastatic LNs remains unclear.
AIM
To evaluate the clinical utility of CNPs in identifying metastatic LNs in CRC.
METHODS
A single-center observational study was conducted, enrolling patients between January 2022 and March 2024. CNP suspension was injected preoperatively. All patients underwent radical resection with main (D3) LN dissection. Regional LNs and selected non-regional LNs were harvested following clinical guidelines. Stained and non-stained LNs were identified by microscopic examination.
RESULTS
A total of 146 CRC patients were enrolled, yielding 3212 regional LNs. Of these, 2789 (86.8%) were stained with CNPs and 423 (13.2%) were non-stained, predominantly located in the pericolic/perirectal region (79.7%). The metastatic rate in stained LNs was 2.8%, significantly lower than in non-stained LNs (13.9%, P < 0.001). Across all T stages, non-stained LNs had a significantly higher proportion of metastases compared to stained LNs (P < 0.001). Additionally, left-sided colon cancers showed a higher proportion of metastatic LNs among non-stained LNs than among stained LNs (P < 0.001).
CONCLUSION
CNP staining effectively traces LNs; however, metastatic involvement is more likely in non-stained LNs, particularly in left-sided colon cancer.
Core Tip: The clinical value of carbon nanoparticles (CNPs) staining for identifying metastatic lymph nodes (LNs) in colorectal cancer remains unclear. Our study demonstrates that while CNPs staining can effectively trace LNs, tumor metastasis is more frequently observed in non-stained LNs.
Citation: Bai BJ, Xu DY, Chen L, Ding LY, Song ZF. Clinical value of carbon nanoparticles in identifying metastatic lymph nodes in colorectal cancer: A single-center observational study. World J Gastrointest Oncol 2026; 18(8): 122077
Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide[1]. Despite advances in comprehensive treatment strategies, radical surgery remains the primary therapeutic approach for CRC. Standard lymphadenectomy is essential for achieving favorable oncological outcomes. There is broad consensus regarding the importance of lymphadenectomy techniques, including complete mesocolic excision and total mesorectal excision. These procedures aim to increase lymph node (LN) yield and improve survival outcomes[2,3]. Consequently, accurate identification of LNs is crucial for performing high-quality LN dissection.
Carbon nanoparticles (CNPs) have been widely used for LN tracing because of their unique physical properties. CNPs are nanoparticles with a diameter of approximately 150 nm that selectively enter the lymphatic system (diameter: 120-500 nm) rather than the vascular system (diameter: 20-50 nm). In addition, CNPs can migrate through lymphatic vessels after being phagocytosed by macrophages[4]. CNPs are primarily used for sentinel LN identification in breast cancer surgery and for parathyroid gland preservation during thyroid cancer surgery[5,6]. A recent review concluded that CNPs represent a safe and effective approach for LN tracing and tumor localization in CRC surgery[7]. Most previous studies have compared CNP-treated patients with control groups, focusing primarily on the number of harvested LNs or metastatic LNs[8-10]. These studies generally reported that the use of CNPs increases LN yield and may facilitate the detection of metastatic LNs. However, limited attention has been paid to LNs that remain unstained after CNP administration. Furthermore, few studies have performed a detailed analysis of individual LNs according to their staining status. The distribution and characteristics of CNP-stained and non-stained LNs across different LN groups remain inadequately investigated, resulting in an unclear understanding of the relationship between CNP staining and LN metastasis.
Therefore, this prospective clinical study enrolled patients who received preoperative CNP injection and analyzed individual LNs within each LN group according to their staining status. The findings may provide a deeper understanding of the clinical value of CNPs in CRC surgery and LN tracing.
MATERIALS AND METHODS
Study design
This study was a single-center, observational trial conducted at Sir Run Run Shaw Hospital between January 2022 and March 2024. The study protocol was approved by the Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (No. 2024-0110). Written informed consent was obtained from all participants.
Patients and procedure
The inclusion criteria were as follows: (1) Pathologically or clinically diagnosed CRC; (2) Preoperative endoscopic injection of CNP suspension; (3) Planned radical CRC resection; and (4) Age ≥ 18 years. The exclusion criteria were: (1) The presence of other malignancies; and (2) A history of colorectal surgery.
Each patient received an endoscopic injection of 0.5 mL of CNP suspension (Chongqing LUMMY Pharmaceutical Co., Chongqing, China) into the submucosal layer at a single site located at the distal margin of the tumor one day before surgery. The injection protocol consisted of sequential administration of 1 mL normal saline, followed by 2.5 mL of diluted CNP suspension (0.5 mL CNP mixed with 2 mL normal saline), and a final 1 mL normal saline flush. The procedure was performed by an experienced endoscopist. All patients underwent laparoscopic or robotic radical resection with main (D3) LN dissection. Tumor tissues and LNs were isolated from surgical specimens by the same pathologist. LN dissection and harvesting were performed according to the Japanese Classification of Colorectal Carcinoma[11]. Individual LNs from the three regional groups, including pericolic/perirectal (D1), intermediate (D2), and main (D3) LNs, were collected and analyzed separately. All specimens underwent formalin fixation, paraffin embedding, and hematoxylin and eosin (HE) staining for pathological evaluation. LN metastasis and CNP staining status were subsequently analyzed.
Statistical analysis
Continuous variables are presented as mean ± SD and were compared using Student’s t-test. Categorical variables were analyzed using Pearson’s χ2 test or Fisher’s exact test, as appropriate. Bonferroni correction was applied for multiple comparisons. A two-sided P value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25.0.0.2 (SPSS Inc., Chicago, IL, United States).
RESULTS
Clinicopathologic characteristics of patients
A total of 146 patients were recruited for the study. The baseline characteristics of the patients are summarized in Table 1. The proportion of males was 62.3%. The mean age of all patients was 64.9 ± 9.7 years. Each patient had sufficient LNs removed (mean ± SD, 22.0 ± 9.2).
Table 1 Clinicopathologic characteristics of patients, mean ± SD/n (%).
Clinical and pathological results of retrieved LNs
LNs were harvested according to the Japanese classification of colorectal carcinoma and divided into three groups: D1 region (pericolic/perirectal), D2 region (intermediate), and D3 region (main LNs) (Figure 1A and B). Stained and non-stained LNs were recorded and confirmed by microscopic observation (Figure 1C and D). As shown in Figure 1C, black particles were microscopically visible in the pathological sections of stained LNs. Further analyses were conducted based on CNP staining status and LN metastasis. Among 3212 retrieved regional LNs, 2789 (86.8%) were stained by CNPs, while 423 (13.2%) were non-stained, predominantly located in the D1 region (79.7%). The metastasis rate in stained LNs was only 2.8%, significantly lower than that in non-stained LNs (13.9%, P < 0.001) (Table 2). Metastatic LNs were primarily concentrated in the D1 region, regardless of staining status. Nine patients underwent non-regional LN dissection, resulting in the collection of 32 para-aortic LNs, among which 5 metastatic LNs were identified from 7 non-stained LNs (71.4%). In contrast, no metastatic LNs were found in the stained group. Among 38 tumor deposits, the vast majority (94.7%) were non-stained. Further analysis indicated that patients with tumor lymphovascular invasion (LVI) had a higher proportion of non-stained than stained LNs (21.5% vs 14.8%, P < 0.001).
The distribution of LNs in different T stages was analyzed in 137 patients, excluding 9 patients who underwent preoperative chemotherapy. As T stage increased, the proportion of patients with LN metastasis significantly increased from 10% in T1 stage to 70% in T4 stage (P < 0.001) (Supplementary Table 1). Correspondingly, the proportion of metastatic LNs also increased (P < 0.001) (Supplementary Table 1). Two patients in T3 stage and one patient in T4 stage (total 2.2%) had LN skip metastases (LNSM), defined as LN metastasis occurring at D2 or D3 regions without D1 region metastasis. Across all T stages, a higher proportion of metastatic LNs were observed in non-stained compared to stained LNs, specifically in T1 stage (14.2% vs 0.7%, P < 0.001), T2 stage (34.9% vs 1.3%, P < 0.001), T3 stage (8.5% vs 3.6%, P < 0.001), and T4 stage (24.5% vs 2.7%, P < 0.001) (Table 3). Similar results were observed in stratified analyses by anatomical subsites (Table 4). In left-sided colon cancer, the proportion of metastatic LNs in non-stained LNs was significantly higher than in stained LNs (P < 0.001). Moreover, the proportion of non-stained LNs from different LN groups varied depending on the tumor location (Supplementary Table 2).
Table 3 The distribution of lymph nodes according to different T stages, n (%).
As a foreign substance, CNPs were suspected of inducing inflammation in the intestinal wall or abdominal cavity. As shown in Figure 2A, an unskilled injection technique might result in CNP leakage into the abdominal cavity. In contrast, the “Sandwich” method effectively contained CNPs within the submucosal layer (Figure 2B). Next, intestinal mucosa stained with CNPs was processed into paraffin sections, alongside adjacent non-stained intestinal mucosa. A large number of CNPs were evenly distributed in the submucosal layer, with the crypt architecture remaining intact and well-organized. Compared to mucosa without CNPs, no evidence of interstitial edema was observed, and there was no significant increase in the infiltration of inflammatory cells or mononuclear cells (Figure 2C and D). Additionally, blood loss during surgery, operative time, and postoperative complications were recorded. Postoperative complications occurred in 18 (12.3%) patients (Table 5).
Figure 2 Submucosal injection of carbon nanoparticle suspension.
A: Traditional injection technique: 0.5 mL carbon nanoparticle (CNP) suspension mixed with 4 mL normal saline is endoscopically injected into the submucosal layer at four points. Leakage of CNP into the abdominal cavity is observed; B: “Sandwich” injection technique: 1 mL normal saline, followed by 2.5 mL CNP suspension (0.5 mL CNP mixed with 2 mL normal saline), and another 1 mL normal saline. The abdominal cavity remains clean, with no CNP suspension leakage; C: Hematoxylin and eosin (HE) staining of intestinal wall stained with CNPs; D: HE staining of intestinal wall without CNP staining.
Table 5 Intra-operative characteristics and post-operative complications, mean ± SD/n (%).
To our knowledge, this is the first study to comprehensively characterize CNP staining in individual LNs and evaluate its clinicopathological significance in CRC. By investigating the relationship between CNP staining and LN metastasis, we found that tumor metastases were more frequently detected in non-stained LNs.
The primary role of CNPs is LN tracing. In thyroid and breast cancers, CNPs are widely used in sentinel LN dissection to guide subsequent treatment based on the pathological status of sentinel LNs[12,13]. Several studies have also explored the application of CNPs in gastric and cervical cancers[14,15]. In CRC, CNPs can visualize both regional and non-regional LNs, enabling more accurate lymphadenectomy. Because of their LN-tracing capability, some researchers have suggested that CNPs facilitate the retrieval of more LNs and, consequently, more metastatic LNs, potentially leading to improved prognosis[9,10,16,17]. In these studies, the CNP group included more patients with positive LNs than the control group. Furthermore, a recent meta-analysis showed that CNPs increased the proportion of cases with more than 12 harvested LNs[18]. Based on these findings, it was concluded that the LN-tracing ability of CNPs facilitated the identification of metastatic LNs. However, this conclusion may be inaccurate. Our results suggest that CNP staining does not confer an advantage in identifying or removing metastatic LNs. Both stained and non-stained LNs were observed in patients who received CNP injection. Therefore, analysis at the individual LN level is necessary to clarify the relationship between CNP staining and LN metastasis.
To address this issue, every retrieved LN was collected and analyzed individually. Interestingly, we observed the opposite pattern. Our data showed that metastatic LNs were less likely to be stained than non-metastatic LNs. Among regional LNs, the proportion of metastatic LNs in the stained group was very low (2.8%), whereas the metastasis rate reached 13.9% in non-stained LNs. Stratified analyses further demonstrated that the proportion of metastatic LNs was consistently higher in non-stained than in stained LNs across all T stages and in left-sided colon cancer. This difference was even more pronounced in non-regional LNs, where all stained LNs were free of metastasis. Therefore, the assumption that LN dissection guided by stained LNs results in the removal of more metastatic LNs may be incorrect. Instead, non-stained LNs warrant greater attention. This seemingly counterintuitive finding may represent the clinical value of CNPs in identifying metastatic LNs. Compared with patients without LVI, those with positive LVI exhibited a higher proportion of non-stained LNs. We hypothesize that tumor cells may occupy the space within LNs or lymphatic vessels, thereby impeding CNP transport or macrophage migration. However, this hypothesis requires further pathological validation. Therefore, CNP staining itself cannot be used to identify metastatic LNs. Conversely, the absence of staining may serve as a positive predictor of LN metastasis.
The distribution of non-stained LNs may be associated with lymphatic drainage patterns and the distance between the LN and the primary tumor. According to our findings, non-stained LNs were predominantly concentrated in the D1 region (79.7%). In general, the proximal resection margin is located more than 10 cm from the tumor, and therefore LNs in mesenteric areas distant from the tumor may be less likely to be stained. In contrast, because paracolic lymphatics ultimately drain into the D3 region, LNs in the D3 region were frequently stained. In addition, tumor deposits were rarely stained, likely because they arise from LVI and lack recognizable LN structures.
LN metastasis in CRC varies substantially across T stages and LN regions. In the present study, the proportions of LN metastasis across different T stages were consistent with those reported previously[19-21]. Although the metastasis rate in the D3 region is relatively low, routine D3 Lymphadenectomy is still strongly recommended to optimize patient outcomes[22]. LNSM is a relatively rare but clinically important pattern of LN metastasis in CRC. Previous studies have reported that LNSM is an independent prognostic risk factor in stage III colon cancer[23]. Therefore, high-quality lymphadenectomy remains critically important. In this context, the LN-tracing capability of CNPs may improve the accuracy and completeness of lymphadenectomy.
Whether CNPs increase surgery-related complications remains controversial. It has been speculated that leakage into the abdominal cavity may induce inflammation or intestinal adhesions. In addition, residual CNPs at the anastomotic site have been suspected to increase the risk of anastomotic leakage. These concerns are common among surgeons. However, previous studies have shown that the overall complication rate is similar between the CNP and control groups (10%-20%)[7]. First, injection-related adverse events, including inflammation and perforation, were not observed in patients receiving CNPs. Furthermore, no significant differences were reported between the CNP and control groups in postoperative complications, including anastomotic leakage, ileus, abdominal infection, and intraoperative blood loss. Our findings further support these observations through both clinical and pathological assessments. The overall complication rate in our cohort was 12.3%, consistent with previous studies[7]. Moreover, no obvious inflammatory changes were observed on HE-stained sections of intestinal mucosa containing CNPs. Nevertheless, because CNPs are foreign materials, meticulous injection techniques are recommended to prevent intraperitoneal leakage. Residual CNPs at the anastomotic site often indicate an inadequate surgical margin and should also be avoided.
Nowadays, the concept of LN dissection is evolving. The lymphatic system should not be regarded merely as a passive route for metastatic spread. Regional LNs play a critical role in antitumor immunity[24]. Extensive lymphadenectomy may not provide additional benefit for certain patients with CRC. Inamori et al[25] reported that the dissection of more than 12 LNs was associated with worse long-term prognosis in microsatellite instability-high/deficient mismatch repair CRC. Fear et al[26] proposed that LN resection might reduce the efficacy of adjuvant immunotherapy. As the relationship between lymphadenectomy and oncological outcomes becomes better understood, future cancer surgery may move toward selective LN dissection, for which CNPs may serve as a useful adjunct. LNs are secondary lymphoid organs that function as reservoirs of antitumor immune cells[27]. Therefore, in the era of immunotherapy, the role of LNs cannot be overlooked. Despite their importance, the underlying biological mechanisms remain poorly understood. Owing to the LN-tracing capability of CNPs, fresh LN tissues can be harvested more efficiently for subsequent cellular and molecular studies.
Several limitations should be acknowledged. This was a single-center study with a relatively small sample size. Long-term oncological outcomes were unavailable, and the majority of patients had left-sided colon cancer or rectal cancer. Future prospective randomized controlled trials with larger sample sizes are needed to improve the accuracy and reliability of the findings.
CONCLUSION
In conclusion, CNP injection is a feasible and safe method for LN tracing in CRC. Although CNP staining cannot directly identify metastatic LNs, tumor metastasis is more likely to occur in non-stained LNs, especially in left-sided colon cancer.
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Footnotes
Peer review: Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Oncology
Country of origin: China
Peer-review report’s classification
Scientific quality: Grade B, Grade C
Novelty: Grade B, Grade C
Creativity or innovation: Grade B, Grade C
Scientific significance: Grade B, Grade B
P-Reviewer: Yang WY, MD, China; Yang Y, MD, Postdoc, China S-Editor: Fan M L-Editor: A P-Editor: Zhao S
