Effects of low-density lipoprotein cholesterol on lymph node metastasis after radical esophagectomy

Abstract

BACKGROUND

Esophageal cancer (EC) is one of the most common malignancies worldwide, and lymph node (LN) metastasis remains one of the leading causes of EC recurrence. Metabolic disorders critically affect cancer progression, and lipid levels are closely associated with the occurrence of EC and several other tumor types. This study analyzed pretreatment lipid levels to determine their association with LN metastasis.

AIM

To dissect the possible mechanisms underlying LN metastasis and clarify the prognostic role of lipid profiles in EC.

METHODS

Serum lipid levels and clinicopathological information were retrospectively collected from 294 patients, and risk factors for LN metastasis were confirmed using a logistic regression model. Latent factors were explored using information from publicly accessible databases and immunofluorescence and immunohistochemical staining techniques.

RESULTS

High serum levels of low-density lipoprotein (LDL) cholesterol promote LN metastasis in EC, while high-density lipoprotein cholesterol has the opposite role. Information of a public database revealed that LDL receptors LRP5 and LRP6 are highly expressed in ECs, and LRP6 overexpression positively correlated with the infiltration of B lymphocytes and a poor prognosis. Immunofluorescence and immunohistochemical staining revealed that the expression of LRP6 and infiltrated B lymphocytes in patients with ≥ 1 regional LN metastasis, containing N1-3 (N+ group) were significantly higher than those in the N0 group. LRP6 was also highly expressed in the B lymphocytes of the N+ group. There was no difference in CXCL13 expression between the N+ and N0 groups. However, CXCR5 expression was significantly higher in the N0 group than in the N+ group.

CONCLUSION

High serum LDL levels can promote LN metastasis in EC, and the mechanisms may be related to LRP6 expression and the infiltration of B lymphocytes.

Key Words: Esophageal cancer; Lymph node metastasis; Low-density lipoprotein cholesterol; B lymphocytes

Core Tip: This study investigated the relationship between pretreatment serum levels of low-density lipoprotein cholesterol (LDL-c) and lymph node (LN) metastasis in patients with esophageal cancer (EC) undergoing radical esophagectomy. Analysis of data from 294 patients revealed that elevated LDL-c levels significantly promoted LN metastasis, in contrast to the protective role of high-density lipoprotein. Further exploration using public databases and immunohistochemical techniques identified LRP5/6, particularly LRP6, as highly expressed in EC tissues, with LRP6 overexpression correlating with increased B-lymphocyte infiltration and adverse prognosis. Notably, patients with regional LN metastasis (N+ group) exhibited significantly higher expression of LRP6 and infiltrated B lymphocytes than those without regional LN metastasis (N0 group). Although CXCL13 expression remained comparable between the groups, CXCR5 expression was notably higher in the N0 group. These findings suggest that high serum LDL-c levels facilitate LN metastasis in EC, potentially through mechanisms involving LRP6 expression and B lymphocyte infiltration, thereby offering novel insights into the prognostic role of lipid profiles in EC progression.

INTRODUCTION

Esophageal cancer (EC) is one of the ten most common malignancies worldwide, ranking seventh in terms of incidence and sixth in mortality[1]. China has the highest incidence of EC, accounting for over 50% of cases globally. It ranks sixth in incidence and fourth in mortality rates among all cancers[1,2]. With advances in diagnosis and therapeutics, the prognosis of patients with EC has improved; however, the overall survival outcomes remain disappointing. Previous studies have confirmed that bidirectional and skip lymph node (LN) spread is a key feature of EC metastasis and a crucial risk factor for unfavorable prognosis[3]. Even after comprehensive treatment, LN metastasis remains the primary cause of EC recurrence[4]. Therefore, the prevention or intervention of LN metastasis is an important approach for improving the prognosis of patients diagnosed with EC.

Lipids play key roles in various biological processes, such as cell growth. Several studies have shown that serum lipid levels are closely associated with the occurrence of EC and other tumor types[5-7]. Serum lipids usually refer to total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides (TG), apolipoprotein A1 (Apo-A1), apolipoprotein B (Apo-B) and lipoprotein a (LPa). Dyslipidemia is defined as a cluster of lipoprotein abnormalities, usually characterized by the presence of high levels of TG, TC, and LDL; low levels of HDL; and abnormal apolipoproteins. Previous studies have shown that high serum levels of TC, LDL, and TG are risk factors for colorectal, breast, prostate, and ECs cancers, and are closely related to tumor progression and metastasis. In contrast, high serum HDL levels are associated with better prognosis[8-11]. Currently, research on Apo-A1, Apo-B, and LPa is mostly limited to cardiovascular diseases. Apo-A1 is also considered a better prognostic marker for EC[12,13]. A previous study investigating EC reported that high serum LDL levels were closely associated with LN metastasis, which may be explained by the evidence that LDL could promote the proliferation and differentiation of EC cells. Another study suggested that low serum LDL levels were associated with worse cancer prognosis[14,15]. Therefore, the diversity of lipid components and the complex tumor microenvironment have made it challenging to characterize the relationship between serum lipids and EC progression and clarify the underlying mechanisms.

Therefore, the present study retrospectively analyzed pretreatment lipid levels in patients diagnosed with EC to determine the association between serum lipid levels and LN metastasis, dissect the possible mechanisms underlying LN metastasis, and clarify the prognostic role of lipid profiles in EC.

MATERIALS AND METHODS

Patients

Data of 294 eligible patients diagnosed with EC who underwent radical esophagectomy at the First Affiliated Hospital of Xingtai Medical College (Hebei, China) between 2014 and 2019 were included in this retrospective study. The inclusion criteria were as follows: Previously diagnosed with EC and underwent radical surgery; TG, TC, LDL, HDL, Apo-A1, Apo-B, and LPa levels examined before surgery [without neoadjuvant chemotherapy (NAC)] or before NAC using a laboratory analyzer (BS2000M, Mindray, Shenzhen, China); and patients who did not take any drugs known to affect lipid levels. Individuals with a history of other cancers, diabetes, or endocrine or metabolic diseases that could influence blood lipid levels were excluded.

Gene expression profiling interactive analysis (GEPIA; http://gepia.cancer-pku.cn/) based on The Cancer Genome Atlas (TCGA), which contains a large amount of normal and cancer tissue RNA sequencing data, was performed to examine the mRNA expression levels of LDL receptor family members in EC and adjacent normal tissues and to determine the impact of LDL receptors on the prognosis of EC. The name of cancer was ESCA, and genes were labeled as LDLR, LRP8, LRP1, LRP2, LRP5, LRP6, LRP4, and LRP3, respectively, with Log2FC cut-off 1 and value cut-off 0.01 as normal TCGA and GTEx data were selected as the matched controls. Subsequently, the Tumor Immune Estimation Resource Version 2 (TIMER2) database (http://timer.cistrome.org/) was used to analyze the correlation between LDL expression and six types of infiltrating immune cells [B cells, dendritic cells (DC), CD4+ T cells, CD8+ T cells, macrophages, and neutrophils] in terms of tumor purity.

Follow-up assessment

All patients diagnosed with EC were followed up, with the last follow-up completed in August 2022. The primary outcome of this study was overall survival, which was defined as the interval between diagnosis and patient death or the last follow-up. Survivors were defined as those alive during the previous follow-up, whereas nonsurvivors were defined as those who died at any time during the study period. The tumor and clinical characteristics of patients with EC, including age, sex, T-staging, and N-staging, were obtained from medical records and pathology reports (Figure 1). Following the 8^th Edition of the TNM classification for EC, patients were divided into the following groups: N+ [patients with ≥ 1 regional LN metastasis (which contains N1-3)]; N0 [patients with no regional LN metastasis (as well as N0)]; T1 (tumor invasion of the lamina propria, muscularis mucosae, or submucosa); T2 (tumor invasion of the muscularis propria); T (tumor invasion of the adventitia); and T4 (tumor invasion of adjacent structures).

Figure 1 The process for the retrospective study. EC: Esophageal cancer; OS: Overall survival.

This study was approved by the Ethics Committee of the First Affiliated Hospital of Xingtai Medical College (No. 2022-01). Informed consent was obtained from all participants or their families if they could not undergo the informed procedure, and their survival status was verified through telecommunication.

Histological examination and immunofluorescence staining

Tumor tissues were harvested, embedded in paraffin blocks, and cut into tissue sections 4 µm thick. The presence of tumor cells was confirmed by hematoxylin and eosin staining. For immunofluorescence staining, the tumors were embedded and cut as described above. After antigen retrieval, the tissue sections were incubated with the primary antibody against LRP6 [sc-25317 (dilution 1:50), Santa Cruz Biotech, Dallas, TX, United States] and CD20 [ab64088 (dilution 1:100), Abcam, Cambridge, MA, United States] at 4 °C overnight, followed by incubation with FITC-conjugated secondary antibody and counter-staining with DAPI. Confocal fluorescence images were acquired using a laser scanning microscope (BX43; Olympus, Tokyo, Japan) equipped with a 20 × objective and analyzed using ISCapture software. Three different fields of view were examined for each specimen at 200-fold magnification, and immunofluorescence optical density (IOD) was quantified. Mean density was determined using the following equation: Mean density = IOD/area.

Statistical analysis

Statistical analyses were performed using SPSS software (version 21.0; IBM Corp., Armonk, NY, United States). Univariate and multivariate logistic regression models were used to analyze the high-risk factors. The cut-off value was determined using receiver operating characteristic (ROC) curve analysis and Kaplan-Meier survival curves. Log-rank tests were used for survival analysis. Differences with P < 0.05 were considered of minimal statistical significance for each comparison.

RESULTS

High serum LDL level was independent risk factor for LN metastasis in EC

Data from 294 patients (219 males and 75 females; 217 patients aged > 60 years) diagnosed with EC were retrieved and analyzed. All patients underwent surgical treatment, including Ivor-Lewis and McKeown esophagectomies. Mediastinal LNs, particularly those of the left and right recurrent laryngeal nerves, were removed according to R0 standards. Among them, 261 patients had squamous cell carcinoma, 29 had adenocarcinoma, and 4 had small cell carcinoma. There were 139 patients without LN metastases (N0 group): 21 with T1, 31 with T2, 57 with T3, and 30 with T4 disease. The 155 patients with positive LN metastases (N+ group) were classified as follows: T1 (n = 3), T2 (n = 16), T3 (n = 61), and T4 (n = 75). A total of 98 patients received neoadjuvant treatment with paclitaxel or platinum. The follow-up success rate was 97.3% (286/294; two patients refused to communicate and six were lost to follow-up), and data regarding the lipid characteristics of each group were collected (Table 1).

Table 1 Lipids characteristics of each group.

	Age		Sex		N0	N+	T1	T2	T3	T4
	≥ 60	< 60	Male	Female
TG (mmol/L)	1.17 ± 0.68	1.11 ± 0.72	1.17 ± 0.75	1.12 ± 0.49	1.17 ± 0.76	1.14 ± 0.63	1.28 ± 0.88	1.23 ± 0.66	1.17 ± 0.72	1.09 ± 0.63
CHOL (mmol/L)	4.49 ± 0.97	4.42 ± 0.84	4.36 ± 0.87	4.80 ± 0.15	4.40 ± 0.88	4.54 ± 0.98	4.38 ± 0.87	4.30 ± 0.97	4.48 ± 0.81	4.57 ± 1.05
HDL (mmol/L)	1.20 ± 0.28	1.17 ± 0.28	1.17 ± 0.27	1.26 ± 0.30	1.22 ± 0.29	1.16 ± 0.27	1.15 ± 0.22	1.21 ± 0.29	1.20 ± 0.29	1.18 ± 0.27
LDL (mmol/L)	2.53 ± 0.74	4.98 ± 2.30	3.36 ± 1.32	2.36 ± 0.83	2.42 ± 0.75	3.85 ± 1.57	2.50 ± 0.85	2.31 ± 0.78	2.52 ± 0.64	4.46 ± 1.91
Apo-A1 (g/L)	1.25 ± 0.23	1.23 ± 0.23	1.23 ± 0.23	1.29 ± 0.24	1.26 ± 0.23	1.23 ± 0.23	1.25 ± 0.21	1.24 ± 0.24	1.25 ± 0.24	1.24 ± 0.22
Apo-B (g/L)	0.84 ± 0.19	0.83 ± 0.20	0.83 ± 0.18	0.88 ± 0.23	0.83 ± 0.19	0.85 ± 0.20	0.87 ± 0.23	0.79 ± 0.17	0.85 ± 0.15	0.84 ± 0.21
Lpa (nmol/L)	236.39 ± 216.24	255.52 ± 233.29	233.40 ± 212.01	264.75 ± 244.13	231.67 ± 200.92	250.12 ± 237.25	166.26 ± 165.06	231.78 ± 224.21	254.76 ± 222.84	247.86 ± 226.81

TG: Triglyceride; TC: Total cholesterol; LDL: Low-density lipoprotein cholesterol; HDL: High-density lipoprotein cholesterol; Apo-A1: Apolipoprotein A1; Apo-B: Apolipoprotein B; LPa: Lipoprotein a.

Univariate logistic regression analysis was used to assess the high-risk factors affecting LN metastasis and T-staging, and the risk factors (P < 0.1 were analyzed using a multivariate logistic regression model. The results revealed that LDL level was an independent risk factor for LN metastasis, whereas HDL level was a protective factor (Figure 2A and B). ROC curve analysis revealed cut-off values of 2.64 and 1.20 for LDL and HDL levels, respectively. Patients were divided into two groups according to the cut-off value, and survival curves were plotted using the Kaplan–Meier estimator and compared using the log-rank test. However, there were no significant differences between the groups (Figure 2C and D). There were no significant correlations between lipid components and T-staging (Table 2). These results indicate that the progression of EC is accompanied by disordered lipid metabolism, and that high LDL levels are independent risk factors for LN metastasis in patients with EC; however, HDL plays the opposite role.

Figure 2 Risk factors analysis of lymph node metastasis and survival. A: Univariate Logistic regression analysis for risk factors of lymph node metastasis; B: Multivariate Logistic regression analysis for risk factors of lymph node metastasis; C: Overall survival curves of low-density lipoprotein; D: Overall survival curves of high-density lipoprotein. LDL: Low-density lipoprotein; HDL: High-density lipoprotein; TG: Triglycerides; Apo-A1: Apolipoprotein A1; Apo-B: Apolipoprotein B.

Table 2 Univariate Logistic regression analysis for risk factors of T-staging.

	T1 (n = 24)	T2 (n = 47)	T3 (n = 118)	T4 (n = 105)	χ/Z	P value
Sex (male, %)	17 (70.5%)	30 (63.8%)	87 (73.7%)	85 (81%)	5.323	0.15
Age (years)	61 (54, 66.5)	65 (51, 69)	64 (59, 70)	65 (59, 70)	5.026	0.170
TG (mmol/L)	1 (0.70, 1.63)	1.06 (0.77, 1.32)	0.99 (0.75, 1.45)	0.95 (0.73, 1.26)	2.095	0.553
TC (mmol/L)	4.20 (3.87, 4.99)	4.22 (3.73, 5.01)	4.56 (4.07, 4.88)	4.55 (3.80, 5.30)	2.726	0.436
HDL (mmol/L)	1.12 (0.99, 1.29)	1.15 (0.95, 1.41)	1.15 (1.00, 1.37)	1.16 (0.99, 1.38)	0.436	0.933
LDL (mmol/L)	2.29 (1.95, 3.01)	2.22 (1.68, 2.83)	2.52 (2.14, 2.92)	2.48 (2.06, 3.08)	4.369	0.224
Apo-A1(g/L)	1.30 (1.10, 1.40)	1.20 (1.00, 1.40)	1.20 (1.10, 1.40)	1.30 (1.10, 1.40)	0.319	0.956
Apo-B (g/L)	0.80 (0.70, 1.05)	0.80 (0.60, 0.90)	0.80 (0.70, 1.00)	0.80 (0.70, 0.93)	3.386	0.336
Lpa (nmol/L)	123.00 (61.25, 208.75)	147.00 (74.00, 304.00)	176.50 (92.60, 350.75)	172.00 (78.70, 351.50)	4.308	0.230

TG: Triglyceride; TC: Total cholesterol; LDL: Low-density lipoprotein cholesterol; HDL: High-density lipoprotein cholesterol; Apo-A1: Apolipoprotein A1; Apo-B: Apolipoprotein B; LPa: Lipoprotein a.

The LDL receptor LRP6 is highly expressed in EC and affects the infiltration of immune cells. Because LDL transports cholesterol from the liver to peripheral tissues, it has been hypothesized that LDL may carry more cholesterol to tumor tissues by binding to its receptors on tumor cells. The LDL receptor family comprises of four subgroups: LDLR, LRP8, LRP1, LRP2, LRP5, LRP6, LRP4, and LRP3. Accordingly, the levels of LDL in 182 EC tissues and 286 normal esophageal tissues from the GEPIA database were analyzed to determine their prognostic role in EC. The results revealed that LRP5 and LRP6 Levels were higher in EC than in normal esophageal tissues (Figure 3A) and that LRP6 was correlated with a poor clinical prognosis for EC (P = 0.017) (Figure 3B). Further analysis of the relationship between LRP6 and immune cells using the TIMER2 database suggested that LRP6 Levels were positively correlated with B lymphocyte infiltration (r = 0.255, P = 5.72 × 10^-4) but negatively correlated with DC infiltration (r = -0.242, P = 1.06 × 10^-3) (Figure 3C). These data revealed that there was high expression of LRP6 in EC cancers and that this level affected the prognosis of patients with EC, with the infiltration of B lymphocytes and DCs in the EC microenvironment.

Figure 3 Gene expression profiling interactive analysis and tumor immune estimation resource version 2 analyses based on the cancer genome atlas database. A: Expression of low-density lipoprotein receptors in esophageal cancer (EC). The levels of LRP5 and LRP6 are higher in EC than those in normal esophageal tissues; B: LRP5 and LRP6 are correlated with a poor clinical prognosis. Overall survival curves of LRP5 and LRP6; C: Correlation between LRP6 and immune cell infiltration. LRP6 is positively correlated with B lymphocyte infiltration and negatively correlated with DC infiltration.

The N+ group exhibited high LRP6 Levels and a high density of B-lymphocytes in the EC microenvironment.

Several studies have reported a decrease in the number of DCs in the immunosuppressive microenvironment of EC. However, explanations for increased B lymphocyte infiltration in the EC microenvironment remain controversial. Therefore, the infiltration of B lymphocytes (CD²⁰⁺) and expression of LRP6 were further analyzed by immunofluorescence staining. The results revealed that the infiltration of B lymphocytes [8.97 (6.23, 12.60) E-3 vs 3.61 (1.89, 7.00) E-3; Z = -3.91, P < 0.01] and the level of LRP6 [2.35 (1.94, 2.97) E-3 vs 1.77 (0.66, 2.60) E-3; Z = -2.51, P = 0.012] in the N+ group were significantly higher than those in the N0 group. The expression of LRP6 in B lymphocytes in the N+ group was also significantly higher than that in the N0 group [5.35 (1.00, 6.28) E-4 vs 1.30 (0.41, 1.80) E-4; Z = -2.61, P = 0.009] (Figure 4).

Figure 4 Expression of LRP6 and infiltration of B lymphocytes in N+ and N0 group. A: Immunofluorescence of LRP6 and B lymphocytes in N+ and N0 group respectively; B: Quantitative analysis with Mann-Whitney U test of CD20. Infiltration of B lymphocytes are significantly higher in the N+ group; C: Quantitative analysis with Mann-Whitney U test of LRP6. Expression of LRP6 is significantly higher in the N+ group; D: Quantitative analysis with Mann-Whitney U test of Merg. Infiltration of LRP6+B lymphocytes are significantly higher in the N+ group.

DISCUSSION

Cancer progression is worsened by different metabolic disorders, such as abnormalities in glucose and amino acid metabolism. In addition to tumor cells, tumor-infiltrating immune cells are involved in reprogramming metabolism, which can result in the generation of a compromised immune system. Our results revealed that high serum LDL level was an independent risk factor for LN metastasis; however, HDL played the opposite role. We then analyzed the levels of LDL from the GEPIA database and found that high LRP6 expression was negatively correlated with EC prognosis. Further analysis revealed that LRP6 was positively correlated with the infiltration of B lymphocytes but negatively correlated with DC abundance in the EC microenvironment. Furthermore, immunofluorescence staining revealed that the LRP6 Levels in the N+ group were significantly higher than those in the N0 group, and the number of infiltrated B lymphocytes in the N+ group was significantly higher than that in the N0 group. These results suggest that LDL may promote LN metastasis in patients with EC and that the underlying mechanisms may be related to the expression of LRP6 and altered infiltration of immune cells.

LDL is a crucial carrier of cholesterol, plays a role opposite to that of HDL, and exerts a substantial effect on the occurrence and development of cancers; however, its role varies among different types of tumors. Several studies have revealed that high serum LDL levels may promote the progression of breast cancer and that LDL can be used as a prognostic biomarker. Moreover, radiotherapy and chemotherapy can modify LDL metabolism in the tumor microenvironment[16]. However, a study investigating small-cell lung cancer suggested that increased serum levels of LDL and LDLR were associated with better survival outcomes, which is related to the fact that lipoproteins in tumor cells can enhance the efficiency of chemotherapeutics[17]. A preclinical study reported that mice with high serum LDL levels are more likely to develop EC[18]. Another clinical study suggested that a low serum LDL level was related to a later T stage and poor prognosis of EC, probably because of the increased uptake of LDL or cholesterol by tumor cells[15]. Our results revealed that high serum LDL level was an independent risk factor for LN metastasis in EC; in contrast, HDL played the opposite role. Therefore, we speculate that LDL transports more cholesterol into the EC tumor microenvironment, thereby promoting LN metastasis. However, neither LDL nor HDL affected the survival of patients with EC, which may be due to the small number of studies or because they could maintain the efficacy of adjuvant therapy. Similar to our findings, a study investigating gastric cancer demonstrated that high serum LDL levels were a risk factor for gastric cancer without affecting survival[19].

LDLR plays a significant role in LDL uptake, and LRP6 is the major LDLR responsible for LDL uptake and the regulation of cell metabolism[20,21]. Many studies have confirmed that LRP6 is highly expressed in various tumors and plays a prominent role in tumorigenesis and tumor progression. The underlying mechanism may be related to the activation of Wnt signaling[22,23]. Based on the GEPIA data, LRP6 was highly expressed in EC and related to poor prognosis, which may be modulated by Wnt signaling. TIMER2 data analysis revealed that the expression of LRP6 was positively correlated with the number of infiltrated B lymphocytes, but negatively correlated with DC infiltration. LRP6 is also expressed in B-lymphocytes and DCs. High LRP6 expression can stimulate the proliferation of malignant B lymphocytes in B lymphocytic leukemia; however, the absence of LRP6 can promote the antitumor activities of DCs. The underlying mechanism may be related to the activation of Wnt signaling[24,25]. Based on the above results, we speculated that overexpression of LRP6 in EC, through interaction with its receptor LRP6, not only promoted the invasion of tumor cells but also facilitated the formation of an immunosuppressive microenvironment characterized by increased B lymphocytes and decreased DC infiltration.

B-lymphocytes are the main participants in humoral immunity and exert antitumor effects through antigen presentation, immunoglobulin secretion, and direct lysis of tumor cells. However, in the tumor microenvironment, regulatory B lymphocytes (Bregs), a subgroup of B lymphocytes, play an immunosuppressive role by secreting interleukin (IL)-10, IL-35, or transforming growth factor-β[26,27]. To date, only a few studies have characterized the exact role of tumor-infiltrating B lymphocytes, with conflicting results, which may be explained by the ambiguous definitions of B lymphocyte subtypes or their unpredictable functions. Several studies have shown that in the lymphoid organs of mice with hypercholesterolemia, there is a significantly increased number of Bregs, accompanied by a high level of IL-10 secretion[28]. Therefore, Bregs can also regulate acquired immune responses, such as the differentiation and function of T cells and DCs, thereby affecting antitumor immunity[29,30]. However, we found that LRP6 Levels and infiltrated B lymphocytes were significantly higher in the N+ group, with greater LRP6 expression in B lymphocytes. Therefore, we speculated that LDL may induce an immunosuppressive B-lymphocyte subtype by binding to LRP6, thereby promoting LN metastasis in EC. Although there is no supporting evidence to confirm the regulatory role of LDL in B lymphocytes, the secretion of IL-10 in B lymphocytes depends on cholesterol metabolism and high cholesterol levels lead to the depletion of CD8+T cells, supporting our hypothesis[31,32]. Overall, our results suggest that patients with EC and high LDL levels are more likely to develop LN metastasis. This may be related to the high expression of LRP6, infiltration of B lymphocytes, and a decrease in DCs in ECs.

Limitations

This study provides important evidence for the metabolism-immune regulatory network of EC. However, this study has some limitations. The use of CD20 markers to detect the total number of B cells could not differentiate between key subpopulations such as Bregs, and it needs to be verified whether the function of B lymphocytes and overexpression of LRP6 are related to Bregs. Ninety-eight patients in this study received NAC, which may have affected the tumor microenvironment, lipid metabolism, and immune cell function, leading to biased results. The relationship between high cholesterol levels in the tumor microenvironment and LRP6 expression requires further validation. Unmeasured confounding factors such as dietary habits, lifestyle, and detailed genetic background are also important issues to consider when causally linking the results of this study. In addition, the number of patients was small, and a subgroup analysis was not performed. Due to the relevant conditions, animal testing was not performed to validate this study. In future studies, we will use flow cytometry (FACS) in combination with multiple B cell subpopulation-specific markers such as CD24, CD38, and IL-10 for an in-depth analysis of Bregs. A high-cholesterol EC mouse model was constructed to observe the effects of the LRP6 inhibitor. Moreover, we are actively seeking collaborations to initiate a multicenter, large-sample prospective cohort study.

CONCLUSION

High LDL levels are an independent risk factor for LN metastasis in patients with EC. These mechanisms may be related to LRP6 expression, B lymphocyte infiltration, and a reduction in the number of DCs in ECs.