Introduction

Hepatocellular Carcinoma (HCC) is a highly malignant tumor of the digestive tract with a high mortality rate [1]. According to recent surveys, the incidence of HCC has increased rapidly among all cancers and the relative survival rate of HCC is much lower than that of other cancers, except pancreatic cancer [2]. The high recurrence and metastasis rate are important reasons for hardly maintaining a long-term prognosis in patients with HCC [3]. Although HCC treatment strategies have improved dramatically with the rapid development of medical technology, current research on the treatment of HCC remains a hot spot for all humans [4]. In particular, studies have shown that various mechanisms in the microenvironment of HCC can improve the ability to escape and tolerate immune responses and decrease effector function of immune cells [5,6]. Therefore, it is imperative to clarify the microenvironment of HCC.

HCC is characterized by uncomplicated distant metastases and rapid progression, mainly considering the blood supply of HCC tissues [7]. Angiogenesis is an early characteristic of solid tumors, which refers to how pre-existing endothelial cells form new blood vessels under suitable conditions [8]. Furthermore, angiogenesis plays a facilitating factor for tumor growth, progression, invasion, and metastasis. Tumor and neovascular ECs may form a highly integrated system that can promote each other’s development [9]. Tumor cells stimulate the proliferation of vascular ECs by secreting specific cytokines, and ECs can also provide rich oxygen and nutrients to promote tumor cells growth [10]. Vascular ECs are reported to be found in glioblastoma derived from tumor cells [11]. Furthermore, some ECs were found to have typical human chromosomes instead of murine chromosomes in mouse transplanted tumors of human glioblastoma [12], suggesting that stem cell-like tumor cells have tumor angiogenesis during tumor formation. Thus, we predicted that angiogenesis inhibition is a potentially effective strategy for HCC therapy.

ENG is mainly enriched on the surface of adult vascular ECs, and its primary function is to promote tumor tissue angiogenesis [13]. ECs cultured in vitro can detect a higher level of ENG expression during the proliferation process [14]. In tumor tissues, ENG is highly expressed in the process of angiogenesis and vascular remodeling [15-17], and other studies have confirmed that ENG is a specific marker of angiogenesis [18]. Studies showed that ENG was only expressed in the neovascular ECs of HCC tissues but not in normal tissues [19]. Therefore, ENG can be used as a specific target for identifying the neovascularization of HCC. Sorafenib, as an anti-angiogenesis targeted drug, is one of the drugs currently used in the treatment of advanced liver cancer. It acts mainly by indirectly inhibiting the expression of VEGFR, PDGFR, and C-kit expression, and then prevents the formation of tumor neovascularization [20-22]. However, the role of ENG in sorafenib inhibited tumor progression is poorly understood.

In this article, we demonstrate that: (i) eight subpopulations of tumor cells were found in HCC by single-cell RNA sequencing assay, and tumor cells were retrodifferentiated to vascular endothelial-like cells; (ii) ENG, overexpressed in the progression of tumor infiltration, predicted aggressive clinicopathological characteristics and poor prognosis; (iii) ENG expression could upregulate COL1A1 expression to promote HCC angiogenesis; (iv) ENG expression promoted the immune response and immune cell infiltration; (v) ENG expression promoted antitumor therapy of sorafenib in HCC patients. These data suggested that ENG expressed in neovascular derived from tumor cells promoted antitumor therapy of sorafenib through activation of the immune response and immune cell infiltration in HCC patients, which highlighted a new scientific strategy for antitumor therapy of HCC.

Materials and Methods

Cell culture

HCC cells (Huh7 and HepG2) and HUVECs were obtained and authorized from the China Center for Type Culture Collection and were cultured in Dulbecco’s modified eagle medium DMED added with 10% fetal bovine serum (Solarbio, Beijing, China), 100X Penicillin-Streptomycin (Solarbio). Huh7 cells were infected with EGFP-Lv for stably exogenous expression of EGFP.

Patients and animals

Seventy-six paired tumor tissues, and adjacent HCC tissues were collected from the Affiliated Hospital of Qingdao University from January 2020 to September 2021 (Table S1). Eighteen HCC patients with sorafenib treatment, were collected for ENG stain and CT films (Table S2). All of these studies were conducted under the supervision of the Research Ethics Committee of the Affiliated Hospital of Qingdao University (Approval NO: QYFYWZLL26589) and obtained informed consent forms from patients or family members. Male Balb/c nude mice, aged five weeks, were purchased from SPF (Beijing) Biotechnology Co., Ltd and raised in an IVC system. EGFP-Lv infected Huh7 cells, 5x10⁶, were subcutaneously injected into mice for two weeks. All animal experiments were approved and conducted under the supervision of the Affiliated Hospital of Qingdao University (Approval No: QYFYWZLL26589).

Small animal ultrasound imaging system

The neovascularization density of tumor tissues was recorded using the Small Animal Ultrasound Imaging System. Briefly, mice anesthetized with pentobarbital sodium were fixed on a small animal ultrasound imaging system, and then a 40 MHz probe was installed subsequently. After opening the software, the exposed tumor site was wrapped with conductive glue. The image of neovascularization density was recorded by adjusting the probe to a proper position.

Small animal in vivo laser confocal microscope

Shape and diameter of neovascularization were recorded on Small Animal in vivo Laser Confocal Microscope. Briefly, pento-barbital sodium anesthetized mice were injected with 1% Evans blue through the tail vein. A Probe-Based Confocal Laser was directly injected into tumor tissues for the neovascularization observation in vivo. Finally, an appropriate image was recorded.

Western blot and quantitative real-time polymerase chain reaction (qRT-PCR)

Total protein was extracted with RIPA (Solarbio) from cells and tissues. The extracted protein was separated with 8% SDS-PAGE gels and transferred to the PVDF membrane immediately. After blocking with 5% skim milk, PVDF membranes were blotted with anti-ENG (1:2000; Abcam), anti-COL1A1 (1:1000; Proteintech), anti-CD31(1:1000; Sigma-Aldrich), anti-CD34(1:1000; Sigma-Aldrich), and anti-GAPDH (1:2000; Proteintech) antibodies and incubated with HRP-linked secondary antibodies (1:5000; Proteintech), subsequently. After incubation with ECL solution, the protein band was exposed and imaged by the Tanon-5200 chemiluminescence imaging system. Total RNA from 76 paired tumors and adjacent tissues of HCC was isolated using Trizol reagent (In-vitrogen). qRT-PCR was performed using the SYBR Green PCR kit (ABclonal) on an ABI Prism 7500 Sequence Detection system (Applied Biosystems). The primers used for the qRT-PCR analysis are listed in Table S3.

Immunofluorescence assay

The frozen slices were fixed with 75% alcohol and penetrated with 0.1% Triton X-100. After blocking with 1% goat serum, slices were blotted with anti-EGFP (1:100; Abcam), anti-CD31(1:50; Sigma-Aldrich), and anti-AFP (1:100; Sigma-Aldrich) antibodies. The residual primary antibody was washed 1xPBS and the slices were incubated with FITC or TRITC-linked secondary antibody at 1:200 dilution. Subsequently, 5 μl of DAPI solution was used for DNA detection. After the 10 minutes of staining, the double immunofluorescence staining was photographed under laser scan ning confocal microscopy.

Immunohistochemistry (IHC)

The immunohistochemistry assay was performed following the manufacturer’s protocol (ZSGB, Beijing, China). First, tissue slices were placed in a 56^oC constant incubator for 30 min to dehydrate, and then put them for hydration. After blocking with serum, 100 μL of primary antibody working liquor was supplemented and cultivated at 37^oC for 60 min. The second antibody was labeled with goat anti-rabbit/anti-mouse IgG labeled with biotin. Subsequently, the horseradish enzyme-labeled streptavidin working liquor was supplemented and cultivated. An appropriate amount of freshly prepared DAB solution was added to the tissue section for 5 min. The slides were photographed and recorded under the microscope.

Trans well cell migration experiment

HUVECs (Human Umbilical Vein Endothelial Cells) at logarithmic growth stage were used to suspend the cells in conditioned medium and counted. Then, 200 ul of cell suspension was added to the Trans well chamber, and 200 ul of medium containing 10% FBS was added to the culture plate at the bottom of the chamber. Posterior to the 12 or 48 h cultivation, cells on the chamber film were subjected to fixation, dyeing, imaging, and the counting was finished in 6 stochastic fields for each group via a microscopic device. Those assays were completed for three times.

HUVECs tubule formation experiment

Corning Matrigel was laid on the 96-well plate, and added 60 ul matrigel per hole. Then the 96-well plate was placed in the incubator for 30 min, and the matrigel was fully solidified. HUVECs were suspended in conditioned medium, and then 1 × 10⁴ HUVECs were inoculated in each well with 3 multiple wells in each group. After incubating for 6 hours in the incubator, microscope observation, photographing and counting were performed.

Data collection and analysis

Single cell RNA-seq data set (GSE149614) and nine HCC RNA-seq data sets were downloaded directly from GEO. Thirty-three types of tumor RNA-seq data sets and clinicopathological characteristics were collected from the TCGA. The seurat R package was used for the single-cell RNA-seq assay. Monocle2 R package was performed for the analysis of pseudo-time trajectories to understand the fate of tumor cells. |logFoldChange| > 1 and P.Val < 0.05 were considered for the identification of differentially expressed genes (DEGs) from ENG-highly and weakly expressed LIHC RNA-Seq from TCGA. Subsequently, Gene Set Enrichment Analysis (GSEA) was performed using the MSigDB molecular signatures database and DEGs were identified. Estimate R package was used for the estimation of the tumor microenvironment. CIBERSORT was performed for immune infiltration in LIHC. Two-tailed Student’s t-tests were performed for differences among variables. A log-rank test was performed for survival analysis. Spearman’s rank correlation was used to analyze the correlations of NEG with StromalS-core, ImmuneScore, ESTIMATScore, TumorPurity, immune infiltration, targets of sorafenib, and altered tumor diameter. Overall and disease-free survival was analyzed with Log-rank tests. Data were shown as the average ± SD. Statistically significant was shown as P value <0.05.

Results

Retro differentiation of tumor cells to vascular endothelial-like cells in the process of infiltrating para-carcinoma

We all know that the type of HCC cell directly determines the cancer progress [23]. However, the tumor cell progression in HCC has not yet been elucidated. Therefore, we downloaded the single-cell data set GSE149614 from GEO.

Subsequently, the cluster analysis indicated that there are eight different subtypes of tumor cell (Figure S1A and B) distributed in HCC tumors and adjacent tissues, and each subtype of a tumor cell has specific markers (Figure S1C and D). To understand the evolution of tumor cells, pseudo-time analysis was performed. Interestingly, in the process of infiltration from the tumor to adjacent tissue, tumor cells were retro differentiated with three main distinct branch points (Figure 1A), and specific markers of the subpopulation were reprogramed in this retro differentiation process (Figure 1B). Interestingly, markers of vascular endothelial cells, such as ENG, A2M, and PECAM1, were increased in the retro differentiation process (Figure 1C-E). To further confirm the reliability, we performed immunofluorescence staining on samples from HCC patients, and the result suggested that the vascular ECs marker, CD31, highly expressed tumor cells at the tumor junction (Figure 1F). All in all, these data showed that tumor cells were retro differentiated to vascular endothelial-like cells in the process of invading peripheral normal tissues from the center of the HCC tissues.

Transformation of HCC cells into neovascular endothelial cells in a nude mouse model

As shown above, tumor cells can transform into ECs in the process of outward invasion. To confirm the above results, we stably introduced the exogenous EGFP gene into Huh7 cells by infecting the EGFP-shRNA lentivirus (Figure 2A). Nude mice were subcutaneously injected with 5 × 10⁶ Huh7 cells and then fed in the IVC system for two weeks. Subsequently, anesthetized nude mice were performed under the S-Sharp Prospect small animal ultrasound imaging system for neovascular density, and a large amount of microvascular was found in the subcutaneous tumor (Figure 2B). Consistently, neovascular enriched tumor tissues, pre-injected with 1% Evans blue, were further observed under in vivo laser confocal microscope (Figure 2C). The successfully constructed subcutaneous tumor-bearing animal model is presented in Figure 2D. To further confirm the origin of neovascularity in tumor-bearing tissues, double immunofluorescence staining was performed for exogenous EGFP and CD31, a marker of neovascularity, respectively. Approximately, tumor cells were highly expressed EGFP and

AFP, and endothelial cells were simultaneously expressed EGFP, AFP, and CD31 (Figure 2E and F). Additionally, compared with tumor-bearing tissues, Huh7 cells expressed lower levels of ECs biomarker (Figure 2G). In summary, endothelial cells in neovascular tumor tissues may be derived from the transformation of tumor cells.

Vascular ECs biomarkers highly expressed in adjacent tumor tissues

The above result suggested that microvascular cells were derived from tumor cells in HCC. Therefore, to clarify the expression of the vascular ECs biomarker in HCC progression, transcriptome data was collected, including 33 types of tumor patients and nine GEO datasets. The expression profile data of different cancer patients revealed that ENG, FLT1, A2M, and PECAM1 were highly expressed in 13, 15, 15, and 16 out of 33 types of tumor tissues, respectively (Figure 3A and Figure S2A–C). Similarly, the expression profile data of HCC patients in the GEO data sets found that ENG, FLT1, and A2M were highly expressed in 7, 4, and 9 out of 9 different GEO data sets (GSE102097, GSE12128, GSE22058, GSE25097, GSE36376. GSE46444, GSE57957, GSE76297, and GSE76427) (Figure 3B and Figure S2D and E). Subsequently, the expression level of ENG mRNA was detected in HCC samples using the qRT-PCR assay. The results indicated that ENG was significantly upregulated in adjacent tissues compared to tumor tissues (Figure 3C). Furthermore, we further performed a western blotting assay in 25 paired human HCC samples to detect ENG expression and found that ENG expression was consistent with ENG transcription level (Figure 3D). These results indicated that the biomarkers of vascular ECs were highly expressed in adjacent tumor tissues of HCC.

ENG expression presents combating clinicopathological characteristics and a poor prognosis in HCC patients

The above result showed that ENG was highly expressed in the adjacent tumor tissues. However, the role of ENG expression was poorly elucidated in HCC patients. Here, we analyzed the relationship between the expression level of vascular ECs markers and clinicopathological characteristics (that is, microvascular infiltration, pathological stage, pathological grade, age, gender, BMI, AFP, and HBV). Although ENG expression was not correlated with gender, age, BMI, and HBV infection (Figure 4), ENG expression was positively correlated with tumor grade and microvascular infiltration. Furthermore, A2M expression was positively associated with age (p=0.028) and AFP level (Figure S3, p=0.036), and PE-CAM1 expression was positively associated with tumor grade (Figure S4, p<0.05). Therefore, the vascular ECs markers ENG might be a marker of microvascular infiltration in HCC. Subsequently, the multivariate analysis presented that ENG, together with microvascular invasion, AFP, virus, BMI, age, sex, tumor grade, and tumor stage, was an independent risk factor for OS (HR= 0.795; P= 0.007), DSS (HR= 0.735; P= 0.005), DFI (HR= 0.7618; P<0.001) and PFI (HR= 0.7922; P= 0.001, Figure 5A-D). Consistently, ENG was weakly expressed in tumors and presented poor OS (P= 0.00059), shorter time to DSS (P= 0.046), and PFI (P= 0.0014, Figure 5E-H). On the contrary, the high expression of ENG in the adjacent areas presented poor OS (P= 0.0017) and a shorter time for DSS (P= 0.046, Figure S5). These results suggested that ENG could be used as an independent prognostic factor affecting the progression and outcome of the disease.

ENG can promote the formation of HCC microvessels

As in the previous study, we found that ENG can be used not only as a marker of the vascular endothelium, but also as an independent risk factor that affects the prognosis of HCC patients. However, the effect of ENG on HCC tumor angiogenesis remains unclear. We first analyzed the correlation between ENG and the vascular marker CD34 through the GEPIA database, and the results suggested that there was a significant positive correlation between ENG and CD34 expression (Figure 6A). In addition, we detected microvessel density (MVD) in tumor tissues of 65 HCC patients, and MVD was counted by the number of blood vessels stained with anti-CD34 antibody. The results showed that the MVD of the high ENG expression group was significantly higher than that of the low ENG expression group and the difference was statistically significant (Figure 6B and C). On the basis of the above results, we further explored the effects of ENG on the migration and tubulogenesis of vascular endothelial cells at the cellular level. We first transfected the lentivirus carrying ENG shRNA into Huh7 and HepG2 cells, and the empty vector was used as a control group (Figure 6D). After 72 hours of transfection, the protein was extracted and detected by Western blot assay. The results showed that the expression of ENG in Huh7 and HepG2 cells after interference with the expression of ENG was significantly lower than that in the blank control group. Subsequently, we carried out transwell cell migration experiments and HUVECs tubule formation experiments, and the results showed that the number of tubule formation was significantly reduced (Figure 6E and G), and the migration ability of HUVECs cells was weakened (Figure 6F and H) after silencing the expression of ENG. Taken together, the above results suggested that ENG was involved in the angiogenesis of HCC.

ENG promotes HCC microvascular formation by upregulating the expression of COL1A1

To better understand the role of ENG in promoting HCC angiogenesis, we will further explore the mechanism by which ENG regulates tumor angiogenesis in this chapter. We first performed an enrichment analysis of the pathway in patients with different types of tumors and the results showed that almost all types of cancer are involved in angiogenesis (Figure 7A and B). Subsequently, we analyzed the correlation between ENG and angiogenesis, and the results also showed that ENG was positively correlated with angiogenesis (Figure 7C). Studies have shown that CoL1A1 is related to the occurrence and development of various tumor diseases [24-26]. Therefore, we also analyzed the correlation between ENG and COL1A1 in the TCGA, LIHA, GTEx and CCLE databases. The results indicated that ENG was positively correlated with COL1A1 (Figure 7D-G). To further verify the relationship between ENG and COL1A1, we performed a Western blot assay on a cell line with low expression of ENG and the results showed that the expression of COL1A1 in Huh7 and HepG2 cells significantly decreased after interfering with the expression of ENG (Figure 7H and I). Based on the above results, we speculate that ENG may be involved in HUVECs migration and tubule formation by regulating COL1A1 expression. Subsequently, we infected the Huh7 and HepG2 ENG low expression cell lines with the overexpressed plasmid COL1A1 for 72 h and then immediately performed the transwell cell migration assay and the HUVEC tubule formation assay. The results showed that the migration capacity of HUVECs was significantly reversed (Figure 7J and K), and the number of tubule formations was significantly recovered after up-regulation of COL1A1 compared to the low expression group (Figure 7L and M).

ENG promotes immune cell infiltration

The above result showed that ENG was not only an independent prognostic factor and predicted a poor prognosis but also could promoted angiogenesis in HCC. However, the role of ENG in the tumor immune microenvironment (TIME) remains unclear. Therefore, we performed bioinformatic analysis and found that ENG expression in HCC may participate in the regulation of immune infiltrating cells and extracellular matrix (Figure S6). To clarify the complex mechanism of ENG in TIME, we calculated the immune/stromal/estimated score and tumor purity using an estimate algorithm (Table S4). The result showed that ENG expression was positively correlated with immune scores, stromal scores, and Estimate scores, while negatively correlated with tumor purity (Figure S7A-D). These results suggested that ENG acted as an essential part of the regulation of the tumor immune microenvironment of HCC. Thus, CIBERSORT was performed to quantify the immune cells of patients with HCC (Table S5). Immune cells, CD4 cells of main memory, CD8 cells of memory, natural killer cells, and T cells of the natural killer were infiltrated in high-expressed patients with ENG (Figure 8A and B). Then, Spearman’s correlation analysis was performed to establish the correlation between ENG expression and the level of immune cell infiltration using quantitative data. As shown in Figure 8C-R, ENG expression correlated positively with Type1 T helper cells (r = 0.55), central memory CD4T cells (r = 0.57), activated CD8 T cells (r = 0.2), regulatory T cells (r = 0.51), T follicular helper cells (r = 0.44), central memory CD8 T cells (r = 0.29), gamma delta T cells (r = 0.21), effector memory CD8 T cells (r = 0.5), effector memory CD4 T cells (r= 0.31), CD56dim natural killer cells (r = 0.35), natural killer T cells (r = 0.36), natural killer cells (r = 0.65), plasmacytoid dendritic cells (r = 0.48), immature dendritic cells (r = 0.33), activated dendritic cells (r = 0.23), and macrophages (r = 0.55). In conclusion, the expression of ENG in HCC was involved in the regulation of immune infiltrating cells.

ENG expression levels correlated with the antitumor efficacy of sorafenib in HCC patients

As mentioned above, ENG is a marker of the vascular endothelium of HCC, and its high expression level can promote an increase in tumor tissue hematogenesis in HCC patients. Therefore, antiangiogenic drugs can target patients with high expression of ENG. We analyzed the correlation between ENG and angiogenesis genes (targets of sorafenib: VEGFR, PDGFR, KIT, and VEGFA) in the TCGA, LIHA, GTEx, and CCLE databases. The results indicated that ENG was positively correlated with VEGFR, PDGFR, VEGFA, and KIT, respectively (Figure S8). These results further illustrated that sorafenib was a potential target drug in the treatment of high-expression ENG in HCC. To better understand the above findings in cancer patients, 18 HCC patients treated with sorafenib, ten responders and eight non-responders, were collected for ENG expression. Two representative cases of tumor diameter (red line) to sorafenib therapy were shown in Figure 9A, and four representative cases of ENG were presented in Figure 9B. Importantly, ENG was low expressed in patients without sorafenib response (Figure 9C and D), and ENG expression was positively correlated with the change in diameter (Figure 9E). Together, these results suggested that the property of sorafenib in tumor suppression was altered for the expression of ENG.

Discussion

The high rate of recurrence, high metastasis, and low survival rate are the main characteristics of HCC, which may be contributed by the highly vascularized features of HCC and the unique tumor microenvironment (TME) [27]. In recent years, angiogenesis has been found to play an essential role in the progress of HCC. The antitumor vascular therapy is widely applied in clinics. Unfortunately, due to the heterogeneity of tumor microvessels, the effect of anti-angiogenesis therapy is limited [28]. Actually, bone marrow-derived ECs [29], cancer stem cells-derived ECs [11], and vascular channels-derived cancer cells [30] successfully formulate neovascular in tumors. Here, multiple subpopulations with different profiles were identified in patients with HCC. In the process of infiltration, tumor cells were retrodifferentiated to vascular markers with highly expressed endothelial-like cells. Instead of murine chromosomes, some ECs have typical human chromosomes in tumors from mice transplanted with human glioblastoma [12]. Consistently, in our artwork, neovascular endothelial cells co-expressed the neovascular marker (CD31) and EGFP (exogenously expressed in cultured Huh7 cells) or AFP (an HCC biomarker, Figure 2) in tumors of mice transplanted with Huh7 cells. These results suggest that the ECs of angiogenesis are derived from tumor cells during tumor formation. Thus, neovascular oxygen and nutrients are provided for rapidly formulated tumor tissues and remove harmful metabolites [31].

ENG is expressed primarily in new capillary endothelial cells at the edge of tumor tissue and is weakly expressed on the vascular ECs of normal tissues. More importantly, ENG promotes tumor cell proliferation, so ENG expression is known as an indicator of dynamic observation of ECs proliferation [32,33]. In fact, our findings support previous results that high ENG expression is a significant enrichment in angiogenesis, extracellular matrix organization, and immune cell activation by GO and GSEA analysis (Figure S6). In addition, patients with high expression of ENG were more likely to have lower overall survival. At the same time, the univariate Cox regression analysis showed that ENG could be an independent prognostic factor that affects OS, DSS, DFI, and PFI. In this study, our team found that ENG can be used as a symbol of vascular endothelial cells and can affect the prognosis of patients with HCC. However, the current mechanism to regulate tumor blood vessel production in HCC is unclear. We first confirmed that ENG expression was positively related to CD34 expression through GEPIA database analysis. At the same time, we also conducted an IHC analysis of 65 cases of HCC samples, and the result prompted the ENG high expression group to have a higher microvascular density. Subsequently, we continued to explore the effects of ENG on migration capacity and the formation of small tubes of vascular endothelial cells. Therefore, the migration capacity of HUVECs cells was reduced, and the number of small tubes was significantly reduced after lowering ENG expression. These results showed that ENG participated in the formation of HCC blood vessels. Therefore, ENG can be used as a potential treatment target to inhibit the formation of new blood vessels of HCC. Finally, it is possible to provide new ideas for HCC blood metastasis. To better understand the role of ENG in promoting HCC angiogenesis, we analyzed the correlation between ENG and angiogenesis, and the results also showed that ENG expression was positively correlated with angiogenesis. Our team also found that ENG was positively correlated with COL1A1. COL1A1 is the composition of the extracellular matrix, which is closely related to the growth, proliferation, and differentiation of cells[24]. Studies have reported that COL1A1 is highly expressed in a variety of malignant tumors, promoting tumor invasion and metastasis[26]. Therefore, we hypothesized that ENG might regulate COL1A1 expression and participate in vascular formation. Subsequently, we continued to verify the relationship between ENG and COL1A1 at the cellular level, and Western blot results showed that COL1A1 expression levels obviously decreased after the downregulation of ENG expression. To further explore whether ENG regulates COL1A1 expression and can cause changes in the vascular production function, we found that overexpression of COL1A1 could reverse the reduced migration capacity and the reduced number of tubules in HUVECs induced by the rescue assay. Therefore, we confirmed that ENG is involved in HCC microvessel formation by regulating COL1A1. Furthermore, according to reports in the literature [18,34,35], the high expression of ENG in adjacent tissues is closely related to recurrence after liver transplantation. To sum up the results, we speculate that ENG can predict tumor recurrence after liver transplantation. It is recommended to actively reduce the level of ENG after liver transplantation to inhibit the formation of new blood vessels. Therefore, ENG is expected to be a prognostic indicator of angiogenesis, recurrence, and metastasis in patients with HCC.

Tumor tissues are enriched with abundant tumor blood. To this end, angiogenesis inhibitors, such as blocking pro-angiogenic factors, specifically inhibit tumor angiogenesis in anti-angiogenesis therapy. Sorafenib, targeted therapy in the first-line treatment of HCC, inhibits angiogenesis through the targeted inhibition of VEGFR, PDGFR, KIT, and VEGFA in tumor suppression. Interestingly, our results showed that the expression level of ENG is positively correlated with the mRNA expression of the target genes for sorafenib treatment, such as VEGFR, PDGFR, KIT, and VEGFA, by analyzing the liver tissues, TCGA, LIHC, CCLE, GTEx, and TCGA (Figure S8). It is widely accepted that the tumor suppressor effect of sorafenib is widely believed to be achieved by regulating the expression of specific target genes. Therefore, to illustrate that sorafenib was a potential targeted drug to treat high-expression ENG in HCC, we screened 18 patients treated with oral sorafenib for HCC and then determined ENG expression in HCC by immunohistochemistry of pathological tissues and evaluated the therapeutic effect by CT. The results show that ENG expression levels were correlated with the efficacy of sorafenib in patients with HCC. Although the mechanism by which ENG directly regulates the expression of sorafenib-specific target genes has not been effectively elucidated, the expression of ENG can indeed effectively promote the antitumor effect of sorafenib in patients with HCC. Therefore, our research is expected to provide new potential targets for tumor anti-angiogenesis therapy in clinics.

Immune cells are infiltrated into the tumor tissues. Unfortunately, immunomodulatory proteins and immune checkpoint molecules suppressed the activation of immune cells infiltrated by tumor tissue. Tumor immunotherapy reactivates immune cell function by blocking overexpressed immunomodulatory proteins in solid tumors, thereby inhibiting tumor progression of many tumors. However, tumor immunotherapy failed to achieve a stable and sustained antitumor effect. Our results showed that the expression level of ENG was related to the degree of infiltration of immune cells in tumor tissues, suggesting that a large number of immune cells are infiltrated in tumor tissues rich in blood vessels. However, abnormally abundant tumor blood vessels lead to immunosuppression in the tumor microenvironment, which dramatically interferes with the effect of immunotherapy. Anti-angiogenesis therapy reprogrammed tumor blood vessels by blocking pro-angiogenic factors, which effectively improved blood perfusion, reduced hypoxia and acidosis, and relieved the tumor microenvironment’s suppression of immune cells. Thus, a completely new antitumor strategy, combined application of targeted therapy and immunotherapy, reconstructs the number and function of immune cells in tumor tissues, which is extremely attractive to patients.

Conclusion

In general, this study has clarified four points. First, neovascular in HCC patients was formed by tumor-derived vascular endothelial cells. Second, the high expression of ENG in HCC adjacent tissues was significantly positively correlated with the level of activated tumor immune cell infiltration, which indicated that ENG had an immunomodulatory effect on tumor immunity. Third, ENG promotes the formation of HCC microvessels by upregulating the expression of COL1A1. Fourth, ENG promotes the antitumor property of sorafenib in HCC. Finally, ENG can be used as a biomarker for the diagnosis, therapy, and prognosis of HCC (Figure 10). Although our study did not indicate the exact molecular mechanism by which ENG promotes the formation of new blood vessels and regulates the recruitment of tumor-specific immune cell populations in tumors, the advantage of this research was that it promotes the development of new precision-targeted immunotherapy research and prognostic and diagnostic biomarker.

Declarations

Data availability statement: All the original data and information in this study are included in the article/Supplementary Material. If you need more information, you can contact the corresponding author.

Ethics statement: Research protocols involving humans and animals have been reviewed and approved by the ethics committee of Qingdao University Hospital. All participating patients have signed written informed consent. All research processes are carried out in accordance with the Declaration of Helsinki.

Author contributions: TY contributed to designing and supervising the study. CZ wrote the manuscript. YS and SS collected and analyzed the data. YJ, XJ, and PJ raised the mouse and checked the statistical method. QG and JD improved the correction manuscript. The final submitted manuscript is approved by all authors.

Funding: The capital health research and development of special (2020-2-1152) and the National Science and Technology Major Project (2018ZX10302205-005) provided this research fund.

Acknowledgments: The authors acknowledge Hua Guo and Shuo Han for their technical assistance. The authors also thank Professor Dexi Chen (Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China) for sharing his small animal in vivo laser confocal microscope and ultrasound imaging system for these studies.

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