Targeting tumor vascular endothelial cells for hepatocellular carcinoma treatment

Abstract

Liver cancer is the sixth most common cancer and the third leading cause of cancer death worldwide. The predominant type of primary liver cancer is hepatocellular carcinoma (HCC). Tumor vascular endothelial cells (VECs), a major component of cells in the microenvironment of HCC, play multifaceted roles in contributing to tumor angiogenesis, proliferation, and migration, as well as therapeutic resistance by attracting myeloid-derived suppressor cells and suppressing cytotoxic CD8 T cell differentiation and function. Recently, Wu et al reported that apatinib, an inhibitor of vascular endothelial growth factor receptor 2, can inhibit tumor VEC glycolysis by regulating phosphatidylinositol 3-kinase/protein kinase B/6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 signaling pathway to suppress HCC progression. With great interest, this editorial paper aims to review the function and key molecular signaling pathways of tumor VECs in HCC initiation and progression and summarize potential treatment options in clinical trials.

Key Words: Hepatocellular carcinoma; Vascular endothelial cells; Angiogenesis; Molecular signaling pathways; Therapy

Core Tip: Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, without effective treatment strategies for unresectable HCC. Tumor vascular endothelial cells have distinct features and functions from liver sinusoidal endothelial cells, lining the interior layer of cancer vascular vessels, and they contribute to tumor angiogenesis, proliferation, migration, and therapeutic resistance. Targeting liver tumor vascular endothelial cell molecular signaling pathways in the promotion of liver fibrosis, immunosuppressive cell infiltration, angiogenesis, and secretion of pro-metastatic factors is a promising strategy for HCC therapy or as a synergistic treatment.

INTRODUCTION

Liver cancer is the sixth most common cancer worldwide[1]. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, which is the third leading cause of cancer death in 46 countries worldwide[1]. Tumor vascular endothelial cells (VECs) have distinct features and functions from liver sinusoidal endothelial cells (LSECs), and they constitute a major component of cell populations in the microenvironment of HCC, which play multifaceted roles in contributing to cancer cell angiogenesis, proliferation, and migration[2]. Tumor VECs line the luminal side of blood vessels within the tumors, whereas LSECs are fenestrated endothelial cells and liver gatekeeper cells, lining hepatic sinusoids to mediate immune tolerance and protect against liver inflammation, injury, and fibrogenesis, with the presence of transcellular pores[3]. Tumor VECs have high expression of vascular endothelial growth factor (VEGF) receptors (VEGFR)1 and VEGFR2 and matrix metalloproteases compared to LSECs to support their proliferation and migration[4]. Tumor VECs also impact immunotherapeutic efficacy by attracting myeloid-derived suppressor cells and suppressing cytotoxic CD8 T cell differentiation and function[5]. Single-cell RNA sequencing data have also demonstrated that tumor VECs can be subclassified into different subtypes. For example, Sun et al[6] reported that there were two populations of tumor VECs. One population played an important role in tumor cell proliferation and inflammation. In contrast, the second population contributed to both cell proliferation and metabolism. The expression of genes such as platelet-derived growth factor receptor-beta, placental growth factor, JUN, and nuclear receptor subfamily 4 group A member 1 changes in these two populations during HCC progression[6]. Analysis of single-cell RNA sequencing data also revealed that VECs interacted with cancer-associated fibroblasts to promote tumor progression and metastasis[7]. Given the important roles of VECs in the tumor microenvironment, they can be targeted to improve the efficacy of immunotherapy and chemotherapy[2].

Recently, Wu et al[8] reported that apatinib can inhibit tumor VEC glycolysis by regulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 signaling pathway to suppress HCC progression. With great interest, this editorial paper aims to review the key molecular signaling pathways of tumor VECs in HCC initiation and progression and summarize potential treatment options in clinical trials.

FUNCTIONS OF VECS IN THE PATHOGENESIS OF HCC

The role of VECs in inflammation

The expression of intercellular adhesion molecule 1 (ICAM-1) is upregulated in VECs in HCC, which regulates the recruitment and migration of inflammatory cells in the tumor microenvironment[9]. Similarly, injured LSECs regulate the infiltration of immune cells in chronic liver disease and HCC by expressing chemokines such as C-X-C motif chemokine ligand 9 (CXCL9) and CXCL16[3]. The spatial transcriptomic data demonstrated that the densities or cell numbers of VEGF A (VEGFA+) macrophages and CD34+ endothelial cells were increased within HCC tumor tissues compared to the paired nontumor tissues, and the ligand-receptor interactions such as VEGFA-FLT1/VEGFR1 or VEGFR2/KDR, CXCL12/C-X-C chemokine receptor type 4 (CXCR4), and interleukin-1A/interleukin 1-R1 mediated their communication to regulate inflammation and tumor cell growth[10]. In addition, high fluid shear stress was able to increase the adhesion of HCC cells to inflammatory endothelial cells by regulating the expression of ICAM-1 and vascular adhesion molecule 1, resulting in liver cancer metastasis[11].

The function of VECs in fibrosis

In an in vitro spheroid model, treatment with free fatty acid can increase pro-collagen type I alpha-1 production in the triple-culture spheroid (primary human hepatocytes, nonparenchymal cells, and LSECs) but not in the coculture system (primary human hepatocytes plus nonparenchymal cells), as well as the mRNA expression of genes lysyl oxidase and fibronectin 1[12]. The expression of platelet-derived growth factor produced by endothelial cells and macrophages can stimulate the activation of hepatic stellate cells by interacting with its receptor platelet-derived growth factor receptor to promote liver fibrosis[13].

The effect of VECs on angiogenesis

Some VECs are derived from bone marrow-derived endothelial progenitor cells and have high expression of ICAM-1, vascular adhesion molecule 1, and VEGF, which contribute to tumor vascular formation in HCC[14]. In addition, tumor VECs (e.g., exportin 1/XPO1-expressing cells) can interact with other cells such as monocytes/macrophages, T cells/natural killer cells, and endothelial cells through MIF-CD74 + CXCR4/CD44 and VEGFA-VEGFR1R2 ligand-receptor interactions. Cell-cell interaction or communication induces the recruitment of immunosuppressive cells and promotes angiogenesis[15].

The induction of T cell exhaustion

With chronic peptide-induced stimulation, LSECs can promote antigen-specific CD8+ T cell exhaustion by increasing the expression of immune checkpoint markers such as programmed cell death protein 1 (PD-1), inhibitory molecules such as T cell immunoglobulin and mucin-domain containing-3, and T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domain[16].

The contribution of VECs in carcinogenesis

The spatial transcriptomic profiling analysis has demonstrated numerous ligand-receptor interactions between tumor cells and endothelial cells, such as VEGFA-VEGFR1 and LGALS9-CD44 interactions, which modulate the local tumor biology[17]. Upregulation of ephrin receptor A1 protein in HCC tumor cells can increase stromal cell-derived factor 1 or CXCL12 expression to recruit CXCR4-expressing endothelial progenitor cells into the tumor microenvironment to promote vascularization and HCC progression[18]. The expression of CXCR4 in tumor VECs was regulated by monocytes and macrophages in the perivascular areas, which can function as a therapeutic target and a predictive marker for sorafenib treatment in HCC patients[19].

MicroRNAs (miRNAs) play a pivotal role in angiogenesis by modulating the expression levels of growth factors. For example, the expression of miR-200b-3p in HCC tissues was increased compared to adjacent non-tumor parts, which decreased endothelial cell migration, proliferation, and tube-forming capacity to suppress angiogenesis in the HCC microenvironment[20]. Another study showed that miR-199a-3p treatment inhibited HCC cell growth, migration, invasion, and angiogenesis by suppressing the expression of VEGF in cancer cells and the expression of VEGF receptors VEGFR1 and VEGFR2 in endothelial cells[21].

Pre-metastatic vascular niche

In metabolic dysfunction-associated steatohepatitis, a choline-deficient L-amino acid-defined diet treatment upregulated adhesion molecule ICAM-1 to facilitate the metastasis of melanoma cells[22]. Platelet endothelial cell adhesion molecule-1 (or CD31) can also increase HCC metastasis by inducing the epithelial-mesenchymal transition via activation of integrin beta-1/focal adhesion kinase/AKT signaling pathway[23]. In conclusion, VECs contribute to inflammation, fibrosis, and angiogenesis in the HCC microenvironment (Figure 1), leading to cancer cell growth and metastasis.

Figure 1 Vascular endothelial cells play multiple roles in the hepatocellular carcinoma microenvironment. All cartoons in this figure were prepared using Biorender (Supplementary material). A: Inflammation. The ligand-receptor interactions, such as vascular endothelial growth factor (VEGF) A-FLT1 [VEGF receptor 1 (VEGFR1)] or VEGFR2/KDR, stromal cell-derived factor 1 (or C-X-C motif chemokine ligand 12)-C-X-C chemokine receptor type 4 (CXCR4), and interleukin-1A/interleukin-1R1, mediate vascular endothelial cell communication with other cells (e.g., macrophages) to regulate inflammation and tumor cell growth; B: Fibrosis. Platelet-derived growth factor produced by endothelial cells can stimulate hepatic stellate cell activation by binding its receptor platelet-derived growth factor receptor to promote liver fibrosis; C: Angiogenesis. Tumor vascular endothelial cells can interact with monocytes/macrophages, and T cells/natural killer cells through MIF-CD74 plus CXCR4/CD44 and VEGFA-VEGFR1/2 ligand-receptor interactions to induce the recruitment of immunosuppressive cells and promote angiogenesis; D: Hepatocellular carcinoma (HCC) progression. For example, overexpression of ephrin receptor A1 protein in HCC tumor cells can increase C-X-C motif chemokine ligand 12 expression to recruit CXCR4-expressing endothelial progenitor cells into the tumor microenvironment to promote vascularization and HCC progression. VEGFA: Vascular endothelial growth factor A; CXCL12: C-X-C motif chemokine ligand 12; IL: Interleukin; ICAM-1: Intercellular adhesion molecule 1; HCC: Hepatocellular carcinoma; LSEC: Liver sinusoidal endothelial cell; LOX: Lysyl oxidase; FN1: Fibronectin 1; PDGF: Platelet-derived growth factor; PDGFR: Platelet-derived growth factor receptor; HSC: Hepatic stellate cell; CXCR4: C-X-C chemokine receptor type 4; VEGFR: Vascular endothelial growth factor receptor; VACAM-1: Vascular adhesion molecule 1; VEGF: Vascular endothelial growth factor; NK: Natural killer; VEC: Vascular endothelial cell; EphA1: Ephrin receptor A1; SDF-1: Stromal cell-derived factor 1.

MOLECULAR TARGETS OF VECS IN HCC TREATMENT

Wu et al[8] reported that apatinib treatment inhibited tumor VEC glycolysis by regulating PI3K/AKT/6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 signaling pathway to suppress HCC progression. With great of interest, we reviewed other molecular targets of VECs in HCC therapy.

Treatment with natural hormone melatonin (MLT) secreted primarily by the pineal gland suppressed hypoxia-induced VEGF and reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs) in vitro. It also had a synergistic effect with hypoxia inducible factor (HIF)-1α inhibitor KC7F2 in the reduction of ROS and VEGF secretion, cell viability, and tube formation of HUVECs[24]. Furthermore, MLT therapy inhibited the viability of HUVECs in a dose-dependent manner and reversed the increased cell viability and tube formation that were induced by hypoxia/VEGF/H₂O₂. Additionally, treatment with HIF-1α inhibitor KC7F2 and MLT synergistically reduced the release of ROS and VEGF and inhibited the cell viability and tube formation of HUVECs. The function of MLT was evaluated in vitro using cultured HUVECs under normoxic (21% of O₂, 5% of CO₂ and 74% of N₂) or hypoxic conditions (1% of O₂, 5% of CO₂ and 94% of N₂), and the tube formation of cultured HUVECs on primary Matrigel was assessed through microscopy observation.

Treatment with Toll-like receptor 4 inhibitor TAK-242 reduced the proportions of liver endothelial cells, dendritic cells, Kupffer cells, and neutrophils, but increased hepatocytes to prevent metabolic dysfunction-associated steatohepatitis-associated HCC development[25]. Genetic depletion of PI3Kγ in haematopoietic/endothelial cells inhibited neutrophil recruitment and hepatocyte proliferation induced by diethylnitrosamine administration in mice with a high-fat diet[26]. The expression of fatty liver binding protein 4 (FABP4) in endothelial cells was increased in HCC samples from human patients with metabolic syndrome compared to other HCC samples from patients with other chronic diseases, such as hepatitis and alcohol consumption. FABP4 was highly expressed in peritumor endothelial cells, which showed a pro-angiogenetic effect in cancer cells[27].

One study revealed that CXCL12-expressing HCC-associated endothelial cells can inhibit the differentiation of cytotoxic CD8+ T cells and chemoattract CXCR4-expressing myeloid-derived suppressor cells into the tumor microenvironment to induce immunosuppression and promote HCC growth[5]. This study further showed that treatment with a bispecific antibody inhibiting both CXCL12 and PD-1 significantly improved anti-HCC immune response and therapeutic efficacy[5]. Another study showed that anti-VEGFR2 treatment delayed HCC growth but did not improve animal survival, as anti-VEGFR2 induced programmed-death ligand 1 expression in HCC cells partly through the expression of interferon-γ in endothelial cells[28]. Therefore, dual anti-PD-1/VEGFR2 therapy enhanced the efficacy of each therapy alone[28].

LSEC-targeted delivery of simvastatin can restore the quiescence of activated hepatic stellate cells by inhibiting LSEC capillarization through activation of Kruppel-like factor 2 (KLF2)-nitric oxide pathway, and enhance natural killer T cell recruitment to improve HCC therapy[29]. Silencing circular RNA hsa_circ_0003575 can increase oxidized low-density lipoprotein-induced VEC proliferation and angiogenesis[30]. UBE2CP3 overexpression in HCC cells promoted HUVEC proliferation, migration, and tube formation by activating extracellular signal-regulated kinase/HIF-1α/ribosomal protein S6 kinase beta-1/VEGFA signaling pathway[31]. Intratumor delivery of exosomes containing high levels of miR-200b-3p suppressed tumor growth by reducing erythroblast transformation-specific-related gene and VEGF expression and angiogenesis[32].

Among these molecular targets or therapies (Table 1), some show promising effects in clinical trials. For example, the combination therapy of apatinib with transarterial chemoembolization[33], and hepatic arterial infusion chemotherapy[34], PD-1 inhibitors (camrelizumab)[35] and sintilimab[36] have been evaluated in phase 2 clinical trials (e.g., https://ClinicalTrials.gov, trial number NCT04411706, accessed on June 15, 2025). Apatinib provides beneficial effects for unresectable HCC. Treatment with an anti-VEGF monoclonal antibody (bevacizumab) significantly reduced tumor growth and circulating levels of VEGFA and stromal cell-derived factor-1, or CXCL12[37]. The plasma levels of VEGF and HIF-1α in HCC were negatively and significantly associated with the treatment response of sorafenib, and patients with high tumor expression of VEGF and HIF had a poor outcome to treatment[38]. In addition to immunotherapy and targeted therapy, nanoparticle-mediated therapy provides promising clinical benefits in HCC therapy in the future[39]. Moreover, some targets such as circular RNAs (e.g., hsa_circ_0036683) and KLF2 are potential biomarkers for HCC prognosis[40,41]. Targeting FABP4 and KLF2 shows effective responses for HCC therapy in cultured cells and animal models[42-44]. For current HCC treatments, the effect of each single treatment varies at different stages of HCC and on different patients. Therefore, combinational therapy should be developed for the treatment of unresectable HCC.

Table 1 Treatments targeting endothelial cells in hepatocellular carcinoma.

Category	Treatments	Signaling pathways	Ref.
Tyrosine kinase inhibitor	Apatinib	Suppression of PI3K/AKT/PFKFB3 signaling pathway by selectively inhibiting VEGFR2	[8]
Natural hormone	Melatonin	Restricting the release of ROS and VEGF secretion in human umbilical VECs	[24]
TLR4 inhibitor	TAK-242	Reduction of the endothelial cell population, as well as dendritic cells, Kupffer cells, and neutrophils	[25]
PI3Kγ inhibition	Genetic depletion	Genetic depletion of PI3Kγ in haematopoietic/endothelial cells inhibited neutrophil recruitment and hepatocyte proliferation induced by diethylnitrosamine administration in mice with a high-fat diet	[26]
FABP4 inhibitor	BMS309403	Suppression of tumor growth	[27]
CXCL12 and PD-1 inhibition	A bispecific antibody	Improvement in CD8+ T cell differentiation and suppression of MDSC cell infiltration in the tumor microenvironment	[5]
Anti-PD-1/VEGFR-2 therapy	Anti-PD-1 and anti-VEGFR2 antibodies	Overcoming therapeutic resistance, induction of cytotoxic CD8 T cell infiltration, and reduction of regulatory T cell and monocyte infiltration	[28]
Kruppel-like factor-nitric oxide pathway inhibition	Simvastatin	Inhibiting LSEC capillarization and enhancing natural killer T cells recruitment to suppress HCC	[29]
Circular RNA regulation	Loss of function study	Silencing circular RNA hsa_circ_0003575. Circular RNA can increase oxidized LDL-induced VEC proliferation and angiogenesis	[30]
Long non-coding RNA UBE2CP3	Overexpression	UBE2CP3 overexpression in HCC cells promoted HUVEC proliferation, migration, and tube formation by activating ERK/HIF-1α/p70S6K/VEGFA signaling pathway	[31]
Exosomal delivery of miRNA	MiR-200b-3p	Intratumor delivery of exosomes containing high levels of miR-200b-3p suppressed tumor growth by reducing ERG and VEGF expression and angiogenesis	[32]

PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; VEGFR2: Vascular endothelial growth factor receptor 2; ROS: Reactive oxygen species; VEGF: Vascular endothelial growth factor; VEC: Vascular endothelial cell; TLR-4: Toll-like receptor 4; FABP4: Fatty liver binding protein 4; CXCL12: C-X-C motif chemokine ligand 12; PD-1: Programmed cell death protein 1; MDSC: Myeloid-derived suppressor cell; LSEC: Liver sinusoidal endothelial cell; LDL: Low density lipoprotein; UBE2CP3: Ubiquitin conjugating enzyme E2C pseudogene 3; HUVECs: Human umbilical vein endothelial cells; ERK: Extracellular signal-regulated kinase; HIF: Hypoxia inducible factor; P70S6K: Ribosomal protein S6 kinase beta-1; VEGFA: Vascular endothelial growth factor A.

CONCLUSION

VECs in the tumor microenvironment and LSECs in liver tissues play crucial roles in HCC initiation and progression. VECs contribute to inflammation, fibrogenesis, angiogenesis, pre-metastatic niche in the tumor microenvironment, and therapeutic resistance. Treatments by regulating VEC function and population, such as tyrosine kinase and Toll-like receptor 4 inhibitors and genetic and epigenetic modulation, can inhibit HCC progression.