Outgrowth of blood vessels to form neovasculature is a fundamental process in solid tumor progression as tumors larger than 2 mm in diameter depend on blood vessels for nutrients and oxygen. Moreover, myriad angiogenic factors are secreted by tumors to induce angiogenesis and establish a nutrient metabolism network for tumor growth. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) constitute the primary activators of a critical pro-angiogenic signaling pathway. This pathway has attracted vast attention as a possible target for anti-tumor strategies, including inhibiting VEGF expression, blocking tumor cell signal transduction, and exhausting VEGF produced by tumor cells. Bevacizumab was the first humanized monoclonal antibody to inhibit neovascularization by binding soluble VEGF A and, subsequently, blocking the VEGFA/VEGFR interaction. In fact, bevacizumab has been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) as an anti-tumor agent 1. Neoadjuvant therapy with bevacizumab combined with chemotherapy has been applied to various types of solid tumors, including non-small cell lung cancer 2, breast cancer 3, glioblastoma 4, and ovarian cancer 5. However, the main challenge facing anti-VEGF therapy is drug resistance, which significantly limits long-term treatment 6.
Increasing evidence indicates that activation of alternative proangiogenic factors, such as b-FGF, PIGF, HGF, EGF, VEGF C, and angiopoietin 7,8 is associated with bevacizumab resistance. Furthermore, several mechanisms of bevacizumab resistance have been reported in glioblastoma 7–10, ovarian cancer 11, and non-small cell lung cancer 12, including: activation of the alternative pathway; (2) increased proliferation, migration, and invasion of tumors 13; vascular disorders caused by tumor hypoxia 14 and tumor metastasis 12; recruitment of vascular progenitor cells and regulatory factors 15; promotion of myeloid-derived suppressor cells, tumor-associated macrophages, cancer-associated fibroblasts, and non-cellular components, such as the extracellular matrix and cytokines in the microenvironment 10,16,17; production of vesicles containing proangiogenic molecules, such as VEGF, matrix metalloprotease-9 (MMP9), and hypoxia-induced factor-1α (HIF1α) 18,19. Based on these potential targets, angiogenin-2 (Ang-2) appears to be the most promising candidate for overcoming bevacizumab resistance. Indeed, an Ang-2/VEGF bi-specific antibody (vanucizumab) was developed by Genentech (South San Francisco, United States); however, the clinical trials were terminated before phase III, due to insufficient efficacy compared to bevacizumab alone. Therefore, intensive characterization of the molecular mechanisms that mediate anti-angiogenesis resistance may provide potential strategies to improve therapeutic efficacy and prolong patient prognosis.
Endothelial cell-specific molecule-1 (ESM1) is a secreted proteoglycan comprising a 20 kDa protein core and a dermatan sulfate 20, that was first cloned from HUVEC cells 21. ESM1 is highly expressed in vascular endothelial cells, i.e., epithelial cells of the distal renal tubules, bronchial tubes, and submucosal lung glands in normal tissues 22. ESM1 expression is also upregulated in lung cancer 23, gastric cancer 24, breast cancer 25, bladder cancer 26, and other malignant tumors and is associated with the inflammatory response and tumor progression. Moreover, ESM1 participates in tumor growth, migration, invasion, and angiogenesis 27. In this context, Scherpereel et al. found that tumors did not develop via subcutaneous injection of normal HEK293 cells but were formed by ESM1-overexpressing HEK293 cells in mice 28. Additionally, inhibiting ESM1 mRNA with siRNA reduces MMPs and epithelial mesenchymal transition (EMT)-related gene expression, thus, inhibiting tumor invasion in a colorectal cancer model 29. ESM1 is also highly expressed in “tip cells,” which mediate vascular growth, highlighting the potential role of ESM1 in vascular network formation 30. However, direct evidence to demonstrate the role of ESM1 in tumor angiogenesis, particularly in anti-angiogenesis therapy resistance, requires further investigation.
Angiogenesis requires multifaceted adjustment and balance during embryonic development and tumor growth. Gene-targeting approaches have confirmed that vascular system development is impaired without VEGF in embryonic mice, suggesting a fundamental role for VEGF in embryonic development and tumor angiogenesis 31. The Delta/Jagged–Notch system also regulates cell fate and, thus, influences cell proliferation, differentiation, and apoptosis. Delta-like ligand-4 (DLL4) is a membrane ligand of Notch1 and is required for normal vascular development and arterial formation in mice 32,33. Heterozygous DLL4 mutations can be lethal to embryos by causing a lack of well-defined major arteries and an increased number of vessel branches and vascular sprouts 32,33. Moreover, DLL4 is strongly expressed in renal cell carcinoma, stomach cancer, colorectal cancer, and metastatic breast cancer 34–36. High levels of DLL4 is associated with a reduced efficacy of bevacizumab in advanced colorectal cancer 37. DLL4 is normally induced by VEGF and is a negative-loop feedback regulator that inhibits vascular sprouting and branching 31. VEGF blockade attenuates the formation of new tumor vessels, and normalization of the remnant vessels leads to tumor recession. Similarly, DLL4 blockade decreases tumor growth as DLL4 inhibition increases the density of poorly functional vascular sprouts and branches, inducing disordered tumor vessels. Thus, coordination between DLL4 and VEGF may promote functional blood vessel formation. In humans, it has been found that VEGF and DLL4 are strongly co-expressed in tumor tissues 38. The DLL4/Notch pathway is being explored as an alternative target for antiangiogenic therapies. Given that several bispecific antibodies target DLL4 and VEGF, including dilpacimab 39 and ABL001 38, solid tumor development requires abundant functional blood vessels to provide proper nutritional support.
Our group previously reported that the curative effect of bevacizumab is significantly improved by neutralizing M2b macrophage-related TNFα with an anti-TNFα nanobody 16, however, elucidating the underlying mechanism of TNFα-induced bevacizumab resistance remains unclear. Herein, we analyze the unique expression patterns of genes from bevacizumab-sensitive or bevacizumab-resistant cancer cells using RNA-seq analysis. We also explore the potential role of ESM1 in mediating TNFα-induced resistance to bevacizumab, and investigate the associated signaling pathways.