The development of vasculature in vivo is an extremely complex process that requires temporal and spatial coordination between multiple cell types to produce an effective vessel. The formation of vasculature from preexisting blood vessels, known as angiogenesis, plays important roles in several physiological and pathological processes, including wound healing, organ development and growth, ischemia, inflammatory disorders, fibrosis, and cancer. Means to deconstruct these complicated biological systems are necessary to gain mechanistic insight into their development, function, and modulation that can be tested in in vivo models and ultimately the clinic. In this chapter, we will first review the classical in vitro techniques to study angiogenesis. Next, we will explore the exciting recent advances that rely on 3D multicellular systems to more accurately mimic vasculature development in vitro. Finally, we will discuss the applications of in vitro angiogenic methods to study related vasculature phenomena, such as vasculogenic mimicry.

The resistance to radiotherapy in lung cancer can be attributed to vasculogenic mimicry (VM) to some extent. Celecoxib (CXB), a selective inhibitor of cyclooxygenase-2 (COX-2), is reported as a radiosensitizer in non-small cell lung cancer (NSCLC). However, whether CXB can regulate VM formation via an off-target effect to radiosensitize NSCLC remains unclear. This study aimed to elucidate the mechanism underlying the radiosensitizing effect of CXB on NSCLC, i.e., whether CXB can inhibit VM formation via binding to newly identified targets other than COX-2. CXB radiosensitivity assay was performed in BALB/c mice bearing H460 xenografts and C57 mice bearing Lewis lung cancer (LLC) xenografts, which were divided into the control, CXB, irradiation (IR) treatment, and IR plus CXB groups. VM formation was observed using 3D Matrigel, periodic acid solution (PAS) staining, and immunofluorescence staining. The potential off-targets of CXB were screened using Protein Data Bank (PDB) database, MGLTools 1.5.6, and AutoDock Vina 1.1.2 and confirmed by Western blotting, enzyme activity assay, and RNA interference in vitro experiments and by immunohistochemistry in vivo experiments. CXB treatment almost eliminated the enhancement of VM formation by IR in vitro and in vivo, partially due to COX-2 inhibition. Four potential off-targets were predicted by molecular docking. Among them, aminopeptidase N (APN) and integrin alpha-V (ITAV) were remarkably inhibited in protein expression and enzyme activity in vitro or in vivo, consistent with the remarkable reduction of VM formation in H460 xenografts in BALB/c mice. In conclusion, CXB dramatically blocked VM through inhibiting newly identified off-targets APN and ITAV, other than COX-2, then radiosensitizing NSCLC.

The term \"vasculogenic mimicry\" (VM) refers to the phenomenon in which vascular-like channels, which are not lined by endothelial cells, are formed in tumors. Since its discovery in 1999, it has been observed in several tumor types and is proposed to provide blood perfusion to tumors in absence of co-apted or neo-angiogenic blood vessels. Pituitary tumors are generally slow growing, benign adenomas which are less vascularized than the normal pituitary gland. To date, VM in pituitary adenomas has not been described. In this histological study, we assessed the presence of VM in a series of surgically resected clinically non-functioning pituitary adenomas (NFPAs) using CD34 and Periodic Acid-Schiff (PAS) double staining. To identify VM, slides were assessed for the presence of CD34-negative and PAS-positive channels indicating that they were not lined by endothelial cells. The histological staining pattern suggestive of VM was noted in 22/49 (44.9%) of the specimens studied. VM was observed in both recurring and non-recurring NFPAs. The incidence of VM present varied from case to case and within groups. There was no association between the presence of VM and gender, tumor size, Ki-67 index, recurrence or cavernous sinus invasion. VM was not noted in cases of non-tumorous pituitaries. Our findings suggest the existence of a complementary perfusion system in pituitary adenomas, implying potential clinical implications with respect to response to therapy and clinical course. Further research is warranted to confirm the presence of VM in pituitary adenomas to elucidate its clinical relevance in patients diagnosed with a pituitary adenoma.