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We read with interest the article by Hilbe et al concerning the contribution of endothelial progenitor cells (EPCs) to the vasculature in non-small cell lung cancer (NSCLC).1 In their study, the authors conclude that “increased numbers of CD133 positive EPCs can be found in NSCLC tissue and these cells seem to contribute to the formation of capillaries”. Although it is interesting and worthy of further study, in our view, the evidence presented in their paper is unconvincing. However, the problems are not apparent to readers unfamiliar with the background or pitfalls of this specialised topic.
The development of a vascular network plays a crucial role in the development and function of normal tissues and organs, in addition to tumour growth and metastasis. Understanding how tumours acquire their vasculature is indispensable for developing novel therapeutic approaches. However, the vascularisation of tumours is very complex, consisting of sprouting, vessel cooption, glomeruloid angiogenesis, mosaic vessel formation, vascular mimicry, and intussusceptive angiogenesis.2–5 Furthermore, there is emerging evidence that putative angioblasts, also known as EPCs, might persist in adult life and contribute to the vascularisation of tumours.6
EPCs have been isolated from peripheral blood and bone marrow. Similar to embryonic angioblasts, EPCs have the capacity to proliferate and differentiate into mature endothelial cells (ECs). To date, no clear definition exists as to when an EPC turns into a mature, fully differentiated endothelial cell in vivo. Early EPCs (localised in the bone marrow or immediately after migration into the circulation) are CD133+/CD34+/VEGFR-2+ (vascular endothelial growth factor receptor 2 positive) cells, whereas circulating EPCs are positive for CD34/VEGFR-2/CD31/VE-cadherin, lose CD133, and begin to express von Willebrandt factor. In general, it is widely accepted that the loss of CD133 reflects the transformation of early circulating EPCs into more mature endothelial-like cells.7
Hilbe et al identified early EPCs by CD133 labelling not in peripheral blood or bone marrow, but in the endothelial tubes of NSCLC tissue. The key evidence for their identity came from immunohistochemical studies.1
In our view, there are three problems with the arguments put forward by Hilbe et al.1 First, the presumed localisation of EPCs on serial frozen sections is not convincing because neither multiple microvessel labelling for CD133 and EC markers nor immunoelectron microscopical examination was performed. Because the cellular boundaries cannot be seen in the figures provided, it is unclear what types of cells are CD133+.
Second, CD133 is not exclusively expressed on early—but not circulating or committed—EPCs. In addition to being expressed on haemopoietic stem cells, CD133 also serves as a marker for non-haemopoietic progenitor cells, such as neural stem cells, embryonic stem cell lines, and adult stem cells with a pluripotent differentiation capacity.8 Furthermore, CD133 was found to be expressed on tumour cells of epithelial origin.9 The possibility that the CD133+ cells in the NSCLC tissue are not ECs was not explored.
Third, a convincing argument for the presence of EPCs in the NSCLC tissue depends on the unequivocal identification of this cell type. It is not clear why Hilbe et al did not use more than one early stem cell marker to detect EPCs.1 Their method differs from several earlier studies that used different antibody combinations.10
The involvement of alternative vascularisation mechanisms—including vasculogenesis—in the tumour blood supply has broad biological and medical importance. We found the message emerging from the Hilbe study a valuable contribution to our knowledge of the vasculogenesis in tumour tissue. Our critical comments are intended simply as a reminder that the extent of these phenomena is still unclear, and can only be determined by rigorous examination.
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