A number of recent reports have suggested a role for bone marrow-derived cells in tumor vasculogenesis. The relevant subsets of the adult wbm that appear to functionally participate in neovascularization are the angioblast-like EPC and the facilitative HSC or HPC cells [29,3,4,6,7,30,8
]. However, the degree of recruitment of these cells has not been effectively quantitated, the mechanisms of recruitment are not understood, and a specific role for these recruited cells in tumor vessel growth and development has not been fully elucidated. Thus, we employed a primitive lin-c-kit+Sca-1+
stem cell population (BMlin-
) that is phenotypically inclusive of these specific neoangiogenic subsets, in a syngeneic model of stem cell recruitment to tumors, to both quantify BMlin-
recruitment and address the question of BMlin-
function in tumor neovascularization.
From our biodistribution data (), we have determined quantitatively that BMlin-
cells are recruited to subcutaneously implanted LLCs, and that these cells are present in the tissue and not simply circulating through the tumor vasculature. However, the fraction of injected cells that are recruited to the tumor environment is far lower than that observed for recruitment of human CD34+
cells to the bone marrow in immunodeficient mice [31
], for recruitment of antigen-specific CD8+
T cells to antigen-expressing tumors [32
], or for the recruitment of neuronal progenitors to the same LLCs in vivo
]. These data suggest that BMlin-
cells exhibit a limited potential for the contribution to actively and aggressively growing tumors. However, it could be argued that very few long-term repopulating BMlin-
cells are actually required to initiate these events, just as very few true HSC are required to repopulate the bone marrow. It is possible that a few BMlin-
cells are recruited, and then go on to expand and differentiate in the tumor environment. The biodistribution data alone are not capable of determining the degree of cell proliferation, and thus we developed the bone marrow EGFP-chimeric animals to address these questions specifically.
Fluorescence imaging of live LLCs implanted into EGFP-chimeric mice at two different time points following bone marrow transplantation, coupled with indirect immunofluorescence staining of the harvested tumor sections, revealed a number of resident EGFP cells within the tumor mass. However, other than the presence of a few arrested EGFP cells within the tumor circulation, our data do not support a role for the recruited hematopoietic cells in tumor neovascularization, evident by the lack of coregistration of CD31/SMA staining and EGFP expression in tumors carried by EGFP-chimeric animals. However, in EGFP-transgenic mice that express EGFP ubiquitously, all tumor vessels identified exhibited both EGFP expression and staining for CD31 and SMA. Together, these data support the hypothesis that tumor vessel growth in LLCs occurs through sprouting of the existing host circulation—not through spontaneous vasculogenesis of circulating progenitor cells. Our observations are supported by a recent elegant study that examined the recruitment of Tie2-expressing mononuclear EPC to tumors [10
]. The authors observed Tie2-expressing cells in the tumor mass and in close proximity to tumor vessels after long-term growth (12 weeks) of recipient tumors, but did not observe any contribution of these cells to the tumor vasculature directly.
Although the present study was supported by recent reports, there remains a significant controversy as to the functional incorporation of hematopoietic cells in sites of neovascularization, and how these conflicting reports may be reconciled. There are specific and significant differences in the tumor models exploited in these studies, which may go some way toward explaining the differing observations reported. Probably of most critical consequence is the type of tumor used. Recent reports that demonstrated vascular incorporation of EPC or other hematopoietic cells have used either colon cancer cell lines [3
] or tumors of neuronal origin [4,21
], which may represent a significant departure from the LLC model employed in the current study. Even more interesting are the reports indicating that tumor growth is significantly retarded in the absence of a normally functioning hematopoietic system, or when hematopoietic subsets have been modified to limit their recruitment or potential [4,6,9
]. At initial interpretation, these data would suggest that hematopoietic cells do contribute directly to tumor vessel growth; however, recently, it has been hypothesized that HSC or HPC represent a “helper” subset that directs and stabilizes neovascularization within tumors, but does not itself participate in vasculogenesis [19
]. This hypothesis is supported not only by the retarded growth of tumors in the absence of functional HSC, but also by the location of these cells, in close proximity to new vessels in the developing tumor mass.
In conclusion, we have demonstrated that whereas long-term repopulating stem cells are recruited in low levels to subcutaneously implanted LLCs in syngeneic animals, neither them nor their progeny contributes directly to vasculo-genesis in this model. Together, these data would suggest caution in interpretation of the role of these cells in any site of spontaneous or induced neovascularization.