While the contribution of BM-derived EPCs to tumor neovessel formation has been reported in mice and humans (12
), the inability to deliver transgenes specifically to EPCs in vivo
has precluded the analysis of their biological function, and assessment of their therapeutic potential. In this study we show for the first time using LV Id1 reporter constructs, and Id1+/GFP
fluorescent reporter mice (24
), that the Id1 gene is selective for EPCs, and can be used to track EPCs in the BM, blood, tumor-stroma, as well as incorporated in tumor vasculature. This identification of luminally incorporated mature EPCs in the tumor vasculature expressing the Id1 gene, also validates that Id1 marks cells that are true EPCs. Furthermore, this finding validates that EPCs do incorporate into tumor vasculature. To the best of our knowledge this study provides the first direct in vivo
evidence of EPCs being marked by a single unique marker in each of these tissues.
To confirm the selectivity of Id1 for BM-EPCs, the LV-Id1pr/p construct was used to deliver the suicide transgene HSV-tk
. Following administration of GCV, EPCs were specifically ablated resulting in a 60–70% reduction in CEPs, as well as angiogenesis inhibition and impaired tumor growth. This Id1 directed specific EPC ablation also did not significantly affect hematopoiesis or other hematopoietic cell populations. These findings are consistent with observations of Id1 knockout mice and their WT littermates, which showed no difference in hematopoietic progenitors (HPCs) (39
). Furthermore, although, recent studies have shown that continuous serial transplantation of Id1 knockout BM results (ultimately) in impaired engraft potential (due to a reduction in long term repopulating hematopoietic stem cells/HSCs), Id1 was found to be dispensable for short term recovery of HSCs (24
). This is supported by the observation in this study that acute suppression of Id1 in the adult BM compartment, while resulting in tumor angiogenic defects was not associated with HSC defects in our HSV-tk
/GCV treated animals.
The LV-Id1pr/p construct was also designed to express shRNAi designed after an endogenous miRNA and determine the function of endogenous EPC-specific genes, Id1 and VEGFR2 in tumor angiogenesis. ShRNA-mediated suppression of Id1 resulted in EPC mobilization defects (a 70–80% reduction in CEPs), associated with severe angiogenesis inhibition and impaired tumor growth, with no significant change in cells of the hematopoietic lineage (B-cells, T-cells, CD11b+
myeloid cells and Tie2+
monocytes). This result is consistent with the observations made following the HSV-tk
delivery in Id1 transgenic mice (14
). However, our result differs slightly from that of Jankovic et al
), who reported reduced number of circulating lymphocytes in the PB of resting Id1 knockout mice. Possibly, in our study, acute and short term suppression of Id1 in the BM compartment is devoid of the developmental compensations in the hematopoietic system associated with the Id1 knockout animal. In another study, Lyden et al
) showed that angiogenesis inhibition in the Id1 mutant was due to defects in mobilization of both VEGFR2+
EPCs and VEGFR1+
hematopoietic cells. However, the use of the combined Id1+/−
genotype in their study may have resulted in defects in VEGFR1+
cell mobilization, as a result of Id3 loss; as described (41
). Furthermore, we have observed that Id1 silencing (either by shRNAi or in Id1 knockout mice) specifically affects VEGFR2+
EPCs, and not VEGFR1+
). In Gao et al
) we showed that suppression of Id1 in the whole BM by shRNAi leads to EPC and tumor angiogenic defects. In this study, restricted delivery of shRNAi to Id1+
expressing cells using the Id1 promoter provides further compelling and direct evidence for the role of Id1 in EPC mobilization in the context of tumor challenge.
Similarly, EPC-specific VEGFR2 knockdown also resulted in loss of EPC function, associated with vessel loss and impaired tumor growth. Notably, administration of VEGFR2 blocking antibody has been previously shown to have anti-angiogenic effects (19
), however, anti-VEGFR2 antibody is not specific to EPCs and also recognizes VEGFR2 expressed on endothelial cells in nascent blood vessels (42
). Therefore, it has been difficult to discern whether the anti-angiogenic phenotype observed in these studies are due to targeting EPCs, mature vessels or both. Given that VEGFR2 suppression was strictly confined to the EPCs in the BM microenvironment, our results provide the most direct evidence for the role of VEGFR2 in EPC-mediated tumor angiogenesis.
Even though, luminally incorporated BM-derived Id1pr/p+
ECs represent a small fraction of the total tumor vasculature, specific ablation demonstrated that EPCs play a critical role in angiogenesis-mediated tumor growth. We have previously shown that tumor recruited EPCs secrete proangiogenic factors (20
), suggesting that in addition to providing structural support to nascent vessels EPCs have paracrine function in vessel recruitment. This makes them uniquely important targets for antiangiogenic cancer therapy. However, our results do not discount the perivascular role other tumor recruited BM-derived hematopoietic cells (2
). Conceivably, each component of the tumor-stroma plays a distinct role in tumor progression, and elimination of specific cell populations may drastically impact tumor growth. Furthermore, variability in reported contribution of EPCs across different mouse tumor models/strains [0% Purhonen et al
); 2–20% Nolan et al
); to 90% Lyden et al
); summarized in review Gao et al
)], means there is a need for a method to dynamically track and stage EPC involvement. Therefore, given that the role of tumor associated BM-EPCs in cancer progression remains the subject of debate, the selectivity of Id1 for EPCs, and its study through gene manipulation in vivo
, provides a key tool for further investigation. Furthermore, as the BM-contributes to the tumor microenvironment, LV-delivery of tissue-specific promoters driving RNAi may be used to understand the role of BM-derived cells in tumor biology.