Early reports examining the effects of VEGF blockade and other antiangiogenic therapies raised the hopes that these agents may substantially slow or stop tumor growth, and that therapeutic resistance to these agents would be less likely to occur, at least in part, because the target was diploid and not prone to the same genetic instability as tumor cells (37
). However, both preclinical studies and clinical experience in lung cancer and other solid tumors (12
) indicate that the vast majority of solid tumors either exhibit primary (intrinsic) resistance or will eventually acquire resistance to the effects of antiangiogenic therapy. Although to date most studies of therapeutic resistance to anticancer drugs have focused on the role of tumor cells, recent studies have suggested that host factors, including tumor stroma, may play an important role in resistance to angiogenesis inhibitors (14
In this study, we used mouse- and human-specific profiling of human NSCLC xenografts in mice to investigate stromal and tumor cell changes occurring in tumors that acquired resistance to BV. This analysis revealed that changes in gene expression, and particularly changes in angiogenesis-related genes, occurred predominantly in stromal and not tumor cells. This observation reinforces the notion that tumor stroma may play an important — and potentially dominant, in at least some circumstances — role in VEGF inhibitor resistance.
Pathway analyses highlighted that among these stromal changes, there were multiple genes in the Egfr
pathways that were upregulated in resistant tumors (e.g., Epgn
, and Fgfbp1
) and that the EGFR pathway appeared to be a central gene interaction pathway. EGFR and FGFR2 upregulation was confirmed using species-specific RT-PCR as well as IHC. As noted below, upregulation of the bFGF/FGFR2 pathway has previously been observed by our group and others in VEGF inhibitor resistance (16
), but to our knowledge, a role for stromal EGFR has not been reported previously. We therefore investigated this pathway in greater detail using 3 models: subcutaneous and orthotopic models of acquired resistance (H1975 and H441, respectively) and a model of relative primary resistance (A549). Tumor cells from all 3 models are known to be relatively resistant to EGFR blockade in vitro (26
In both models of acquired BV resistance, there was a significant increase in activated EGFR that largely, but not exclusively, localized on VSCs, which were predominantly pericytes (Figures , , and ). No significant p-EGFR was detectable on VSCs of control tumors. This was accompanied by an increase in pericyte coverage and a pattern of less tortuous, normalized revascularization in the BV-resistant tumors. Dual inhibition of VEGFR and EGFR pathways reduced pericyte coverage of tumor vessels compared with BV alone, which indicates that EGFR signaling plays a functional role in pericyte coverage of tumor vessels in the models studied. Dual targeting also significantly delayed the emergence of resistance and prolonged survival in the H441 model, with similar trends observed in the H1975 model (Figures and ). To our knowledge, this is the first evidence demonstrating a potential role for EGFR signaling in pericytes or other stromal cell populations of the tumor microenvironment in resistance to VEGF pathway inhibition in murine models of NSCLC.
Consistent with our observation regarding EGFR in tumor pericytes, a recent study found that the EGFR TKI gefitinib significantly suppressed tumor-associated pericyte function (46
). During the revisions of this manuscript, other investigators reported a role for the stromal heparin-binding Egf/Egfr (Hb-Egf/Egfr) signaling pathway in the progression of a pancreatic neuroendocrine tumor model of EGFR-targeted inhibition (47
). The authors demonstrate that stromal cell–derived Hb-Egf activates the EGFR pathway in perivascular cells, contributing to increased pericyte coverage and angiogenesis. These reports provide further support for a role of EGFR signaling in pericyte function in tumor revascularization.
A recent study has identified a role for PDGF-C expressed by tumor-associated fibroblasts in VEGF inhibitor resistance (25
) and in attenuating tumor response to anti-VEGF treatment in a model of glioblastoma (48
). PDGFR signaling in pericytes has also been implicated in vessel maturation, and recent evidence indicates that VEGF signaling suppresses pericyte PDGFR signaling, inhibiting vessel maturation (49
). Somewhat surprisingly, we did not observe upregulation of any PDGFRs or ligands. In contrast, we noted modest but statistically significant downregulation of the stromal genes Pdgfa
, and Pdgfrb
. Given the role of the PDGF family in multiple tumor processes, including pericyte recruitment and function (50
), it appears that pericyte-expressed EGFR may play a complimentary or compensatory role in the increased pericyte coverage observed in the acquired resistance models. Although the current study does not address this issue, it will be interesting to determine whether increased pericyte EGFR signaling in the H441 model (in the absence of increased EGFR gene or protein levels) is driven by increased ligand production or by reduced VEGFR-driven inhibition of signaling, as observed for pericyte PDGFR.
In the A549 model, stromal EGFR was also upregulated in BV-resistant tumors, but was localized exclusively to tumor endothelium, not VSCs. As expected, dual VEGFR/EGFR inhibition did not reduce pericyte coverage in this model, but did significantly delay the emergence of resistance compared with BV alone (Figure ). This observation highlights that a signaling pathway may play different roles in tumor stroma depending on the cellular context. Studies examining EGFR distribution on endothelium suggest that it is restricted to blood vessels supplying pathologic tissues (52
), where it activates angiogenic programs (53
). Others have reported that EGFR is activated on endothelium when tumor cells express EGFR ligands, such as TGF-α or EGF (54
Activation of the bFGF/FGFR2 pathway has previously been shown to be a critical regulator of the angiogenic switch (56
) and to be upregulated in response to antiangiogenic therapy (17
). We observed an approximately 6-fold increase in stromal Fgfr2
gene expression in tumors with acquired resistance and, consistent with this finding, an increase in the number of FGFR2-expressing cells in these tumors. This immunoreactivity appeared to be largely, but not exclusively, on tumor endothelium. This suggests that the FGFR2 pathway may promote VEGF-independent endothelial survival, as previously observed in other preclinical models (57
), although we cannot rule out the possibility that it plays a role in other nonendothelial stromal cells. Circulating levels of bFGF were also elevated in the plasma of mice bearing BV-resistant tumors. This observation is notable in light of our recent observation that acquired resistance to chemotherapy and BV in colorectal cancer patients is associated with an increase in circulating bFGF (45
), which suggests that similar mechanisms may be occurring in cancer patients.
The mechanisms underlying regulation of tumor stromal genes altered in resistant tumors remain to be established and are likely to differ in the various stromal cell types. Expression of many of the genes, including CAIX
), and EGFR
family members, is known to be regulated by hypoxia or to correlate with expression of HIF1α, as previously reviewed (60
). One possible explanation is that BV therapy initially triggers a substantial decrease in tumor MVD and increases tumor hypoxia (61
), inducing upregulation of hypoxia-dependent pathways. It is worth noting, however, that BV resistance was not associated with significant increases in many stromal genes known to be upregulated by hypoxia, and many of the genes upregulated in BV resistance are not known to be regulated by hypoxia. Hypoxia is therefore likely to be only one of many factors — both host and tumor cell dependent — likely to affect the resistant tumor and its microenvironment. These regulators of the stromal response merit further investigation.
Resistance to VEGF inhibition was also associated with different patterns of vascular remodeling in the models of acquired and primary resistance. In the H1975 model of acquired resistance, short-term treatment with BV during the sensitive phase initially induced a reduction in MVD; an increase in EC apoptosis, as observed in other studies (29
); and tumor shrinkage. This was followed by the development of resistance, marked by a pattern of normalized revascularization with increased MVD, reduced EC apoptosis, and a higher degree of pericyte coverage (Figure and Supplemental Figure 1). These effects appeared to be VEGFR2 independent, as VEGFR2 phosphorylation remained inhibited in resistant tumors. Similar normalized revascularization was observed in the H441 orthotopic model (Figures and ). Prior studies have indicated that pericyte coverage may exert a protective effect on tumor endothelium (66
), potentially through the production of factors promoting endothelial survival and VEGF independence. Our findings were consistent with this hypothesis and revealed pericyte EGFR signaling to be a potential mediator of this effect.
In the A549 model of relative primary resistance, a distinct pattern of disorganized sprouting revascularization was observed in resistant tumors. This was marked by decreases in pericyte coverage with BV treatment and increased vessel tortuosity in resistant tumors. Unlike the acquired resistance models, stromal p-EGFR was upregulated in tumor endothelium, which suggests that the endothelium may be able to switch its dependence from VEGFR- to EGFR-driven endothelial proliferation and angiogenesis in the BV-resistant A549 xenografts, resulting in sprouting revascularization. It is worth noting that in an earlier study, we observed a similar switch (from EGFR- to VEGFR-dependent tumor endothelium) in a melanoma model (68
), supporting the feasibility of this proposed mechanism. Endothelial EGFR signaling may explain, at least in part, the intrinsic relative resistance of these tumors to VEGF blockade, as well as our prior observation that A549 cells display EGFR TKI resistance in vitro, but show moderate sensitivity to EGFR inhibition when grown as xenografts (26
). Other pathways that may contribute to this vascular phenotype are currently under investigation, including regulators of EC motility (e.g., HGF/c-MET) and vessel maturation (e.g., Ang-2/Tie-2). Nevertheless, this model provides evidence that there are distinct patterns of vascular remodeling that can accompany VEGF inhibitor resistance–associated tumor revascularization.
This study has a number of clinical implications for the use of VEGF inhibitors in NSCLC and other tumor types. First, it suggests that dual inhibition of the VEGFR and EGFR pathways may delay the emergence of therapeutic resistance in NSCLC. Consistent with this possibility, a recent phase III study (ATLAS) comparing the use of BV combined with erlotinib versus BV alone as maintenance therapy after chemotherapy demonstrated a significant, but modest, PFS improvement with an observed hazard ratio of 0.72 (P
= 0.001; refs. 69
). Combined VEGFR/EGFR inhibition (via BV with erlotinib or vandetanib) has also demonstrated significantly improved PFS compared with EGFR inhibition alone (71
). These studies showed a significant delay in tumor progression while treatment with VEGFR/EGFR inhibition was ongoing; however, significant improvements in overall survival were not observed. The explanation for this lack of durable clinical benefit is not known, but it is possible that once the dual inhibition is discontinued, these 2 pathways, or other alternative escape pathways, rapidly emerge.
The results of the present study may not be broadly generalizable to other tumor types or regimens containing chemotherapy. In a randomized phase III trial in colorectal cancer, the addition of the EGFR monoclonal antibodies panitumumab (74
) or cetuxumab (75
) to BV and chemotherapy showed trends toward worse clinical outcomes. Furthermore, in a recent study of colorectal cancer patients treated with BV plus chemotherapy, we observed increases in plasma bFGF, HGF, PDGF, and several myeloid factors prior to development of progressive disease (45
); in contrast, in our model, bFGF was the sole factor that significantly increased. This suggests that resistance mechanisms may be disease or regimen specific.
Second, these findings raise the possibility that combinations of VEGF inhibitors with drugs targeting other potential stromal resistance pathways — such as FGFR2 — may improve treatment efficacy. Third, they suggest that the analysis of both tumor cell and stromal markers — not just tumor cell markers alone — may provide important clinical information. Fourth, they suggest that analysis of vascular patterns in VEGF inhibitor–resistant tumors may provide information regarding the underlying mechanisms of resistance.
In summary, our findings suggest that in NSCLC models, gene expression changes associated with VEGF inhibitor resistance occur predominantly in tumor stromal cells, not tumor cells, providing further evidence that tumor stroma may play an important — and potentially dominant — role in VEGF inhibitor resistance. Primary and acquired resistance may be associated with distinct patterns of vascularization, described here as normalized and sprouting patterns, and distinct patterns of stromal signaling. Finally, we identify what we believe to be a novel role for pericyte EGFR signaling in VEGF inhibitor resistance. It is worth noting, however, that although combinations of VEGF and EGFR pathway inhibition have shown promise in NSCLC, therapeutic resistance nevertheless continues to emerge, which indicates that additional resistance mechanisms remain to be uncovered.