Twenty-four hours after a single injection of CA4P, mammary tumors in MMTV-PyMT mice were significantly more hypoxic/necrotic than those in controls (P
< 0.05; Figure , A and B) and contained CD31+
blood vessels with smaller lumens (Figure C). This is consistent with the vascular-disrupting effects of CA4P reported in various mammary tumor models (4
CA4P induced vascular damage, increased tumor hypoxia, and increased TEM numbers in MMTV-PyMT mammary tumors.
The number of F4/80+
TAMs was also significantly increased in CA4P-treated tumors (Figure , D and E). F4/80, TIE2, and CD31 immunofluorescence staining of tumors showed that infiltration of CD31–
TEMs significantly increased 24 hours after CA4P injection (Figure , D and E), while the number of F4/80+
TAMs was not significantly altered (data not shown). These data were confirmed by flow cytometric analysis of dispersed tumors (Supplemental Figure 1; supplemental material available online with this article; doi:
). Virtually all F4/80+
TEMs expressed the proangiogenic enzyme MMP9 in both untreated and treated tumors, whereas far fewer TIE2–
TAMs were MMP9+
(Supplemental Figure 2, A and B). CA4P treatment also significantly increased tumor infiltration by MMP9-expressing TEMs in N202 mammary tumors grown subcutaneously in syngeneic mice (Supplemental Figure 2, C and D).
Tumor expression of CXCL12 (SDF1) by both tumor and stromal cells (identified on the basis of cell morphology) increased 24 hours after CA4P treatment (Figure A and Supplemental Figure 3) and was consistent with the hypoxic upregulation of CXCL12 (16
) and raised plasma levels of CXCL12 seen in CA4P-treated patients (17
). Ninety percent of tumor TEMs and 60% of TIE2–
TAMs expressed the CXCL12 receptor, CXCR4, in MMTV-PyMT tumors (Figure B). CXCR4 expression per cell was significantly higher in TEMs than in TIE2–
TAMs and was augmented further by CA4P treatment (Figure C). We then used the CXCR4 inhibitor, AMD-3100, to investigate the role of the CXCL12/CXCR4 axis in TEM recruitment to tumors after CA4P. This significantly reduced TEM but not TIE2–
TAM recruitment to CA4P-treated N202 tumors (Figure D). Of note, AMD-3100 plus CA4P treatment elicited a significant increase in tumor necrosis compared with CA4P treatment alone (Figure D).
Inhibition of tumor recruitment of CXCR4+ TEMs increases the therapeutic efficacy of CA4P.
In order to study the effects of combined CXCR4 blockade and CA4P treatment on tumor responses to CA4P, we used the subcutaneous N202 mammary tumor model, which allows more reliable tumor measurements and monitoring of tumor volume than the MMTV-PyMT model. AMD-3100 alone had no effect on N202 tumor growth (Figure E), consistent with previous studies in other tumor models (14
). The combined drug treatment (AMD-3100 and CA4P) induced a significant inhibition of N202 tumor growth compared with CA4P alone (Figure E). These results are consistent with the necrosis data obtained in MMTV-PyMT tumors (Figure D).
Because CXCR4 is not only expressed by TEMs in tumors, we also used a conditional suicide gene–based strategy for specific TEM elimination (8
). This involved transplanting BM cells from Tie2
-HSV-thymidine kinase transgenic mice into syngeneic, wild-type mice. Six weeks after BM transplant, ganciclovir (GCV) was used to specifically eliminate TEMs in mice carrying established N202 tumors. CA4P induced an increase in necrosis (Figure C) and vessel narrowing (Supplemental Figure 4A) in the N202 mammary tumors of transplanted mice. Coadministration of GCV to deplete TEMs resulted in significant TEM depletion in CA4P-injected tumors (Figure , A and B) accompanied by a striking increase in tumor necrosis in CA4P-treated tumors compared with non-TEM–depleted CA4P-treated tumors (Figure C). Tumor necrosis was most marked at 24 hours after CA4P in the TEM-depleted mice, indicating that TEMs are protective at early stages after treatment (Figure C). Interestingly, the number of tumor-infiltrating F4/80–
neutrophils increased significantly at 24 hours after CA4P treatment and increased further in TEM-depleted mice that received CA4P (Supplemental Figure 4B). However, very few F4/80–
neutrophils were seen at 72 hours in any of the treatment groups (Supplemental Figure 4B). Contrary to CXCR4 blockade (Figure D), GCV completely abolished TEM recruitment to tumors (Figure B) and — in agreement with previous findings (8
) — markedly inhibited tumor growth (Supplemental Figure 5). It was therefore not possible to assess the additive effects of CA4P treatment and TEM elimination on tumor growth in this model.
Conditional depletion of TEMs increases the efficacy of CA4P treatment.
Taken together, our results show that TEMs are rapidly recruited into CA4P-treated mammary tumors, where they have a protective effect against vascular damage, necrosis induction, and growth retardation. At present, it is not possible to distinguish whether TEMs are acting to protect against the damaging cascade of events that lead to CA4P-induced tumor necrosis or whether they are promoting rapid revascularization and tumor repopulation of necrotic regions. Considering the proangiogenic roles of TEMs in tumors (8
) and our finding that TEMs avidly express MMP9, we speculate that a reparative role is highly likely. Either way, our data indicate that targeting these cells is a promising approach to improving the efficacy of a VDA like CA4P.
Endothelial progenitor cells (EPCs) were previously shown to play an important role in tumor recovery following VDA treatment (17
). Shaked et al. (19
) depleted EPCs using DC101, a mouse monoclonal antibody to VEGFR-2. When administered to tumor-bearing mice along with a CA4P derivative, OXi-4503 (CA1P), DC101 decreased circulating EPC numbers and tumor blood flow and increased tumor necrosis, compared with OXi-4503 alone. However, DC101 would also have blocked VEGFR-2 signaling in the viable tumor blood vessels and therefore had direct effects on tumor angiogenesis that were independent of EPCs (18
). Because BM-derived EPCs are very rare cells in mouse blood and tumors (8
), it is likely that other BM-derived cell types, such as monocyte-lineage cells, are primarily involved in regulating tumor responses following VDA treatment. Although our findings do not eliminate a potential contribution of EPCs to tumor revascularization/recovery after CA4P, they indicate that TEMs play a crucial role in this process.
Interestingly, TEM depletion stimulated an increase in neutrophil infiltration into tumors — even in the absence of subsequent treatment with CA4P, suggesting that TEMs may inhibit tumor infiltration by Gr1+
). CA4P treatment alone also increased neutrophil infiltration, possibly in response to tumor necrosis, and TEM depletion, in combination with CA4P, increased it even further. It is noteworthy that tumor Gr1+
neutrophils had significantly dropped by 72 hours, illustrating that any participation in CA4P-induced changes in tumors was time dependent. Clearly, the enhanced recruitment of neutrophils at 24 hours after treatment failed to protect tumors. Rather, neutrophils may have contributed to the increased level of necrosis present by exhibiting a cytotoxic phenotype (24
). Interestingly, levels of neutrophil-derived myeloperoxidase (MPO), indicative of such a phenotype, are associated with CA4P-induced vascular injury in tumors (25
In summary, our data show that TEMs are rapidly recruited to CA4P-treated mammary tumors and protect against the vascular damaging effects of this archetypal VDA. This suggests that targeting TEMs could increase the long-term efficacy of CA4P.