Recruitment of myeloid but not endothelial precursor cells facilitates tumor regrowth after local irradiation

Abstract

Tumor neovascularization and growth might be promoted by the recruitment of bone marrow-derived cells (BMDC), which include endothelial precursor cells and "vascular modulatory" myelomonocytic (CD11b+) cells. BMDCs may also drive tumor regrowth after certain chemotherapeutic and vascular disruption treatments. In this study, we evaluated the role of BMDC recruitment in breast and lung carcinoma xenograft models after local irradiation (LI). We depleted the bone marrow by including whole-body irradiation (WBI) of 6 Gy as part of a total tumor dose of 21 Gy, and compared the growth delay with the one achieved after LI of 21 Gy. In both models, the inclusion of WBI induced longer tumor growth delays. Moreover, WBI increased lung tumor control probability by LI. Exogenous delivery of BMDCs from radiation-naïve donors partially abrogated the WBI effect. Myeloid BMDCs, primarily macrophages, rapidly accumulated in tumors after LI. Intratumoral expression of stromal-derived factor 1alpha (SDF-1alpha), a chemokine that promotes tissue retention of BMDCs, was noted 2 days after LI. Conversely, treatment with an inhibitor of SDF-1alpha receptor CXCR4 (AMD3100) with LI significantly delayed tumor regrowth. However, when administered starting from 5 days post-LI, AMD3100 treatment was ineffective. Lastly, with restorative bone marrow transplantation of Tie2-GFP-labeled BMDC population, we observed an increased number of monocytes but not endothelial precursor cells in tumors that recurred following LI. Our results suggest that an increase in intratumoral SDF-1alpha triggered by LI recruits myelomonocytes/macrophages which promotes tumor regrowth.

Figure 1. Anti-tumor effect of local (L) irradiation with or without whole-body (W) irradiation

A, Experimental design: Tumors received the same total radiation dose (21Gy) over the same period of time, either by L and W irradiation or by two fractions of L irradiation. B,C, W irradiation significantly delayed tumor re-growth after radiation therapy in both 54A (B) and MCa8 (C) models, and this effect was abrogated by infusion of bone marrow cells from non-irradiated donors (n=7-9, *denotes p<0.05). D, At higher L irradiation doses, addition of W irradiation (n=21) significantly enhanced the local control probability (>90 days) of 54A tumor compared to L irradiation alone (n=22, p<0.05).

Figure 2. The role of SDF-1α-CXCR4 pathway in tumor re-growth after local irradiation

A, Representative fluorescence confocal microscopy images of tumor immunostaining for SDF-1α and quantification of SDF-1α expression before and 2 days after 20Gy of radiation. Irradiation induced a significant increase in SDF-1α. B,C, Effect of CXCR4 blockade using AMD3100 on tumor re-growth after irradiation of 54A (B) and MCa8 (C) tumors. CXCR4 blockade delayed tumor growth when commenced immediately after but not 5 days after irradiation (n=6-8). (*denotes p<0.05).

Figure 3. BMDC infiltration into tumors 3 days post-irradiation

A, Immunohistochemistry for CD11b and CD31 expression in 54A tumors. Local (L) irradiation significantly increased the number of CD11b⁺ myeloid BMDCs compared to control tumors, whereas inclusion of whole-body (W) irradiation abrogated this effect. No significant difference was seen in microvascular density measured by CD31 expression. B,C, Flow cytometry analysis of whole tumor lysates confirmed the changes in myeloid BMDCs after L and W irradiation (n=5). The majority of BMDCs were F4/80⁺ macrophages in both 54A (B) and MCa8 (C) tumors. (*denotes p<0.05).

Figure 4. Analysis of BMDCs in tumors recurring after local irradiation in WT/Tie2-GFP-BMT mice

A, Representative confocal microscopy images of fluorescence immunohistochemistry in tumors recurring after 20Gy of radiation versus non-irradiated size-matched (control) tumors. Note localization of GFP expression (green) in CD11b⁺ cells outside vessels (blue, see arrows) but not in CD31⁺CD11b⁻ vascular endothelial cells (red). B,C, Quantification of BMDCs: Overall myeloid cell infiltration (CD11b⁺) and CD31⁺ microvascular density were not significantly changed, but the total number of Tie2⁺ BMDCs increased in irradiated tumors (B). These represented mostly Tie2⁺CD11b⁺ TEMs, the majority of which were also CD31⁺ but had peri-vascular location; in contrast the number of vessel-incorporated Tie2⁺CD31⁺CD11b⁻ EPCs was negligible and not different in tumors grwoing after irradiation (C). (*denotes p<0.05).