(A) H&E staining shows a marked increase in tumor necrosis (N) 24 hours after 50 mg/kg CA4P. (B) Staining with anti-PIMO shows increased hypoxia (red). (C) Tumor sections stained with anti-CD31 antibodies show reduced lumen of blood vessels (white) 24 hours after 50 mg/kg CA4P. (D) Tumors stained with DAPI (to show cell nuclei; blue), anti-F4/80 (green), and anti-TIE2 (red). Yellow arrows denote F4/80⁺TIE2⁺ TEMs; F4/80^–TIE2⁺ structures represent blood vessels. (E) Number of all F4/80⁺ cells/field and F4/80⁺TIE2⁺ TEMs/field (by immunofluorescent staining; see D above) and proportion (%) of F4/80⁺TIE2^– and F4/80⁺TIE2⁺ cells (TEMs) in viable cells obtained from dispersed tumors (by flow cytometric analysis; see Supplemental Figure 1). Scale bars: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared with relevant control group. NS, no statistical difference (P > 0.05).

(A) CXCL12 expression, determined by immunofluorescence staining (see Supplemental Figure 3), was increased in MMTV-PyMT tumors 24 hours after injection with 50 mg/kg CA4P. (B and C) Flow cytometric analysis of dispersed MMTV-PyMT tumors shows that (B) the majority of F4/80⁺TIE2⁺ TEMs express CXCR4 and the MFI for CXCR4 is higher for TEMs than TIE2^– TAMs and (C) CXCR4 expression/TEM is upregulated after CA4P treatment. (D) The CXCR4 inhibitor, AMD-3100, inhibits recruitment of TEMs, but not TIE2^– TAMs, to MMTV-PyMT tumors and increases CA4P-induced tumor necrosis. n > 5 mice per group. *P < 0.05; **P < 0.01. (E) Twice daily i.p. injections of 5 mg/kg AMD-3100 (for the period indicated) enhanced the effects of 3 daily i.p. injections of 50 mg/kg CA4P on the growth of N202 tumors. Statistical analysis in E employed the nonlinear mixed effects model described in the Supplemental Methods.

(A) N202 tumors excised 24 hours after the last of 3 daily i.p. injections of CA4P or saline in the presence or absence of GCV to deplete TEMs were stained with DAPI (blue), anti-F4/80 (green), anti-TIE2 (red), and anti-MMP9 (white). TEMs are highlighted with yellow arrows; non-TAMs that express MMP9 are shown with white arrows. Scale bar: 50 μm. (B and C) Changes in TEM numbers and tumor necrotic area 24 hours and 72 hours after last CA4P injection. n = 6 mice per group. *P < 0.05.