Figure S1: 5-FU-induced increase in lung metastasis of TS/A. Figure S2: Effects of 5-FU treatment on cells infiltrating into lungs. Figure S3: Characterization of CD11b+Ly6G+ cells infiltrated into lung. Figure S4: 5-FU effects to expression of Cxcl1 and Cxcl2 in lung. Figure S5: Molecular mechanisms underlying 5-FU, CTX and DOX-induced Cxcl1 and Cxcl2 expression in 4T1 and TS/A cells. Figure S6: Molecular mechanisms underlying 5-FU-induced CXCL1, CXCL2, and CXCL8 expression in a human breast cancer cell line, BT-20 cells. Figure S7: Effects of 5-FU treatment on CD11b+Ly6G+ neutrophils in lungs. Figure S8: Reduction in 5-FU-induced increase in lung metastasis by Prokr1 deletion. Figure S9: Schematic demonstration on presumed cellular and molecular mechanisms underlying 5-FU-enhanced lung metastasis of breast cancer. Figure S10: Effect of 5-FU on cell senescence in 4T1 and TS/A cells. Figure S11: Effect of an ATM inhibitor, KU-60019, on Cxcl1 and Cxcl2 expression in 4T1 and TS/A cells. Figure S12: Effect of H2O2 on Cxcl1 and Cxcl2 expression in 4T1 cells. Figure S13: Relation of PROKR1 and PROK2 expression with the prognosis of breast cancer patients. Figure S14: Effect of Cxcl1, Cxcl2 and G-CSF on Prok2 expression in CD11b+Ly6G+ neutrophils. Figure S15: Effect of 5-FU on G-csf expression in lung tissue.
ARTICLE ABSTRACT
Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, to prevent tumor recurrence primarily as a form of metastasis. However, anticancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of postsurgical administration with 5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU–mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T-cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NFκB-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated 5-FU–mediated enhanced lung metastasis with depressed neutrophil infiltration. Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2 (Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor failed to augment lung metastasis in the mice receiving Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation. Mol Cancer Ther; 17(7); 1515–25. ©2018 AACR.
