Fig. 5.

Inhibition of CXCR4 reduces desmoplasia and increases effector to regulatory T-lymphocyte ratio in the lung metastases. (A–D) Representative histology images of lung metastases derived from orthotopic E0771 breast tumors. Immunofluorescence (IF) images of lung metastases were field-of-view images from the mosaic confocal imaging. After primary tumor resection, the mice were treated with AMD3100 in combination with an ICB mixture of α-CTLA-4 and α-PD-1 for one cycle, and the lung metastases were collected for analysis. (A) H&E stainings of spontaneous lung metastases from E0771 tumors. The metastatic nodules (red arrows) are significantly larger in the control, compared with all three treatment groups. (Scale bars, 100 μm.) (B) Representative images of IF staining of hyaluronan. Treatment with AMD3100 reduces the hyaluronan fraction in the metastatic TME. (C) Representative images of IF staining of collagen I. Treatment with AMD3100 reduces collagen I in the metastases. (D) Representative IF images showing aSMA (red), collagen I (white), and CD3 (green) in the metastases. Combination of AMD3100 and ICB increases infiltration of CD3+ cells into the TME. (E and F) Flow cytometry analysis of lung metastases derived from orthotopic E0771 breast tumors. (E) CD3+CD4+Foxp3+ regulatory T cell population. Both the monotherapy and combination therapy decrease Foxp3 T cells in the tumors (*P = 0.017, by one-way ANOVA). (F) CD8+ to Foxp3+ Treg ratio. Both AMD3100 groups increase the ratio of effector CD8+ T cells to Treg, and the combination of AMD3100 with immunotherapy mixture further extends the ratio (*P = 0.026, by one-way ANOVA). n = 5. Error bars indicate SEM. (Scale bar, 100 μm.)