Figure 6.

Targeting HSP47-mediated COL1 biosynthesis inhibits BrM

(A) Molecular formula of Col003.

(B) 4T1-BMT5 or LLC1-BMT5 cells were treated with the indicated concentration of Col003 for 24 h and COL1 level in the CM was quantified by ELISA (mean ± SD, n = 3 independent experiments). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

(C) CSF was extracted from mice at different time points after intravenous injection of Col003 (20 mg/kg) and the concentration of Col003 was determined by high-performance liquid chromatography (mean ± SD, n = 3 mice for each time point).

(D) 4T1-BMT5 or LLC1-BMT5 cells were intracardially injected into mice. Seven days later, Col003 was injected (5 or 20 mg/kg) into mice via vein every third day for 1 month. Representative BLI images are shown. BLI photon fluxes in the brain for each mouse were quantified (mean ± SD, n = 8 mice for each group, two-sided Mann-Whitney test). ∗∗∗p < 0.001.

(E) H&E staining of BrM tissues in mice. Representative images are shown (left). Scale bars, 200 μm. Large metastases are >300 mm on the longest axis (mean ± SD, n = 8 mice, two-sided Mann-Whitney test). ∗∗p < 0.01 and ∗∗∗p < 0.001.

(F) The BMFS of mice was evaluated (n = 8 mice for each group, Kaplan-Meier model with two-sided log-rank test). ∗∗∗p < 0.001.

(G) Representative FACS plots and quantification of CD8⁺CD3⁺CD45⁺ cells in the mouse BrM tissues.

(H) Representative FACS plots and quantification of CD206⁺CD11b⁺CD45^low and CD11c⁺CD11b⁺CD45^low cells in the BrM tissues.

(I–L) The populations of Ki-67⁺, CD69⁺, GZMB⁺, IFN-γ⁺, and TNF-α⁺ CD8⁺ T cells and CD25⁺FOXP3⁺CD4⁺ Tregs were analyzed by FACS.

(G–L) Data are expressed as mean ± SEM, n = 5 independent experiments, Student’s t tests. ∗∗∗p < 0.001.

(M) Consecutive mouse BrM tissues were analyzed by immunohistochemistry using the antibodies against GFP, COL1, and CD8α. Representative images are shown. Scale bars, 200 μm.