Figure 4.

COL1 mediates HSP47-induced microglia recruitment and polarization

(A) ELISA quantification of the COL1 levels in the CM of 4T1-BMT5 and LLC1-BMT5 cells compared to the corresponding parental cells.

(B) ELISA quantification of the COL1 levels in the CM of 4T1-BMT5 and LLC1-BMT5 cells expressing HSP47 shRNAs.

(A and B) Data are expressed as mean ± SD of n = 3 independent experiments, Student’s t tests. ∗∗p < 0.01 and ∗∗∗p < 0.001.

(C) BV2 cells were cultured in the CM of 4T1-BMT5 or LLC1-BMT5 cells expressing HSP47 shRNA and then treated with 4 mg/mL COL1 for 24 h. Cells that migrated onto the lower surface of the Transwell chamber were stained by crystal violet. Representative images are shown. Scale bars, 100 μm. Data are presented as mean ± SEM, n = 6 randomly selected microscope fields of two independent experiments, Student’s t tests. ∗∗∗p < 0.001.

(D) BV2 cells cultured in the CM of 4T1-BMT5 or LLC1-BMT5 cells expressing HSP47 shRNA were treated with COL1 for 48 h. The cell lysates were analyzed by immunoblotting using anti-CD206 and anti-Arg1 antibodies.

(E) BV2 cells were treated as in (D) and the expression of CD206 was analyzed by FACS. Representative FACS plots are shown. FACS data were statistically analyzed (mean ± SD, n = 3 independent experiments, Student’s t tests). ∗∗p < 0.01 and ∗∗∗p < 0.001.

(F) 4T1-BMT5 cells expressing HSP47 shRNA were treated with COL1 for 24 h and then intracardially injected into mice. Representative BLI of two mice for each group 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 < 0.05.

(G) The BMFS of mice was evaluated (n = 8 mice for each group, Kaplan-Meier model with two-sided log-rank test). ∗∗p < 0.01.

(H) 4T1-BMT5 cells expressing control shRNA or HSP47 shRNA were engineered to express GFP and then intracardially injected into mice. Consecutive mouse BrM tissues were immunostained with the antibodies against GFP, HSP47, COL1-4, CD206, or CD8α. Scale bars, 200 μm. Insets: high-magnification images corresponding to the areas marked by dotted red lines.

(I) CD8⁺ T cells were cultured overnight in the CM of tumor cells overexpressing HSP47 or 4 mg/mL COL1. Cells that migrated onto the lower surface of the Transwell chamber were counted by FACS.

(J) BV2 cells were treated with the CM of tumor cells expressing HSP47 or 4 mg/mL COL1 overnight. The medium was replaced with fresh medium and then used to treat CD8⁺ T cells. The T cells that migrated onto the lower surface of the Transwell chamber were counted by FACS.

(I and J) Representative FACS plots are shown. FACS data were statistically analyzed (mean ± SD, n = 3 independent experiments, Student’s t tests). ∗p < 0.05 and ∗∗∗p < 0.001.

(K and L) IF double staining of COL1 and CD206 in human BrMs from patients with breast or lung cancer. Scale bars, 100 μm. The staining density of COL1 and CD206 cells in each tissue was analyzed (n = 10 tissues for breast cancer BrMs and n = 16 tissues for lung cancer BrMs, Pearson correlation coefficient).