Figure 3.

HSP47 promotes M2 microglial polarization and immunosuppression in brain metastatic niche

(A) Representative fluorescence-activated cell sorting (FACS) plots and quantification of CD206⁺CD11b⁺CD45^low and CD11c⁺CD11b⁺CD45^low cells in the mouse BrMs (mean ± SEM, n = 5 independent experiments, Student’s t tests). ∗∗∗p < 0.001.

(B) Mouse BrMs derived from 4T1-BMT5 cells were double stained with anti-HSP47 and anti-CD206 antibodies. Representative images are shown. Scale bars, 100 μm. The density of CD206⁺ cells in each microscope filed was counted (mean ± SEM, n = 6 randomly selected fields, Student’s t tests). ∗∗∗p < 0.001.

(C) Representative FACS plots and quantification of CD8⁺CD3⁺CD45⁺ cells in the mouse BrMs derived from 4T1-BMT5 cells. Results are presented as mean ± SEM of n = 5 independent experiments, Student’s t tests. ∗∗∗p < 0.001.

(D) LLC1-BMT5 cells expressing HSP47 shRNAs were intracardially injected into mice. Mice were then injected intraperitoneally with a CD8α monoclonal antibody (mAb; 100 μg/mouse) or immunoglobulin G2b (IgG2b) (100 μg/mouse). Representative BLI images and quantification of BLI photon fluxes in the brain are shown (mean ± SD, n = 8 mice for each group, two-sided Mann-Whitney test). ∗∗∗p < 0.001.

(E) Mice intracardially injected with 4T1 or LLC1 cells expressing HSP47 were kept on a standard diet or fed with PLX3397 (290 mg/kg) for 3 weeks. Representative BLI images are shown. BLI photon fluxes in the mouse brain were quantified (mean ± SD of n = 8 mice for each group, two-sided Mann-Whitney test). ∗p < 0.05 and ∗∗p < 0.01.

(F) H&E staining of BrMs in mice. Scale bars, 200 μm. Large metastases are >300 mm on the longest axis (mean ± SD of n = 8 mice, Student’s t test). ∗p < 0.05 and ∗∗p < 0.01.

(G) The percentages of mice forming brain or bone metastases were quantified.

(H) Representative FACS plots and quantification of CD11b⁺CD45^lowcells in mouse BrMs (mean ± SEM, n = 5 independent experiments, Student’s t tests). ∗∗∗p < 0.001.

(I) Representative FACS plots and quantification of CD8⁺CD3⁺CD45⁺ cells in mouse BrMs (mean ± SEM, n = 5 independent experiments, Student’s t tests). ∗∗∗p < 0.001.

(J–M) The populations of Ki67⁺, CD69⁺, granzyme B (GZMB)⁺, interferon (IFN)-γ⁺, and TNF-α⁺ CD8⁺ T cells and CD25⁺FOXP3⁺CD4⁺ Tregs in (I) were quantified (mean ± SEM, n = 5 independent experiments, Student’s t tests), respectively. ∗∗∗p < 0.001.

(N) Immunofluorescence (IF) co-staining of HSP47 and CD206 in brain metastatic lesions of patients with breast cancer. Representative images of two tumors are shown. Scale bars, 100 μm. The expression correlation between HSP47 and CD206 was analyzed (n = 10 randomly selected fields, Pearson correlation coefficient).