HSP47 is overexpressed in brain metastasis and high-level HSP47 predicts poor survival in cancer patients
(A) Heatmaps showing the differential expression of ECM genes in breast cancer BrMs (n = 97) compared to primary tumors (n = 137) (GEO: GSE184869, GSE12276, GSE29431, GSE46928, and GSE100534; GitHub: JNCI_2018) and in lung cancer BrMs (n = 144) compared to primary tumors (n = 166) (GEO: GSE14108 and GSE13213; OMIX: OMIX575; BioProject: PRJNA681304; ArrayExpress: E_MTAB_8659).
(B) Venn diagram showing the overlapping genes that are co-upregulated in BrMs in (A).
(C) Diagram showing the in vivo selection procedure of brain metastatic cell populations (BMT5) from 4T1 breast cancer cells and LLC1 lung cancer cells, respectively.
(D) 4T1-BMT5 and the parental 4T1 cells (5 × 104 cells/mouse) expressing the firefly luciferase were intracardially implanted into immunocompetent BALB/c mice, while LLC1-BMT5 and the parental LLC1 cells (5 × 104 cells/mouse) were implanted into C57/BL6 mice, respectively. BrMs were monitored by bioluminescence imaging (BLI) at 28 (4T1 and 4T1-BMT5 cells) or 35 days (LLC1 and LLC1-BMT5 cells) after cell implantation. Representative BLI images are shown (left). BLI photon fluxes in the brain for each mouse were quantified (right, mean ± SD of n = 8 mice, two-sided Mann-Whitney test). ∗∗∗p < 0.001.
(E) Quantification of the percentages of mice that developed bone metastases or BrMs after intracardial implantation of the tumor cells.
(F) RT-qPCR assays detected the expression of the 15 co-upregulated genes in 4T1-BMT5 and LLC1-BMT5 cells compared to the parental cells (mean ± SEM of n = 3 independent experiments, two-tailed Student’s t test). GAPDH was used as an internal control. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
(G) Immunostaining of HSP47 in BrMs and the paired primary breast (top) or lung cancer tissues (bottom). Representative images of two patients for each cancer type are shown. Scale bars, 100 μm. The expression scores of HSP47 in BrMs were compared with those of the paired primary tumors (n = 12 for BRCA and n = 26 for LUAD, paired Student’s t test).
(H) Kaplan-Meier analysis showing the correlation between HSP47 mRNA levels and overall survival of patients with breast cancer (top) and lung cancer (bottom).