Metastasis remains the most common cause of death in most cancers with limited therapies for combating disseminated disease. the combined depletion significantly reduced brain metastasis expression at the primary site and organ-specific metastasis-free survival (MFS) using a dataset of locally advanced primary breast cancer with clinical annotation20. Patients were separated into three equal tertiles of low medium and high expression as described in the methods. Kaplan-Meier analysis was used to assess MFS for brain bone and lung. Interestingly the high expression group Acolbifene (EM 652, SCH57068) was associated with decreased MFS only for the brain and not bone or lung (Fig. 3c). This was further evident in a complementary Cox proportional hazards model analysis (Supplementary Fig. 4c). We similarly determined whether other tumor genes that were differentially expressed in the experimental model (Fig. 1c) were associated Mouse monoclonal to KLHL13 with differences in patient survival (Supplementary Table 2). In addition to in brain metastasis together with and in bone metastasis were the only genes that showed similar stage-dependent and cell type-specific expression changes to in brain metastasis (Fig. 3a Supplementary Fig. 4a). Given that we did not observe an association of expression with patient bone MFS and neither nor expression associated with brain and bone MFS respectively (Supplementary Fig. 4a b) we chose to further investigate the potential role of cathepsin S specifically in brain metastasis a function not previously ascribed to this protease or any cathepsin family member. The patient expression data above was derived from whole tumor samples thus precluding cell type-specific expression analyses. We therefore stained a set of patient samples of brain metastases with matched primary breast tumors in a subset of cases (Supplementary Table 3). Across all samples (breast cancer and brain metastases) we found the major cell types contributing to the tumor mass were cytokeratin (CK)+ tumor cells and CD68+ macrophages with a minor fraction representing CK-CD68- cells (Fig. 3d e Supplementary Fig. 5a-d). CTSS levels were highest in CD68+ macrophages with expression also in CK+ tumor cells albeit at lower levels than Acolbifene (EM 652, SCH57068) in macrophages in both primary tumors and matched brain metastases (Fig. 3d f Supplementary Fig. 5a b e). CTSS expression in tumor cells was observed in all molecular subtypes of breast cancer analyzed (Fig. 3d f Supplementary Fig. 5a b e Supplementary Table 3). Combined depletion of cathepsin S in tumor and stromal cells reduces experimental brain metastasis We investigated the stromal cell source of Ctss in the experimental Acolbifene (EM 652, SCH57068) brain metastasis model. Seeding and outgrowth of brain metastasis induced a stromal response characterized by accumulation of astrocytes and macrophages/microglia in metastatic lesions (Supplementary Fig. 1d). Detection of cathepsin S using an antibody that recognizes both mouse and human homologs in combination with cell-type specific markers identified macrophages as the predominant stromal cell type expressing Ctss in brain metastases and normal brain (Fig. 4a). We observed a gradual increase of Ctss expression in Iba1+ macrophages from normal brain to early- and late-stage metastases. CTSS expression was also detectable in tumor cells though at lower levels than in macrophages mirroring the patient analyses. At late stages CTSS expression was undetectable in the majority of the tumor cells. We found a similar expression pattern in an immunocompetent brain metastasis model (Supplementary Fig. 3g). These data confirm the stage- and cell type-dependent expression changes at the protein level as predicted by the HuMu array. Figure 4 Macrophages are Acolbifene (EM 652, SCH57068) the predominant source of stromal-derived cathepsin S and only combined depletion of tumor- and stromal-derived cathepsin S reduces experimental brain metastasis Given the reciprocal cell type-specific expression pattern of cathepsin S we sought to investigate if tumor and stromal sources play important perhaps complementary roles in the seeding and outgrowth of experimental brain metastases. To address this we performed short hairpin (sh)-RNA-mediated knockdown (KD) in Br-M cells achieving a 90% reduction of CTSS.