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Analysis of tumour- and stroma-supplied proteolytic networks reveals a brain-metastasis-promoting role for cathepsin S

Abstract

Metastasis remains the most common cause of death in most cancers, with limited therapies for combating disseminated disease. While the primary tumour microenvironment is an important regulator of cancer progression, it is less well understood how different tissue environments influence metastasis. We analysed tumour–stroma interactions that modulate organ tropism of brain, bone and lung metastasis in xenograft models. We identified a number of potential modulators of site-specific metastasis, including cathepsin S as a regulator of breast-to-brain metastasis. High cathepsin S expression at the primary site correlated with decreased brain metastasis-free survival in breast cancer patients. Both macrophages and tumour cells produce cathepsin S, and only the combined depletion significantly reduced brain metastasis in vivo. Cathepsin S specifically mediates blood–brain barrier transmigration through proteolytic processing of the junctional adhesion molecule, JAM-B. Pharmacological inhibition of cathepsin S significantly reduced experimental brain metastasis, supporting its consideration as a therapeutic target for this disease.

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Figure 1: HuMu ProtIn array enables simultaneous acquisition of gene expression changes in tumour and stromal cells.
Figure 2: Independent validation of differentially expressed genes in experimental brain, bone and lung metastases.
Figure 3: Cathepsin S shows highly regulated stage- and cell-type-specific expression changes in experimental brain metastases, and that cathepsin S expression in primary breast tumours is inversely correlated with brain MFS in patients.
Figure 4: Macrophages are the predominant source of stromal-derived cathepsin S and only combined depletion of tumour- and stromal-derived cathepsin S reduces experimental brain metastasis.
Figure 5: Cathepsin S deficiency in tumour cells and macrophages impairs metastatic seeding and outgrowth.
Figure 6: Cathepsin S mediates BBB transmigration of brain metastatic cells.
Figure 7: Cathepsin S cleaves tight junction proteins that regulate BBB integrity.
Figure 8: Pharmacological inhibition of cathepsin S reduces brain metastasis formation in a preclinical trial.

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Acknowledgements

We thank K. Simpson and X. Chen for excellent technical support, and members of the J.A.J. laboratory for insightful discussion. We thank L. Akkari, O. Olson and D. Yan for reading the manuscript. We are grateful to P. Bos for advice on the BBB assays and experimental brain metastasis model. We thank the MSKCC Core Facilities of Genomics, Molecular Cytology, Small Animal Imaging and Monoclonal Antibody Production for technical assistance. We thank H. Chapman (UCSF) for providing cathepsin S KO mice. This research was supported by the following: US National Cancer Institute program grants of the Integrative Cancer Biology Program (CA148967; J.A.J., C.S.L.) and Tumour Microenvironment Network (CA126518; J.A.J., J.M.), the Health Research Science Board of New York, and the Alan and Sandra Gerry Metastasis Research Initiative (J.A.J.), Deutsche Forschungsgemeinschaft (SE2234/1-1; L.S.), US National Cancer Institute F31 fellowship CA167863 and Gerstner Sloan Kettering graduate program (R.L.B.), US National Cancer Institute F32 fellowship CA130329 (S.D.M.), and Canadian Institutes of Health Research (D.F.Q.).

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Contributions

L.S., S.D.M. and J.A.J. designed experiments. L.S., S.D.M., D.F.Q. and B.T.E. performed experiments and analysed data. R.L.B., F.R. and C.S.L. performed computational analyses. E.B., P.K.B., W.C.H., L.J.H. and J.M. provided patient samples or reagents. J.A.J., L.S. and R.L.B. wrote the manuscript. All authors edited or commented on the manuscript. J.A.J. conceived and supervised the study.

Corresponding author

Correspondence to Johanna A. Joyce.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of the stromal cell types in early- and late-stage brain, bone and lung metastasis.

(ac) Quantification and representative images of in vivo BLI intensity are shown for (a) brain metastases (n = 7 and 9 mice), (b) bone metastases (n = 6 and 7 mice), and (c) lung metastases (n = 4 mice per group) for early and late stages. Circles represent individual mice and horizontal lines represent the mean ± s.e.m. (df) Representative images of control tissue and early- and late-stage brain, bone, and lung metastases stained for GFP (tumour cells) and the endothelial marker CD34 or the macrophage markers CD68 (lung and bone) or Iba1 (brain). DAPI staining was used to visualize cell nuclei. Brain metastasis sections were also stained with the astrocyte-specific marker GFAP. Images are representative of three independent samples per stage. Scale bar indicates 50 μm.

Supplementary Figure 2 Tissue- and stage-specific gene expression changes in tumour and stroma.

(a) Venn diagram of the tumour-derived genes that are significantly different between early and late metastases across different metastatic sites (Fig. 1c). Of the 308 genes significantly different between early and late stages in either brain, bone or lung metastases, 176 genes change by stage in all three sites. (b) Venn diagram depicting the overlap of the 75 stroma-derived genes that are significantly different between early- and late-stage metastases across brain, bone and lung (Fig. 1d). Unlike the tumour-derived genes depicted in a, there were no stromal-derived genes that were significantly different between early- and late-stage metastases in all three tissues investigated. (c) Heatmap depicting tissue-specific gene expression in the brain, bone and lung stroma. No tissue-specific genes were identified for tumour-derived genes. Proteases are denoted in purple, endogenous inhibitors in red, and their interacting partners in black. (d) qPCR confirmed the tissue-enriched expression pattern of Htra1 for brain, Mmp13 for bone, and Mmp11 for lung in control tissue, early- and late-stage metastases. n = 6, 4, 4 samples for control tissues, n = 5, 6, 4 samples for early stages, and n = 6, 6, 4 samples for late stages in brain, bone and lung respectively. Graphs represent mean + s.e.m. P values were obtained using two-tailed unpaired t-test: P < 0.05,P < 0.01, and P < 0.001.

Supplementary Figure 3 Independent validation of differentially expressed genes in experimental brain, bone and lung metastases.

qPCR was used to confirm stage-differential expression changes, and to determine if the expression changes in stromal cells in brain or bone metastasis are a general response to tumour cell colonization of the respective organ, or if the expression changes depend on the tumour cell variant. Hatched bars represent ‘mismatched’ samples (Br-M in bone and Bo-M in brain). Filled bars indicate the ‘matched’ samples. (a,b) qPCR for Ctsh, Cst7, Pcsk1n, Serpini1 (brain) in a and Adamts4, Adamts12, Casp2, Ctse (bone) in b confirmed stage-differential expression changes in a tissue-dependent manner. ‘Mismatched’ samples did not show significant changes. (c,d) qPCR for ADAM21, CTSZ, FAU, TIMP2 (brain) in c and ADAM17, CASP3, DPP8 (bone) in d confirmed stage-differential expression changes in a tissue-dependent manner. ‘Mismatched’ samples (Br-M to bone and Bo-M to brain) revealed that tumour cells underwent similar significant expression changes between early and late stages as identified for matched samples in the respective tissue. (e) qPCR for SERPINE2 confirmed stage-differential expression changes in Lu-M tumour cells in lung metastasis, while those changes were not present in Br-M and Bo-M cells in brain or bone metastasis, respectively. (f) qPCR for Serpina3n and Timp1 confirmed stage-differential expression changes (control versus late) in the stroma of lungs from xenografted animals as well as lungs from the immunocompetent, syngeneic MMTV-PyMT breast cancer model. (g) Immunofluorescence staining for Serpina3n and Timp1 in lungs of MMTV-PyMT breast cancer model (upper panels) and Ctss (red) in co-staining with GFP (tumour cells; green) or Iba1 (macrophages; white) in the syngeneic PyMT-BrM model (lower panels). Images are representative of three independent samples per stage. Scale bar indicates 50 μm. For qPCR validation: n = 11, 15, 15, 13, 7, 15 samples in a, n = 11, 15, 15, 13, 7, 5 samples in b, n = 8, 15, 7, 9, 5, 15 samples in c, n = 8, 15, 7, 9, 5, 5 samples in d, n = 8, 6, 15, 12, 5, 3 samples in e. n = 5 and 9 samples for control lung and late-stage metastases (xenograft model), and n = 6 and 9 for control lung and late-stage metastases (syngeneic model). mRNA expression is depicted relative to early-stage metastases in ae and relative to control tissue in f. All assays were performed in triplicate and gene expression was normalized to Ubc for all stromal genes, B2M for tumour cell-derived genes. P values were obtained using two-tailed unpaired t-test: NS = not significant, P < 0.05,P < 0.01, and P < 0.001.

Supplementary Figure 4 Identification of genes associated with metastasis-free survival (MFS) and differentially expressed between the tumour and stroma.

(ab) As shown in Fig. 3a for brain metastasis, cross-species scatter plots depict expression changes in the tumour and stromal gene space during the transition from early- to late-stage metastasis for (a) bone and (b) lung metastasis. Differentially expressed genes in the stroma (mouse) or in the tumour (human) gene space are shown in pink or black respectively. Genes that are differentially expressed in both the stroma and the tumour gene space are shown in purple. (ce) Differentially expressed genes shown in Fig. 1c were analysed for association with MFS for either (c) brain, (d) bone, or (e) lung metastasis, depending on the tissue in which the gene was differentially expressed. Scaled gene expression values were used in a Cox proportional hazards model as described in the methods. Hazard ratios (HR) and 95% confidence intervals (CI) are shown for each organ site. HR < 1.0 is associated with better patient prognosis, whereas HR > 1.0 is associated with poor patient prognosis. For CTSS, the HR for brain MFS was 1.4 (95% CI: 1.05–1.89; P = 0.0209). (f) Genes depicted in ce are shown in the Venn diagram, where few genes were found to be significantly associated with MFS in multiple tissues. A single gene, SLPI, was significantly associated with MFS in all three tissues, and high expression of levels of SLPI correlated with poor patient prognosis. Hazard ratio significance was determined using Wald’s test with a nominal P value cutoff of <0.05.

Supplementary Figure 5 Tumour cells and macrophages are the major constituent cell types of patient brain metastases and express high levels of CTSS.

(a,b) Representative images of (a) primary breast cancer and (b) brain metastases patient samples stained for CTSS (red) in combination with the macrophage marker CD68 (white) or a pan-cytokeratin (CK) antibody to visualize tumour cells (green). DAPI staining was used to visualize cell nuclei (blue). All patient samples listed in Supplementary Table 3 were stained and analysed and representative images are shown. The patient samples shown here represent different subtypes of breast cancer based on ER/PR/Her2 status. Scale bar indicates 100 μm in the upper panel rows and 20 μm in the lower panel rows. (c) Quantification of proportions of CK+ tumour cells and CD68+ macrophages in brain metastases samples (n = 7 samples from patients for which there was no matched primary breast tumour tissue available). (d) Combined quantification of proportions of CK+ tumour cells, CD68+ macrophages, and the remaining CKCD68 cell population in primary breast cancer (BC) (n = 6 samples) and brain metastasis samples (BrM) (n = 13; either matched to the primary, or unmatched samples). (e) Quantification of the CTSS index as a measure of relative CTSS protein levels in tumour cells and macrophages (n = 7 samples). Data in c and e are presented as box plots. Boxes: values between the 25th and 75th percentile; whiskers: minimum and maximum values; horizontal line: median. Data in d are presented as stacked bars + s.e.m.

Supplementary Figure 6 Cathepsin S deficiency does not affect viability of Br-M cells or vessel formation in mice.

(a) Quantification of CTSS mRNA expression in Br-M Ctrl and Br-M CTSS KD tumour cell lines (n = 7 samples per group). Expression is depicted relative to Br-M Ctrl cells. All assays were run in triplicate and gene expression was normalized to B2M. (b) Western blot analysis of CTSS expression levels in cell lysates and conditioned media (CM) from Br-M Ctrl and Br-M CTSS KD cells. Western blots shows representative results from 3 independent experiments. qPCR and western blotting revealed a 90% knockdown efficiency for CTSS at both the mRNA and protein levels. (c) Representative images of CTSS immunofluorescence staining of Br-M Ctrl and Br-M CTSS KD cell lines. DAPI staining was used to visualize cell nuclei (blue). Scale bar indicates 20 μm. (n = 3 replicates). (d) CTSS KD does not affect cell viability in culture as determined by MTT assays (n = 4 independent experiments). (e) Quantification of tumour cell apoptosis (percentage of TUNEL+GFP+ cells) on day 35 post-tumour cell inoculation. Ctrl; Ctss WT (n = 3 mice), CTSS KD; Ctss WT (n = 4 mice), Ctrl; Ctss KO (n = 6 mice), and CTSS KD; Ctss KO (n = 3 mice). (f) Brain metastases size and vessel density were defined as the area covered by GFP (tumour cells) or CD34 (endothelial cells) respectively, and the GFP-covered area was quantified relative to CD34-covered area using Metamorph image analysis. Ctrl; Ctss WT (n = 4 mice), CTSS KD; Ctss WT (n = 4 mice), Ctrl; Ctss KO (n = 6 mice), and CTSS KD; Ctss KO (n = 4 mice) (g) Quantification of vessel density in the brain, defined as the ratio of Texas red lectin+ area to total DAPI area (n = 4 for WT mice, n = 6 for Ctss KO mice), and (h) assessment of vessel permeability by intraveneous injection of Evan’s blue dye (n = 8 mice for each group). Each western blot shows the representative result of three independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 Cathepsin S deficiency alters the kinetics of brain metastasis formation, though not bone metastasis, but does not affect tumour growth in a regression trial.

(a) Quantification of BLI intensity in vivo at the indicated time points after tumour cell inoculation. Ctrl; Ctss WT (n = 16 mice), CTSS KD; Ctss WT (n = 15 mice), Ctrl; Ctss KO (n = 11 mice), and CTSS KD; Ctss KO (n = 10 mice). (b) Schematic of the regression trial experimental design. (c) Quantification of the BLI intensity from d0-d35 after Br-M tumour cell inoculation (n = 7 mice per group) in the regression trial setting. (d) Quantification of the BLI intensity in the bone and spine region of vehicle-treated and VBY-999-treated animals from the prevention trial, at d35 following Br-M tumour cell inoculation (n = 23 mice for vehicle group and n = 21 mice for VBY-999 group). (e) Quantification of the BLI intensity in the bone and spine region at d35 following Br-M tumour cell inoculation, in the four experimental groups. Ctrl; Ctss WT (n = 10 mice), CTSS KD; Ctss WT (n = 8 mice), Ctrl; Ctss KO (n = 10 mice), and CTSS KD; Ctss KO (n = 11 mice). (f) Quantification of the BLI intensity in the head region (which may arise from skull and/or brain lesions) of vehicle-treated and VBY-999-treated animals from the prevention trial, at d35 following Bo-M tumour cell inoculation (n = 12 mice for vehicle group and n = 13 mice for VBY-999 group). Graphs represent mean ± s.e.m. or circles represent individual mice and horizontal lines represent the mean ± s.e.m. P values were obtained using two-tailed unpaired t-test: NS = not significant, P < 0.05.

Supplementary Figure 8 Cathepsin S deficiency impairs transmigration in an in vitro BBB assay, and sequence analysis identifies a putative cleavage site for cathepsin S.

(a) Pharmacological inhibition of cathepsin S with increasing concentrations of the cathepsin S-specific inhibitor VBY-999 (0 μM (Vehicle) to 100 μM) did not affect Br-M cell viability as determined by MTT assays (n = 3 independent experiments). (b) Quantification of the number of transmigrated Br-M Ctrl and CTSS KD cells. n = 15 samples per condition with 200 fields of view analysed per sample in three independent experiments. (c) Expression of tight junction and adherens junction proteins was confirmed in an independent data set (GSE47067) of FACS sorted endothelial cells. Only Jam-B and Ocln are significantly enriched in brain endothelial cells compared to either lung or bone endothelial cells. n = 3, 3, 6 samples analysed per organ. (d) Western blot analysis of recombinant JAM-A, -B and-C cleavage by CTSS, in the presence or absence of VBY-999 (10 μM), using an antibody that detects the IgG1 domain in the recombinant JAM fusion proteins. (e) Alignment of the amino acid sequence of JAM-A, -B, and-C. Motifs that are conserved in all 3 family members are highlighted in dark purple, and motifs that are conserved in 2 of the 3 JAM family members are depicted in light purple. The putative cleavage location for cathepsin S is indicated by the red box. (f) Quantification of JAM-B cell based cleavage experiments (n = 3 independent experiments). Graphs represent mean ± s.e.m. in a and b, and box plots in c. Boxes: values between the 25th and 75th percentile; whiskers: minimum and maximum values; horizontal line: median. Circles represent individual samples and horizontal lines represent the mean ± s.e.m. in f. P values were obtained using two-tailed unpaired t-test: NS = not significant, P < 0.05,P < 0.01, and P < 0.001. Each western blot shows the representative result of three independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 9 Uncropped western blots.

Uncropped images of immunoblots displayed in the main and supplementary figures. Dashed boxes indicate regions that were cropped.

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Sevenich, L., Bowman, R., Mason, S. et al. Analysis of tumour- and stroma-supplied proteolytic networks reveals a brain-metastasis-promoting role for cathepsin S. Nat Cell Biol 16, 876–888 (2014). https://doi.org/10.1038/ncb3011

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