Fig 2: Identification of SPARC as an extracellular protein cleaved in the TNBC microenvironment at acidic pH. (A) Experimental set-up of the MDA-MB-231 cell-HMF co-culture system. MDA-MB-231 TNBC cells and HMFs were co-cultured in serum-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37 °C for 24 h. The 24 h-conditioned medium was then concentrated to 0.2 mg/mL, and incubated in cleavage buffer with or without pepstatin A (Pepst.) (12.5 µM) at pH 5.5 and 37 °C for 60 min. A representative image of an MDA-MB-231/HMF co-culture is shown in the right panel (x 200). (B) Box and whisker plot of the normalized ratios of the N-terminal peptides identified by TAILS in the MDA-MB-231/HMF co-culture secretome. 3091 peptides quantified at t = 60 min of incubation with cleavage buffer were used to generate the graph. The without/with pepstatin A ratios corresponding to SPARC peptides are highlighted in red; whiskers correspond to the 2.5th and 97.5th percentiles. (C) Validation of SPARC cleavage in the MDA-MB-231/HMF co-culture secretome. Secretome samples (2 µg) from the MDA-MB-231/HMF co-culture incubated in cleavage buffer with or without pepstatin A (Pepst.) (12.5 µM) at pH 5.5 and at 37 °C for 60 min were separated on 13.5% SDS-PAGE followed by immunoblotting with anti-SPARC antibody (clone AON-5031).

Fig 3: Reduced levels of a-granule proteins SPARC, VWF and PF4 during storage. (a) Non-imputed LFQ values of SPARC (blue) and SPARC levels as measured with ELISA (red). (b) Non-imputed LFQ values of VWF (blue) and VWF levels as measured with ELISA (red). (c) Western blot analysis of SPARC, LFQ values (blue) and intensities of Western blot (red). (d) Western blot analysis of VWF, LFQ values (blue) and intensities of Western blot (red). Calmodulin was used as loading control. Bands were normalized according to intensity of calmodulin (loading control), and average intensity per membrane was normalized to 1. (e) Non-imputed LFQ values of PF4 (blue) and PF4 levels as measured with ELISA (red). Data represents mean ± standard deviation (n = 3), *P < 0.05 compared to day 1 from this time point onwards. Uncropped immunoblots of SPARC, VWF and calmodulin can be found in Supplementary Data S5.

Fig 4: Effects of FL SPARC and cath-D-induced cleaved SPARC fragments on adhesion, migration, transmigration and invasion of TNBC cells. (A) Cell adhesion. MDA-MB-231 cells were let to adhere for 30 min on a fibronectin matrix in the absence or presence of recombinant FL SPARC (SPARC), or recombinant cath-D-induced cleaved SPARC fragments (cleaved SPARC) at the final concentration of 240 nM. Left panels, representative images of adherent cells. Right panel, adherent cells were stained with crystal violet, and adhesion was quantified at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n = 3); ***, p < 0.001, ANOVA and Bonferroni's post hoc test. Similar results were obtained in four independent experiments. (B) Cell migration. MDA-MB-231 cells were let to migrate for 16 h on a fibronectin matrix in the absence or presence of recombinant FL SPARC, or cleaved SPARC at a final concentration of 240 nM. Left panels, representative images of migrating cells. Right panel, migrating cells were quantified by MTT staining and absorbance was read at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n = 3); *, p < 0.05; **, p < 0.01; ***, p < 0.001, ANOVA and Bonferroni's post hoc test. Similar results were obtained in three independent experiments. (C) Endothelial transmigration. MDA-MB-231 cells were let to transmigrate for 16 h through a HUVEC monolayer in the absence or presence of recombinant FL SPARC, or cleaved SPARC at a final concentration of 240 nM. Left panels, representative images of transmigrating cells. Right panel, transmigrating cells were stained with MTT and quantified at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n = 3); *, p < 0.05, **, p < 0.01, ***, p < 0.001, ANOVA and Bonferroni's post hoc test. Similar results were obtained in two independent experiments. (D) Cell invasion. MDA-MB-231 cells were let to invade for 16 h on a Matrigel matrix in the absence or presence of recombinant FL SPARC, or cleaved SPARC at a final concentration of 240 nM. Left panels, representative images of invading cells. Right panel, invading cells were stained with MTT and quantified at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n = 3); ***, p < 0.001, ANOVA and Bonferroni's post hoc test. Similar results were obtained in three independent experiments.

Fig 5: Limited proteolysis of fibroblast- and cancer cell-derived SPARC at acidic pH by cath-D secreted by TNBC and mouse breast cancer cells. (A) Time-course of SPARC degradation in MDA-MB-231 / HMF conditioned medium. MDA-MB-231 TNBC cells and HMFs were co-cultured in serum-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37 °C for 24 h. The 24 h conditioned medium from co-cultured MDA-MB-231 / HMF was incubated at 37 °C in cleavage buffer with or without pepstatin A (Pepst.) at pH 5.5 for the indicated times. SPARC cleavage in conditioned medium was analyzed by 13.5% SDS-PAGE and immunoblotting with an anti-SPARC antibody (15274-1-AP). O/N, overnight. (B) Influence of the milieu acidity on SPARC degradation in MDA-MB-231 / HMF conditioned medium. MDA-MB-231 TNBC cells and HMFs were co-cultured as in (A). The 24 h conditioned medium was incubated at 37 °C in cleavage buffer with or without pepstatin A at the indicated pH overnight. SPARC cleavage was analyzed as described in (A). (C and D) Time-course of SPARC cleavage in TNBC cell conditioned medium. HS578T TNBC cells (C) and SUM159 TNBC cells (D) were cultured in serum-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37 °C for 24 h. The 24 h conditioned medium was incubated at 37 °C in cleavage buffer with or without pepstatin A at pH 5.5 for the indicated times. SPARC cleavage was analyzed as described in (A). (E) SPARC cleavage by cath-D secreted by MDA-MB-231 cells. MDA-MB-231 cells were transfected with Luc or cath-D siRNAs. At 48 h post-transfection, siRNA-transfected MDA-MB-231 cells were co-cultured with HMFs as described in (A). Then, the 24 h conditioned media from co-cultured siRNA-transfected MDA-MB-231 / HMF were incubated at 37 °C in cleavage buffer with or without pepstatin A at pH 5.5 for 120 min. Cath-D secretion by siRNA-transfected MDA-MB-231 cells was analyzed with an anti-cath-D antibody (H-75). SPARC cleavage was analyzed as described in (A). (F) SPARC cleavage by cath-D secreted by inducible Ctsd knock-out MMTV-PyMT mammary tumor cells. Inducible Ctsd knock-out MMTV-PyMT breast cancer cells were incubated or not with 4-hydroxytamoxifen (OH-Tam; 3 µM) for 4 days to induce Ctsd knock-out. Then, cells were cultured in FCS-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37 °C for 24 h. The 24 h-conditioned medium was incubated at 37 °C in cleavage buffer with or without pepstatin A at pH 5.5 for 120 min or O/N. Cath-D secretion was analyzed with an anti-cath-D antibody (AF1029). SPARC cleavage was analyzed as described in (A).