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Figure S1. Association between SPARC expression and distant metastasis
free survival (RFS) in breast cancer patients with different PAM50 subtypes.
A, All patients (n=1379, High: 663, Low: 716; p>0,05). B, Patients with Luminal A
(LumA) subtype (n = 323, High: 213, Low: 110; p>0.05). C, Patients with Luminal B
(LumB) subtype (n = 358, High:134, Low: 224, p>0.05). D, Patients with Basal
subtype (n = 251, Hich: 69, Low: 182, p>0.05). E, Patients with Normal subtype (n
= 211, High: 127, Low: 84, p>0.05). The analyses were performed with Gene
Expression-Based Outcome for Breast Cancer Online tool. See also Figure 1.
FigureS2. Downregulation of SPARC expression in 4T1 and LM3 murine
breast cancer cell lines
A, SPARC mRNA levels measured by RT-qPCR in 4T1 cells transduced with a
lentiviral vector expressing a scramble (SCR) siRNA or siRNAs targeted to
different regions of murine SPARC mRNA (i518, i52-1 and i52-2). B and C, RTqPCR SPARC mRNA levels in 4T1 (B) and LM3 (C) cell clones transduced with
the SCR siRNA or i52-1 siRNA. D, Immunoblot of secreted SPARC. The optical
density of the bands was normalized to SYPRO Ruby-stained loading control (LC)
and .expressed as fold change with respect to control 4T1-SCR or LM3-SCR cells.
A, B and C, data are the mean ± SEM of 3 experiments. ***p<0.001, ns not
significant, one-way Anova and Bonferroni multiple comparisons test. D, Data are
representative of 3 experiments. See also Figure 1.
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Figure S3. SPARC targeting inhibits primary tumor growth and lung
metastasis establishment. A, In vivo growth of SPARC-deficient LM3 cell clones
(LM3-C5 and –C12) and control cells (LM3-SCR) in BALB/c mice. B,
Immunohistochemical expression of SPARC in control (4T1-SCR) and SPARCdeficient tumors (4T1-C18). C, Quantification of SPARC intensity. D, Trichromic
Masson staining in 4T1-SCR and 4T1-C18 tumors. E, Quantification of collagen
intensity. Red scale bars, 50 µm. Data were obtained from samples collected 7
days after injection. Data in C and E are expressed as mean ± SEM (t test,
*p<0.05, ***p<0.001). F, Representative microphotographs of lung sections from 30
days-old 4T1-SCR or 4T1-C18 tumor-bearing mice, stained with hematoxilin and
eosin (H&E). The arrows show micrometastases. Black scale bars, 300 µm. G, In
vivo growth of 4T1-SCR and 4T1-C1 tumors. H, Quantification of spontaneous
macroscopic lung foci 30 and 45 days after orthotopic inoculation of 4T1-SCR and
4T1-C1 cells, respectively, when both primary tumors reached 900 mm 3. A, G and
H, data are the mean ± SEM of 3 experiments (n= 3-4 mice per group). **p<0.01,
***p<0.00. See also Figure 1.
Figure S4. SPARC-deficient cells exhibit a delay in cell cycle progression. A,
In vitro cell proliferation of the different SPARC-deficient LM3 clones compared to
control cells (LM3-SCR). Data are the mean ± SEM of 3 experiments. ***p<0.001,
two-way Anova and Bonferroni multiple comparisons test. B, Representative
microphotographs of the BrdU immunostaining assay in 4T1 cells under basal
conditions. See also Figure 2E for quantification. C, Time course of BrdU
immunostaining after hydroxyurea synchronization. D, Percentage of cells at each
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stage of the cell cycle analyzed by flow cytometry at 0, 2, 4, 6, 8, 16 and 22 hours
after hydroxyurea synchronization. E, Immunohistochemical staining of Ki67positive cells in control 4T1-SCR and SPARC-deficient 4T1-C18 tumors. The
arrows show Ki-67-positive cells. Scale bars, 50 µm. F, Immunoblot against p53
and actin. HCT116 colorectal cancer cells were used as a control of positive
staining for p53. C, D, E and F data are representative of 3 experiments. Scale
bars, 20 µm. See also Figure 2.
Figure S5. Transcriptomic analysis of SPARC-deficient tumors revealed
changes in cell-ECM interaction and immune response. A, Representative
microphotographs of tumor sections from 2 day-old 4T1-SCR and 4T1-C18 tumorbearing mice, stained with Hematoxylin and Eosin (H&E). Scale bars, 100 µm. B,
Heatmap displays expression levels of 156 S-PT genes in the 4T1-SCR primary
tumors (4T1-SCR), 4T1-C18 SPARC-deficient tumors (4T1-C18) and 4T1-SCR
metastases (4T1-SCR MTTS) (n= 4 mice per group). C, Heatmap displays
expression levels of 165 S-ESM genes in the 4T1-SCR primary tumors (4T1-SCR),
4T1-C18 SPARC-deficient tumors (4T1-C18) and 4T1-SCR metastases (4T1-SCR
MTTS) (n= 4 mice per group).
Figure S6. Enforced expression of COX-2 in SPARC-deficient 4T1 cells
partially restores cells capacity to grow as primary tumor. A, Western-blot
analysis of COX-2 protein levels in 4T1-SCR, 4T1-C18(E), 4T1-C18(COX2-C7)
and 4T1-C18(COX2-C15) spheroids. The optical density of bands were normalized
to β-actin intensities and expressed as fold change value relative to 4T1-SCR
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spheroids. Experiments were repeated twice with similar results. B, Representative
images of Ki-67 immunostaining of 4T1-SCR, 4T1-C18(E) and 4T1-C18(COX2-C7)
tumors sections obtained at the end of the experiment. The arrows show Ki-67+
cells. See also Figure 4F. C, Representative images of cleaved caspase-3
immunostaining of 4T1-SCR, 4T1-C18(E) and 4T1-C18(COX2-C7) tumors sections
obtained at the end of the experiment. The arrows show cleaved caspase-3
positive cells. Black scale bars, 50 µm. D, Western-blot analysis of cleaved
caspase-3 levels in 4T1-SCR, 4T1-C18(E) and 4T1-C18(COX2-C7) tumoral
extracts. Tumoral extracts from 3 different mice are shown for each condition. The
optical density of cleaved caspase-3 bands (19 and 17 kDa) were normalized to
complete caspase-3 (30 kDa) intensities and expressed as fold change value
relative to the first 4T1-SCR tumoral extract. Experiments were repeated twice with
similar results. E, Representative microphotographs of lung sections from 30 daysold 4T1-SCR, 4T1-C18(E) and 4T1-C18(COX2-C7) tumor-bearing mice, stained
with H&E. The arrows show micrometastases. Red scale bar, 300 µm. See also
Figure 4I.
Figure S7. SPARC targeting impairs invasive capacity of breast cancer cells.
(A) In vitro invasion of LM3 derived clones (% of control LM3-SCR cells). Data are
the mean ± SEM of 3 experiments. ***p<0.001, **p<0.01, one-way Anova and
Bonferroni multiple comparisons test. See also Figure 6.
Figure S8. Association between hS-PT and hS-ESM expression and distant
metastasis free survival (RFS) in breast cancer patients with different PAM50
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subtypes. hS-ESM signature expression in: A, Patients with Luminal A (LumA)
subtype (n = 323, High: 156, Low: 167; p>0.05). B, Patients with Luminal B (LumB)
subtype (n = 358, High: 137, Low: 221, p>0.05). C, Patients with Basal subtype (n
= 251, High: 74, Low: 177, p>0.05). D, Patients with Normal subtype (n = 211,
High: 130, Low: 81, p>0.05). hS-PT signature expression in: E, Patients with
Luminal A (LumA) subtype (n = 323, High: 150, Low: 173; p>0.05). F, Patients with
Luminal B (LumB) subtype (n = 358, High: 105, Low: 253, p>0.05). G, Patients with
Basal subtype (n = 251, High: 114, Low: 137, p>0.05). H, Patients with Normal
subtype (n = 211, High: 140, Low: 71, p>0.05). The analyses were performed with
Gene Expression-Based Outcome for Breast Cancer Online tool. See also Figure
7.
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