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Predictive outcomes for HER2-enriched cancer using growth and metastasis 1
signatures driven by SPARC 2
3
Leandro N. Güttlein,1* Lorena G. Benedetti,1* Cristóbal Fresno,2 Raúl G. Spallanzani,3 Sabrina F. 4
Mansilla,4 Cecilia Rotondaro,1 Ximena L. Raffo Iraolagoitia,3 Edgardo Salvatierra,1 Alicia I. 5
Bravo,5 Elmer A. Fernández,2 Vanesa Gottifredi,4 Norberto W. Zwirner,3,6 Andrea S. Llera,1** 6
and Osvaldo L. Podhajcer1**. 7
8
Affiliations: 9
1 Laboratorio de Terapia Molecular y Celular. IIBBA, Fundación Instituto Leloir, CONICET. 10
Buenos Aires, Argentina. 11
2 UA AREA CS. AGR. ING. BIO. Y S, CONICET, Universidad Católica de Córdoba, Córdoba, 12
Argentina. 13
3 Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina 14
Experimental-CONICET. Buenos Aires, Argentina. 15
4 Laboratorio de ciclo celular y estabilidad genómica. IIBBA, Fundación Instituto Leloir, 16
CONICET. Buenos Aires, Argentina. 17
5 Unidad de Inmunopatología, Hospital Interzonal General de Agudos Eva Perón. San Martín, 18
Provincia de Buenos Aires, Argentina. 19
6 Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de 20
Química Biológica, Buenos Aires, Argentina. 21
22
* These authors contributed equally to this work 23
** Both should be considered senior authors 24
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25
Correspondence to: 26
Osvaldo Podhajcer, PhD. 27
Av. Patricias Argentinas 435. CP 1405. Buenos Aires, Argentina. 28
Phone +541152387500 (int 2107)/ Fax +5401152387501. Email: [email protected] 29
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Running title: Sparking murine breast tumor progression with SPARC 31
Keywords: SPARC; osteonectin; breast cancer; metastasis; immunosuppression 32
Financial Support: This work was supported by AFULIC (Amigos de la Fundacion Leloir para 33
Investigación contra el Cáncer). 34
Conflicts of interest: The authors declare no conflict of interest. 35
Word count: 5954 36
Total number of figures: 7 37
Total references: 51 38
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Abstract 39
Understanding the mechanism of metastatic dissemination is crucial for the rational design of 40
novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular 41
glycoprotein which has been extensively associated with human breast cancer aggressiveness 42
although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC 43
knockdown greatly reduced primary tumor growth and completely abolished lung colonization of 44
murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A 45
comprehensive study including global transcriptomic analysis followed by biological validations 46
confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting 47
a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSCs). The role of 48
SPARC in metastasis involved a COX-2 independent enhancement of cell disengagement from 49
the primary tumor and adherence to the lungs that fostered metastasis implantation. 50
Interestingly, SPARC-driven gene expression signatures obtained from these murine models 51
predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, 52
the results reveal that SPARC and its downstream effectors are attractive targets for anti-53
metastatic therapies in breast cancer. 54
55
Implications: These findings shed light on the prometastatic role of SPARC, a key protein 56
expressed by breast cancer cells and surrounding stroma, with important consequences for 57
disease outcome. 58
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Introduction 59
Breast cancer (BC) is the second cause of cancer death among women in most countries. Only 60
25% of the patients with metastatic disease survive after 5 years (1). Global transcriptomic 61
analysis of primary BC samples has shown the existence of a molecular signature predictive of 62
metastatic development embedded in the primary tumor (2) indicating that the cell clones 63
responsible for cancer dissemination are already present in the primary tumor. 64
Previous studies performed in women who died of metastatic BC showed that more than 80 % 65
of metastasis deposits were in the lungs and pleura (3). SPARC has been heralded as one of 66
the very few genes that acts in a concerted way to promote the establishment of lung 67
metastasis (4). SPARC expression in BC was associated with the less differentiated grade 3 68
samples (5) and with the more aggressive CD44+ basal-like samples (6). SPARC was proposed 69
as an independent marker of poor prognosis for in situ ductal carcinoma (7), invasive ductal 70
carcinoma (8) and BC as a whole (9). mRNA sequencing studies identified SPARC as one of 71
the top 6 transcripts in BC primary tumor samples (10). In silico analysis associated SPARC 72
expression with poor prognosis in patients with HER2-enriched BC (11). While most studies 73
associated SPARC expression with the more aggressive forms of BC and with poor prognosis, 74
other studies seem to contradict this view. The expression of SPARC in combination with 75
TIMP3, FN1 and LOX was significantly associated with distant metastasis-free survival in lymph 76
node-negative patients (12). Additional studies showed that strong expression of stromal cells-77
derived SPARC in BC samples appear to correlate with survival (13). In addition, low SPARC 78
staining has been shown to correlate with poor prognosis in patients with luminal A or triple-79
negative BC (TNBC) (14). 80
This controversy was also observed in in vitro and in vivo studies using murine models. SPARC 81
has been identified as one of the four genes that promotes lung-metastasis (4) and its 82
expression in MCF-7 cells promoted epithelial-mesenchymal transition (EMT), through Snail 83
(15). Knock down of its expression in MCF7 cells impaired motility and invasive capabilities (16). 84
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Moreover, BC cells respond to SPARC by showing increased activity of MMP-2, a potential 85
mechanism that might facilitate cell dissemination (17). On the contrary, enforced 86
overexpression of SPARC in MDA-MB-231 cells diminished bone metastases through the 87
reduction of tumor cell-platelet aggregation (18). 88
Using immunocompromised mice models, it was shown that knock down of SPARC expression 89
in human melanoma cells induced tumor rejection by an IL8/GRO-driven recruitment and 90
activation of polymorphonuclear cells (19,20). SPARC-producing mammary carcinoma cells 91
exhibited a reduced tumor growth in SPARC null mice and an increased infiltration of CD45+ 92
leucocytes compared with wild type mice (21). Moreover, SPARC expressed by tumor-93
infiltrating macrophages increased the dissemination of 4T1 mammary cancer cells in a 94
SPARC-null mice model (22). These reports suggest a link between SPARC and the 95
inflammatory response that cannot be fully assessed in immunocompromised models. Here, we 96
performed a comprehensive study in immunocompetent mice models aimed to clarify the role of 97
SPARC in BC progression and identify downstream mechanisms and effectors. By knocking 98
down SPARC expression in malignant BC cells we show that SPARC promotes primary tumor 99
growth and dissemination through downstream effectors that affect cell cycling/viability, 100
immune response and extracellular matrix remodeling. A SPARC-dependent molecular 101
signature derived from these studies was able to identify patients with worse prognosis in the 102
HER2-enriched human BC subtype. 103
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Materials and Methods 104
Mice and human samples 105
Female BALB/c mice (6–8 weeks) were housed at the animal facility of Leloir Institute (NIH 106
A5168-01). All the in vivo experiments were approved by the Institutional Animal Care and Use 107
Committee (IACUC-FIL, Protocols 51 and 69). Paraffin-embedded tumor sections were obtained 108
from the Service of Pathology, Eva Peron Hospital, San Martín, Buenos Aires, Argentina 109
following institutional guidelines (Table S1). 110
111
Cell culture 112
Authenticated 4T1 and MCF7 cells were purchased from the American Tissue Culture 113
Collection (ATCC) between 2009 and 2011. LM3 cells were kindly provided by Elisa Bal 114
(Institute of Oncology “Angel H. Roffo”, University of Buenos Aires). All the cell lines were grown 115
in the recommended media, supplemented with 10% fetal bovine serum (Natocor, Argentina) 116
and antibiotics, and maintained at 37°C, 5% CO2 for no longer than 5 passages. To generate 117
spheroids, dispersed cells were seeded in 96-well plates pre-coated with 75 µl of 1% agarose at 118
a density of 1x104 cells per well. All the cells were routinely tested for mycoplasma 119
contamination by PCR (23). Stable knockdown of SPARC and stable expression of COX-2 and 120
SPARC was carried out as described in the Supplementary materials and methods (M&M). 121
122
In vivo assays 123
2 x 105 cells were subcutaneously (s.c.) injected in the mammary fat pad of BALB/c mice in 50 124
µL of serum-free PBS. Tumor volume was calculated as V=ds2 dl/2, where ds and dl are the 125
smaller and larger diameters, respectively. When mice showed physiological signs of distress 126
(i.e. piloerection and respiratory failure), they were euthanized and tumors were removed and 127
fixed in 10% buffered formalin for immunohistochemical studies. Metastasis foci were counted 128
after fixing lungs with 10% buffered formalin. For quantification of circulating tumor cells, tumor-129
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bearing mice were bled from the submandibular region 7 and 14 days after cell injection. 130
Erythrocytes were lysed with buffered ammonium chloride solution (ACK buffer) for 5 min and 131
the remaining cells were resuspended in selective media with G418 (Invitrogen) and cultured for 132
4 weeks in 10-cm tissue culture plates. Images were captured under x100 magnification and 133
clonogenic GFP+ colonies were counted with a CellProfiler homemade pipeline (24). For the 134
analysis of lung adhesion capability, 1 x 105 4T1 cells pre-stained with DiR (Molecular Probes) 135
were intravenously (i.v.) injected in 0.05 mL PBS. DiR in vivo tracking on isolated lungs was 136
followed with a Fluorescence Imaging (FI) IVIS Lumina Bioluminometer (Xenogen). Captured 137
images were measured as average photons per second per square centimeter per steridian 138
(p/s/cm2/sr). 139
140
Immunohistology and immunofluorescence 141
After deparaffinization, 5 µm sections of human or murine samples were incubated with 142
antibodies (Ab) against human SPARC (1:10, MAB941; R&D), mouse SPARC (1:10, AF942, 143
R&D), mouse Cleaved Caspase-3 (1:1000, RA15046, Neuromics) and mouse Ki67 (1:400, Cell 144
Signaling). Staining was revealed using the Vectastain Elite ABC kit (Vector). For total collagen 145
detection, deparaffinized sections were stained with Masson’s trichrome according to standard 146
protocols. A homemade Matlab algorithm (The Mathworks, Inc.) was used to quantify SPARC, 147
Ki67 and collagen staining (details provided in supplementary M&M). Cleaved Caspase-3+ cells 148
were counted manually. COX-2 expression in murine spheroids was assessed by 149
immunofluorescence. Briefly, spheroids were rinsed twice with PBS, fixed in 4% PFA for 1 h, 150
cryopreserved overnight (ON) in 30% sucrose, embedded in tissue OCT, and stored at -20°C. 151
Cryostat sections of 9 µm were incubated ON at 4°C with an anti-COX-2 Ab (1:100, ab15191, 152
Abcam) followed by 2 h incubation with 1/200 Cy-5-labeled donkey anti-rat Ab (Jackson). Image 153
J (25) was used to quantify spheroid fluorescence. Corrected total spheroid fluorescence 154
(CTSF) was calculated as Integrated Density – (Area of selected spheroids x Mean 155
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fluorescence of background). All images were captured using BX-60 Olympus fluorescent 156
microscope. 157
158
Microarrays analysis 159
RNA was obtained from cells and tumors (2 days-old primary tumors and 30 days-old 160
metastasis foci) using the RNeasy mini kit (Qiagen). The 2 days-old tumors could be identified 161
as a small but palpable knob of around 1.0-1.5 mm3 that was clearly seen on the everted skin 162
(26). Total RNA amplification and cRNA labeling was performed using the SuperScript Indirect 163
RNA Amplification System (Invitrogen). Profiling using the 38.5 k Murine Exonic Evidence 164
Based Oligonucleotide (MEEBO, Microarray Inc) is described in the supplementary M&M. 165
Microarray profiling data were deposited into GEO (GSE83832). mRNA levels were validated by 166
quantitative RT-PCR as described in supplementary M&M. 167
168
Studies with immune cells 169
Frequency of myeloid derived suppressor cells (MDSCs) (CD11b+Gr-1+ cells) and T 170
lymphocytes (CD4+ or CD8+ cells) was analyzed by flow cytometry. Briefly, cell suspensions 171
from spleen, primary tumor, bone marrow and lungs were labeled with mAb PE/Cy7 anti-Ly-172
6G/Ly-6C (Gr-1, clone RB6-8C5, Biolegend) in combination with PE/Cy5 anti-CD11b (clone 173
M1/70, Biolegend) for MDSCs or with PeCy5 anti-CD4 (clone RM4-5 BD) or Alexa-488 anti-CD8 174
(clone 53-6.7, BD) for T lymphocytes subsets. Cells were analyzed on a FACSCanto II flow 175
cytometer (BD). CD4+ and CD8+ lymphocytes were depleted in vivo by intraperitoneal 176
administration of 0.2 mg of mAbs against CD4 (clone YTS 191.1; ATCC) and CD8 (clone YTS 177
169.4; ATCC) respectively, at days -1, 1, 8, 15, and 22, relative to inoculation of malignant cells 178
(27). Control mice received equivalent amounts of normal mouse IgG at the same days. 179
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Depletions were confirmed in peripheral blood 7 days after tumor challenge by FC using non 180
cross-reactive Abs. 181
182
Statistical and bioinformatics analysis 183
Prism5 (GraphPad Software, Inc., San Diego, CA, USA) was used. Two groups were compared 184
with a Student t test for unpaired data. ANOVA using Bonferroni posttest was used for multiple 185
comparisons. A p-value<0.05 was considered statistically significant. Functional analysis over 186
the Kyoto Encyclopedia of Genes and Genomes (KEGG) (28) and Gene Ontology’s (GO) (29) 187
biological process (BP) were performed using the different candidate gene lists for in vivo and in 188
vitro comparisons in DAVID using the reliably detected genes for each experimental setting. 189
Association studies between candidate genes and clinical outcome in BC patients were carried 190
out by means of survival analyses using distant metastasis free survival (DMFS) as end-point. 191
See supplementary M&M for complete description. 192
193
Other methods 194
Further information about the materials and methods used in this study are provided in the 195
Supplementary M&M section. 196
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Results 197
Increased SPARC expression is associated with human and murine BC dissemination. 198
Our initial studies were performed to assess whether SPARC expression in primary tumor 199
samples from treatment-naïve patients is associated with the presence of axillary lymph node 200
metastases. We observed that increased SPARC staining in primary tumors correlated with the 201
presence of axillary lymph node metastases (Figure 1A-1B and Table S1). Average SPARC 202
intensity staining was significantly higher in primary tumors with positive axillary lymph nodes 203
than those with no metastatic nodes (Figure 1C). Integration of human tumor DNA microarrays 204
and survival data from Gene Expression-Based Outcome for Breast Cancer Online tool (GOBO) 205
(30) also showed that increased SPARC mRNA level was associated with reduced DMFS only 206
in the aggressive HER2-enriched BC subtype (Figure 1D and Figure S1). Taken together, 207
these data confirmed that SPARC expression is associated with increased aggressiveness and 208
dissemination capacity of human BC. 209
4T1 and LM3 cells spontaneously metastasize to the lungs after implantation in the mammary 210
fat pad of immunocompetent BALB/c mice (31,32). Cell clones stably expressing the shRNA 211
i52-1 targeting SPARC exhibited up to 90% decrease in SPARC mRNA and up to 80% 212
decrease in secreted SPARC that appeared as a unique band of 43 KDa (Figure S2B-2D). We 213
confirmed no changes in the expression levels of potential off-targets of the shRNA against 214
SPARC (Table S3). All the 4T1- and the LM3- SPARC-deficient cell clones showed a strongly 215
impaired capacity to grow as primary tumors (Figures 1E and S3A). As expected, primary 216
tumors obtained from SPARC-deficient cell clones showed SPARC expression in stromal cells 217
compared to 4T1-SCR control tumors that expressed SPARC both in the stromal and the 218
malignant cells compartments (Figure S3B and S3C). In addition, SPARC-deficient tumors 219
exhibited a slight but significant reduction in collagen deposition compared to control 4T1-SCR 220
tumors (Figure S3D and S3E). SPARC-deficient cell clones completely lost their capacity to 221
generate spontaneous lung metastases (Figures 1F-1G and S3F); even when SPARC-deficient 222
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4T1-C1 tumors were allowed to reach 1000 mm3, lung metastatic foci were hardly seen (Figures 223
S3G and S3H). Interestingly, SPARC-deficient tumors released reduced levels of malignant 224
cells to the circulation compared to control tumors and these cells were unable to colonize the 225
lung after i.v. administration (see below Figures 6A and C). These data suggest that SPARC 226
promotes primary tumor growth and metastatic dissemination through independent downstream 227
mechanisms. 228
229
Transcriptomic analysis of SPARC-deficient cells and tumors revealed changes in cell 230
cycling/viability, cell-ECM interaction and immune response 231
In order to gain insight into the downstream effectors of SPARC, we compared the 232
transcriptome of SPARC-deficient 4T1-C18 cells with that of 4T1-SCR control cells. Both GO 233
and KEGG pathway analyses using 407 differentially expressed genes (Table S4) highlighted 234
terms related mainly to “Cell cycle” and “cell-ECM interaction” (Figures 2A-2B, Tables S5-S6). 235
Consistent with these results, SPARC-deficient cells exhibited a significantly reduced in vitro 236
proliferative capacity (Figure 2C and Figure S4A). Moreover, SPARC-deficient 4T1-C18 cells 237
showed almost 16% increase in the percentage of cells in G1 (46 ± 3% vs. 30 ± 3%; p<0,01) 238
and 9% decrease in the percentage of cells in the S-phase compared to control 4T1-SCR cells 239
(38 ± 2 vs. 47 ± 5; p > 0,05) (Figure 2D). The lower percentage of 4T1-C18 cells in the S-phase 240
was confirmed by BrdU labelling (Figures 2E and S4B) and by following BrdU staining and cell 241
cycle distribution analysis at different time points after hydroxyurea synchronization (Figure 242
S4C-S4D). Interestingly, 4T1-C18 tumors exhibited more than 50% reduction of cycling Ki67-243
positive cells compared with 4T1-SCR tumors (Figure 2F and S4E), indicating that SPARC 244
enhanced cell cycle entrance also in vivo. 245
To further identify the downstream effectors of the protumorigenic and prometastatic role of 246
SPARC we performed a comparative analysis of the transcriptome of 4T1-SCR primary tumors, 247
4T1-C18 SPARC-deficient primary tumors and 4T1-SCR metastases (4T1-SCR MTTS). For 248
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transcriptomic studies we selected 2 days-old primary tumors (Figure S5A) to avoid tumor 249
necrosis. Metastatic samples were obtained from established 30 days-old 4T1-SCR MTTS. Our 250
previous data as well as the literature suggest the co-existence in the primary tumors of genes 251
that promote primary tumor (PT) outgrowth, with genes responsible for the early stages of 252
metastasis (ESM). These genes might be under SPARC control (S-PT genes and S-ESM 253
genes) or not (PT genes and ESM genes) (Figure 3A). A third group of genes, responsible for 254
metastasis establishment, were not part of these analyses since SPARC-deficient cells did not 255
establish lung metastases. Based on this assumption, S-PT genes correspond to the genes 256
differentially expressed between 4T1-SCR vs 4T1-C18, shared with the genes differentially 257
expressed between 4T1-SCR vs 4T1-SCR MTTS (Figure 3B, up). Using this approach we 258
identified 156 S-PT genes (Table S7 and Figure S5B). GO and KEGG analyses revealed an 259
enrichment in genes associated with inflammatory/immune response (Figures 3C-3D, Tables S8 260
and S9). Technical validation of nine S-PT genes by qPCR confirmed the downregulation of pro-261
inflammatory genes, like interleukin 6 (Il6), GRO-α (Cxcl1) and cyclooxygenase-2 (Ptgs2), in 262
SPARC-deficient 4T1-C18 tumors compared to 4T1-SCR tumors (Figure 3E). The expression of 263
these proinflammatory genes is also downregulated in lung foci compared to the 4T1-SCR 264
primary tumor (Table S7). 265
On the other hand, S-ESM genes are among those differentially expressed between 4T1-SCR 266
vs 4T1-C18 and shared with the genes differentially expressed between 4T1-C18 vs 4T1-SCR 267
MTTS (Figure 3B, down). (Table S10 and Figure S5C). GO and KEGG analysis of the 165 S-268
ESM genes showed enrichment in terms related with ECM organization (Figure 3E, Table S11) 269
and “ECM-receptor interaction”, respectively (Figure 3F, Table S12). We technically validated by 270
qPCR the expression levels of S-ESM genes widely associated with cell-ECM interaction such 271
as fibronectin 1 (Fn1), osteopontin (Spp1) and collagen 6 (alpha1) (Col6a1) (Figure 3G) among 272
others. Thus, SPARC-driven cell-ECM interaction appears to play a key role in BC 273
dissemination and colonization of the lungs. 274
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Expression of COX-2 in SPARC-deficient cells restores primary tumor growth but not the 275
metastatic capacity 276
From the previous analysis COX-2 seems to be a candidate to validate the existence of S-PT 277
genes. Initially, we observed no correlation between SPARC and COX-2 levels in cells growing 278
in 2D layers (Figure 4A). Since the in vivo transcriptome study was performed on 2 day-old 279
tumors, we hypothesized that the in vivo changes occur very early and might be related to the 280
tumor architecture rather than to tumor-stroma interaction. To mimic this scenario we prepared 281
spheroids and observed a dramatic decrease in COX-2 levels in SPARC-deficient spheroids 282
compared to 4T1-SCR spheroids (Figure 4B-4C). COX-2 expression in 4T1-C18 spheroids was 283
rescued upon exogenous SPARC addition (Figure 4C and 4D). Moreover, enforced expression 284
of SPARC in MCF7 spheroids also induced a dramatic increase in COX-2 levels (Figure 4E) 285
confirming that COX-2 expression is under SPARC control. 286
The enforced expression of COX-2 in SPARC-deficient cells (Figure S6A) restored only partially 287
their capacity to grow as a primary tumor (Figure 4F). The partial restoration in tumor growth 288
occurred despite a reduced staining in Ki67 positive cells (Figure 4G and S6B). Besides, these 289
cells showed reduced in vivo apoptosis compared to control and SPARC-deficient tumors 290
(Figure 4H, S6C and S6D), suggesting that reduced in vivo apoptosis is associated with the 291
partial restoration of tumor growth in COX-2 expressing SPARC-deficient cells. Most remarkable 292
was the evidence that SPARC-deficient cells overexpressing COX-2 were unable to establish 293
pulmonary metastases (Figure 4I and S6E), not even after i.v. administration (data not shown). 294
These data suggest that COX-2 is not involved in SPARC-driven capacity of tumor cells to 295
metastasize in the lungs. 296
297
SPARC modulates MDSCs expansion through COX-2 298
Current evidence indicates that 4T1 tumors support its outgrowth in BALB/c mice by inducing a 299
strong immunosuppressive state with systemic accumulation of MDSCS (33,34) mainly due to 300
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tumor expression of COX-2 (35). To assess whether SPARC induces immunosuppression, we 301
compare MDSCs (CD11b+Gr-1+) levels in control and SPARC-deficient tumor-bearing mice. We 302
observed a systemic reduction in the percentage of MDSC in spleens (6,9±3,2% vs. 25,3±2,0% 303
p<0,001) (Figure 5A and B), lungs (0,7±0,2% vs. 6,2±0,9% p<0,001) (Figure 5C and D) and 304
bone marrow (48,0±6,4% vs. 62,1±5,3% p>0,05) (Figure 5E and F), in mice bearing 4T1-C18 305
tumors compared to mice bearing 4T1-SCR tumors. Interestingly, enforced expression of COX-306
2 in SPARC-deficient 4T1-C18 cells restored expansion of CD11b+Gr-1+ cells, demonstrating 307
that SPARC-driven MDSC expansion is mediated by COX-2 (Figure 5G). The reduced systemic 308
levels of MDSCs was associated with an increased infiltration of CD4+ and CD8+ cells in 309
SPARC-deficient primary tumors (Figure 5H-5I). Depletion of both CD4+ and CD8+ T cells 310
partially restored 4T1-C18 primary tumor growth in BALB/c mice (Figure 5J) but had no effect 311
on the establishment of metastatic foci (Figure 5K). Overall, these results suggest that SPARC 312
drives a COX-2-mediated MDSCs expansion to evade immune surveillance affecting only 313
primary tumor growth, but not metastatic dissemination to the lungs. 314
315
SPARC knock down impairs lung foci colonization through the modulation of cell-ECM 316
interaction 317
Gene enrichment analyses indicated that S-ESM genes are associated with cell-ECM 318
interaction. SPARC has been widely associated with the modulation of cell-ECM interactions 319
(36). As mentioned before, SPARC-deficient 4T1-C18 cells exhibited a reduced capacity to 320
enter into circulation compared with control 4T1-SCR cells (Figure 6A) that was coincidental 321
with the reduced in vitro invasiveness of SPARC-deficient cell clones (Figure 6B and S7A). 322
SPARC-deficient cell clones injected i.v. generated less than five experimental metastatic foci 323
per lung compared to more than 100 foci generated by control cells (Figure 6C). This was 324
confirmed by in vivo infrared tracking of DiR-prelabeled cells showing that 4T1-C18 cells were 325
unable to colonize the lungs, suggesting an active role of SPARC in the initial establishment of 326
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the metastatic niche in the lung (Figure 6D-6E). Consistent with this, SPARC-deficient 4T1-C18 327
cells displayed a reduced ability to adhere to a fibronectin matrix (Figure 6F). Further analysis of 328
the S-ESM genes showed that the expression of fibronectin 1 was downregulated in SPARC-329
deficient tumors compared to both the 4T1-SCR tumors and the 4T1-SCR metastases (Figure 330
3G and Table S10). Overall, these data indicate that SPARC regulation of cell-ECM interaction 331
is essential for lung colonization. 332
333
The S-PT and S-ESM molecular signatures predict outcome of patients with HER2-334
enriched breast tumors 335
We next asked whether the orthologous of SPARC-regulated genes in 4T1 primary tumors 336
might play a role in the clinical outcome of human BC patients. We used GOBO tool (30) to 337
stratified samples into two groups based on hS-PT or hS-ESM gene expression quantiles and 338
performed Kaplan-Meier analysis. Interestingly, we found significant differences in DMFS 339
between two groups of worse and better outcome in all BC patients (Figure 7A and 7C) and in 340
HER2-enriched subtype using both the hS-PT and hS-ESM gene sets (Figure 7B, 7D and S8).341
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Discussion 342
Tumor heterogeneity is a hallmark of solid adenocarcinomas including human BC. Most studies 343
have shown that paired primary tumor and asynchronous metastasis are clonally related (37); 344
the frequency of certain mutations associated to specific primary tumor cell clones prevails in 345
the invasive and metastatic carcinoma indicating that a signature of metastasis is present in the 346
primary tumor (2,38). On these bases, we decided to knock down SPARC expression looking 347
for changes that might affect BC progression in an immunocompetent model. 348
Our study showed that SPARC produced by BC epithelial cells plays a fundamental role in 349
primary tumor growth and lung metastatic colonization in immunocompetent mammary tumor 350
models. This comprehensive study, that involved knocking down SPARC expression in 351
malignant epithelial cells followed by transcriptomic analysis, demonstrates that SPARC drives 352
primary tumor growth and metastasis through independent, but not mutually exclusive, 353
mechanisms. SPARC promotes primary tumor growth through the regulation of cell cycling and 354
viability as well as the induction of an immunosuppressive state. The role of SPARC in lung 355
metastasis is mainly associated with the stimulation of the ability of the malignant cells to detach 356
from the primary tumor, invade, and enhance its adhesion in the lung parenchyma. The 357
relevance of SPARC in human BC progression is here highlighted by the predictive capacity of 358
the molecular signatures associated with SPARC on the outcome of patients with the HER2-359
enriched BC aggressive subtype. 360
Both the in vitro and the in vivo transcriptome analyses confirmed that SPARC controls cell 361
cycling. SPARC silencing induced a p53-independent delay in the cell cycle progression of 4T1 362
cells (Figure S4F), with extended G1 and G2 phases; this effect was associated with the 363
induction of Wee1 and Cdc27 expression as well as the down-regulation of Cyclin B1 and B2 364
(Table S4). In coincidence, SPARC-deficient tumors exhibited reduced levels of Ki67 positive 365
cells coupled with increased levels of apoptotic cells; this balance between cell cycling and cell 366
viability could explain at least in part the reduced capacity of SPARC-deficient cells to grow into 367
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a primary tumor. The present data is consistent with previous findings showing that SPARC 368
depletion in melanoma cells promoted G2/M cell cycle arrest and down-regulation of cdk1 and 369
Cyclin B1 expression, through a p53/p21Cip1/Waf1-dependent mechanism (39,40). Notably, the 370
overexpression of COX-2 completely restored the viability of SPARC-deficient cells. However, 371
these changes in cell viability had only a partial effect on SPARC-deficient cells capacity to grow 372
as a primary tumor indicating that additional mechanisms were implicated in this process. 373
COX-2 is a marker for highly aggressive human BC (41) and promotes tumor progression in 374
experimental models by reducing cancer cell apoptosis and inducing immune escape (35,42). 375
We observed the downregulation of COX-2 expression in SPARC-deficient tumors and further 376
confirmed that COX-2 expression levels are under SPARC control. Interestingly, re-expression 377
of COX-2 in SPARC-deficient cells partially restored primary tumor growth capacity but had no 378
effect on lung metastasis establishment, not even when experimental metastases were induced 379
by direct injection through the tail vein. These experiments confirmed that COX-2 belongs to the 380
S-PT genes group. Interestingly, COX-2 was shown to play a role as a downstream mediator of 381
SPARC effect on MDSC expansion. MDSC render the tumor microenvironment less permissive 382
to T cells functionality (43). In close correlation with a more permissive microenvironment, we 383
observed an increased infiltration of CD4+ and CD8+ T cells in SPARC-deficient tumors. 384
However, depletion experiments revealed that T cells were involved only in restricting primary 385
tumor growth with no remarkable role in SPARC-driven lung metastasis establishment. Previous 386
data in immunocompromised mice models suggested that SPARC produced by malignant cells 387
might affect the innate immune response against human xenografted tumors (19,20,44). This is 388
the first work showing that both the innate and the adaptive immune response are under control 389
of SPARC produced by the malignant cells. This immune response, mediated in part by COX-2, 390
played a significant role on primary tumor growth but not on the establishment of metastatic foci. 391
During the time this work was under revision, an article was published showing that the 392
overexpression of SPARC in HER-2-induced SPARC-negative BC cells induced an epithelial 393
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mesenchymal transition accompanied by a COX 2-mediated expansion of MDSC (45). This 394
study confirms our conclusions in terms of COX-2-mediated expansion of MDSC although we 395
were unable to see changes in the expression levels of genes associated with EMT in our 396
model (data not shown). Other matricellular proteins, such as galectin-1 and osteopontin (whose 397
expression is regulated by SPARC), also promote breast tumor evasion from innate and 398
adaptive immune response, suggesting the existence of a shared function of this pleiotropic 399
family of proteins in tumor progression (46,47). 400
Independently from the above-mentioned processes, SPARC promotes metastasis by 401
regulating cell-ECM interaction driving the disengagement of malignant cells from the primary 402
tumor and the adherence to the lung parenchyma. Indeed, the amounts of circulating tumor 403
cells decreased in SPARC-deficient tumors-bearing mice and such changes correlated with the 404
reduced in vitro invasiveness of SPARC-deficient cells. Moreover, i.v. injected SPARC-deficient 405
cells were unable to colonize the lung and establish metastatic foci, mainly because of an 406
impairment in their ability to adhere to the lung. In the article that appeared when this 407
manuscript was under revision it was also suggested that MDSC play a key role in the 408
metastatic capabilities of mammary tumor cells (45). Although we cannot rule out the 409
involvement of MDSC in lung colonization, our data points to SPARC regulation of cell-ECM 410
interaction as the driven mechanism. Consistent with the evidence that SPARC can regulate cell 411
to cell and cell-ECM interaction, a recent report demonstrated that SPARC expressed by 412
melanoma cells induced vascular permeability, extravasation and lung metastasis through direct 413
interaction with VCAM1 (48). 414
A remarkable finding in the present study was that both SPARC and SPARC-driven gene 415
signatures predict the outcome of the HER2-enriched group of BC patients. As we mentioned in 416
the introduction, most studies including the present one, favor the view that SPARC expression 417
is associated with a more aggressive, less differentiated phenotype of human BC. Interestingly, 418
in silico studies found a clear association between high SPARC levels and poor prognosis in the 419
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HER2-enriched BC subtype (11). Here, we confirmed and extended those findings by showing 420
that not only SPARC but also downstream effectors associated with inflammatory/immune 421
responses and cell-ECM interactions have prognostic significance in HER2-enriched BC 422
patients. Interestingly, immune response and tumor invasion –two processes regulated by 423
SPARC in the present studies- were also associated with prognosis in HER2+ BC patients (49-424
51), suggesting a potential link between SPARC-driven effects and HER2-enriched patients 425
outcome. 426
In conclusion, the present study confirms that SPARC plays a protumorigenic and prometastatic 427
role in BC through alternative, non-exclusive mechanisms. The present data highlights the 428
tumor-associated pathways under SPARC control and points SPARC, and its downstream 429
effectors, as attractive targets for the design of rational therapies. 430
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Authors’ Contributions 431
Conception and design: L.N. Güttlein, L.G. Benedetti, V. Gottifredi, N.W. Zwirner, A. Llera, 432
O.L. Podhajcer 433
Development of methodology: L.N. Güttlein, L.G. Benedetti, C. Fresno, R.G. Spallanzani, S. 434
F. Mansilla. 435
Acquisition of data: L.N. Güttlein, L.G. Benedetti, C. Rotondaro, A.I. Bravo, R.G. Spallanzani, 436
S.F. Mansilla, C. Rotondaro, X.L. Raffo Iraolagoitia, E. Salvatierra. 437
Analysis and interpretation of data (statistical analysis, biostatistics, computational 438
analysis): L.N. Güttlein, L.G. Benedetti, C. Fresno, E.A. Fernández. 439
Writing, review, and/or revision of the manuscript: L.N. Güttlein, L.G. Benedetti, V. 440
Gottifredi, N.W. Zwirner, A.S. Llera, O.L. Podhajcer. 441
Administrative, technical, or material support: C. Rotondaro, A.I. Bravo. 442
Study supervision: A.S. Llera, O.L. Podhajcer. 443
444
Acknowledgments 445
We acknowledge the continuous support of AFULIC from Rio Cuarto, Argentina. We thank 446
Maximiliano Neme and Leonardo Sganga for technical assistance with microscopy studies and 447
flow cytometry studies respectively, and G. Mazzolini, F. Pitossi and J. Mordoh for providing us 448
with different antibodies. 449
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Figure legends 604
Figure 1. SPARC expression is associated with breast cancer progression both in 605
patients and experimental models. 606
A, SPARC immunostaining of primary human BC specimens (n=73) classified into low (n=17), 607
moderate (n=27) and intense (n=29) by a pathologist (ABI) at blind. Representative micrographs 608
are shown. Scale bars, 100 µm. B, Percentage of samples with lymph node metastasis as a 609
function of SPARC staining for each group. The fractional number of patients with positive 610
lymph node is shown. C, SPARC staining in BC primary tumors as a function of lymph node 611
status. Results are expressed as mean ± SEM, **p<0.01, t test. D, Association between SPARC 612
transcript levels and clinical outcome of BC patients with HER2-enriched subtype (n=166, High: 613
78, Low: 88; p<0.01). See also Figure S1. E, In vivo growth of SPARC-deficient 4T1 cell clones 614
compared to control cells (4T1-SCR) in syngeneic BALB/c mice. Data are the mean ± SEM of 3 615
experiments (n= 8 mice per group). ***p<0.001, two-way ANOVA and Bonferroni multiple 616
comparisons test. F, Quantification of spontaneous macroscopic lung foci. Results are 617
expressed as mean ± SEM (n= 8 mice per group). ***p<0.001, one-way ANOVA and Bonferroni 618
multiple comparisons test. H, Representative images of the experiment in (F) are shown. See 619
also Figures S2 and S3. 620
621
Figure 2. SPARC modulates cell cycling. 622
A and B, Clusters of GO Biological Process (BP) and KEGG pathway enrichment analysis of 623
differentially expressed genes between 4T1-SCR control and SPARC-deficient 4T1-C18 cells in 624
vitro. Name of clusters in (A) were arbitrary assigned according to their functionality. C, In vitro 625
cell proliferation of the different SPARC-deficient 4T1 clones compared to control cells (4T1-626
SCR) analyzed by MTS assay. Data are the mean ± SEM of 3 experiments. ***p<0.001, two-627
way ANOVA and Bonferroni multiple comparisons test. D, Percentage of 4T1-SCR or 4T1-C18 628
cells in different phases of the cell cycle analyzed by flow cytometry of propidium iodide-stained 629
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29
cells. E, Percentage of cells in S-phase assessed by BrdU immunostaining under basal 630
conditions. F, Quantification of Ki-67 staining in 4T1-SCR and 4T1-C18 tumor sections, 5 days 631
after cells inoculation in the mammary fat pad. E and F, Data are the mean ± SEM of 3 632
experiments. ***p<0.001 **p<0.01, *p<0.05, ns non-significant, t test. See also Figure S4. 633
634
Figure 3. SPARC-dependent genes associated with primary tumor growth and lung 635
metastasis establishment. 636
A, Model depicting the co-existence in primary tumors of two groups of genes involved in 637
primary tumor growth (PT) and early-stage of metastasis (ESM)t that might be or not under 638
SPARC (S) regulation (See the text for details). B, Top, Venn’s diagram showing that S-PT 639
genes are those differentially expressed between 4T1-SCR vs 4T1-C18 primary tumors, shared 640
with the genes differentially expressed between 4T1-SCR primary tumor vs 4T1-SCR MTTS. 641
Bottom, Venn’s diagram showing that S-ESM genes are those differentially expressed between 642
4T1-SCR vs 4T1-C18 primary tumors, shared with the genes differentially expressed between 643
4T1-C18 primary tumors vs 4T1-SCR MTTS. C and D, Clusters of GO Biological Process (BP) 644
and KEGG pathways enrichment analysis of S-PT genes. E, RT-qPCR validation of six S-PT 645
genes identified in the microarray. F and G, Clusters of BP and KEGG pathways enrichment 646
analysis of S-ESM genes. H, RT-qPCR validation of six S-ESM genes identified in the 647
microarray. E and H, data of RT-qPCR are presented as mean ±SEM (n=3). See also Figure 648
S5. 649
650
Figure 4. SPARC-driven COX-2 expression affects primary tumor growth. 651
A, RT-qPCR of COX-2 mRNA levels in cells growing as monolayer. B, RT-qPCR of COX-2 652
mRNA levels in cells’ spheroids. C, COX-2 immunofluorescence in spheroids sections. 653
Spheroids were grown in the presence of 2 µg/mL human purified SPARC (+) or vehicle (-) and 654
fixed after 72 hr. Scale bars, 100 µm. D, Quantification of COX-2 specific fluorescence in the 655
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30
experiment depicted in C. Data are presented as mean ± SEM and are representative of 3 656
experiments. ***p<0.001, ns not significant, one-way ANOVA and Bonferroni multiple 657
comparisons test. E, RT-qPCR of SPARC and COX-2 mRNA levels in spheroids of MCF7 cells 658
transduced with a lentivirus expressing GFP or hSPARC cDNA (MCF7-GFP and MCF7-SPARC 659
respectively).F, In vivo growth of 4T1-SCR, 4T1-C18(E), 4T1-C18 (COX2-C7) and 4T1-C18 660
(COX2-C15) tumors in BALB/c mice (n= 9-13 mice per group). G, Quantification of Ki-67 661
positive area in tumor sections obtained at the end of the experiment depicted in F (n=3). H, 662
Quantification of cleaved caspase-3 positive cells in tumor sections at the end of the experiment 663
depicted in F. I, Number of macroscopic foci in the lungs. Data were obtained when primary 664
tumors reached a size of 1 cm3 (n=4-6). Data are representative of 3 experiments. *** p<0.001, 665
** p<0.01, *p<0.05, ns not significant, one-way ANOVA and Bonferroni multiple comparisons 666
test. See also Figure S6. 667
668
Figure 5. SPARC drives MDSCs expansion through COX-2. 669
Percentage of spleen (A) lung (C) and bone marrow (E) CD11b+Gr-1+ cells in mice harboring 670
different tumor types (n=4 mice per group). (B), (D), (F) Representative dot plots of data 671
depicted in A, C and E, respectively. Numbers within dot plots correspond to the percentages of 672
cells in each marked region. G, Percentage of CD11b+Gr-1+ cells in spleen of mice challenged 673
with different tumor types (n=4 mice per group). H and I, Percentage of intratumoral CD4+ and 674
CD8+ cells in mice harboring the different tumor types (n=4 mice per group). J, In vivo tumor 675
growth of 4T1-SCR or 4T1-C18 cells. Mice were treated with an i.p injection of the indicated 676
mAbs. Control mice received the equivalent amounts of normal rat IgG at the same days (CI) 677
(n=4 mice per group). B, D, F, G, H, I and J, Data are presented as mean ± SEM and are 678
representative of 3 experiments. *p < 0.05; **p < 0.01, ***p<0.001, ns not significant, one-way 679
ANOVA and Bonferroni multiple comparisons test. K, Number of lung macroscopic foci of 4T1-680
SCR and 4T1-C18 tumor-bearing mice. Tumor-bearing mice were treated with the indicated 681
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31
mAbs. Data are presented as mean ± SEM. ns not significant, unpaired t test with Welch’s 682
correction. 683
684
Figure 6. SPARC modulates the early stages of metastatic dissemination and 685
establishment of lung foci. 686
A, Amount of circulating tumor cells. B, In vitro invasion assays. C, Number of macroscopic 687
metastatic foci after tail vein injection of the tumors indicated in each panel (n=3 mice per 688
group). D, Quantification of fluorescence intensity in lungs of naïve mice five hours after i.v. 689
injection of DiR-stained cells. The average radiance of lungs was relativized to the average 690
radiance of spleen (n=3 mice per group). E, Adhesion assays on fibronectin showing cells per 691
field after 20 minutes plating. Data are the mean ± SEM of 3 experiments. ***p<0.001, **p<0.01, 692
*p<0.05, one-way ANOVA and Bonferroni multiple comparisons test. See also Figure S7. 693
694
Figure 7. Human orthologous of S-PT and S-ESM genes predict clinical outcome of 695
HER2-enriched patients. 696
A and B, Association between expression of hS-PT and DMFS of BC patients. A, All patients 697
(n=1379, High: 608, Low: 771; p<0.01). B, Patients with HER2-enriched subtype (n=166, High: 698
56, Low: 110; p<0.05). C and D, Association between expression of hS-ESM and DMFS of BC 699
patients. C, All patients (n=1379, High: 596, Low: 783; p<0.01). D, Patients with HER2-enriched 700
subtype (n=166, High: 60, Low: 106; p<0.05). The analyses were performed with Gene 701
Expression-Based Outcome for Breast Cancer Online tool. See also Figure S8. 702
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PAM50 - Her2-Enriched
0.00
0.25
0.50
0.75
1.00
0 5 10 15
Time (Years)
DM
FS
(%
)
P=0.00453SPARC expression
High: n=78
Low: n=88
A
C B
E
D
Anti hSPARC
Low Moderate Intense
0 10 20 300
200
400
600
800
1000
1200
1400 4T1-SCR
4T1-C1
***
***
***
4T1-C18
4T1-C5
Days after injection
Tum
or
volu
me
(m
m3)
4T1-
SCR
4T1-
C1
4T1-
C5
4T1-
C18
0
50
100
150
******
***
Num
be
r o
f sp
onta
ne
us
lung
macro
me
tasta
se
s
0 10 20 300
20
40
60
80
Low 6/17
Moderate 17/27
Intense 21/29
SPARC staining (AU)
% S
am
ple
s w
ith
po
siti
ve ly
mp
h n
od
e
F G 4T1-SCR 4T1-C18
Negative Positive0
10
20
30 **
n= 29n=44
Lymph node status
SP
AR
C s
tain
ing
(A
U)
Figure 1
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A
0 1 2
Cytoskeleton organization
Cell cycle
Vasculature development
Cell Adhesion
Threshold
Enrichment Score
0 1 2 3
Cell cycle
Oxidative phosphorylation
p53 signaling pathway
Threshold
- Log (p value)
B
C D E F
0 24 48 72 960.0
0.5
1.0
1.5
2.0 4T1-SCR
4T1-C1
***
***
4T1-C18
4T1-C5
Time (hours)
Ab
so
rbance
49
0 n
m
G1 S
G2
Sub
G1
0
20
40
60 4T1-SCR
4T1-C18**ns
ns ns
% C
ells
at cyc
le p
hase
4T1-
SCR
4T1-
C18
0
20
40
60
80
*
Brd
U+ C
ells
(%
)
4T1-
SCR
4T1-
C18
0
20
40
60
80
100**
Ki6
7+ c
ells
(p
er
field
)
Figure 2
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A
C D E
F G H
PT genes
S-PT
ESM genes
S-ESM
4T1-C18
primary tumor
4T1-SCR
primary tumor
4T1-SCR MTTS
Lung metastasis
4T1-C18 vs
4T1- SCR
4T1-SCR vs
4T1-SCR MTTS
4T1-C18 vs
4T1- SCR
4T1-C18 vs
4T1-SCR MTTS
B
Il6
Ptg
s-2
Ptg
r2Ncf1
Ndr
g 1
Cxc
l1
Esa
mBra
fSrc
-6
-4
-2
0
2
4 Micro arrayRT-qPCR
Lo
g2
FC
Fn1
Spp
1
Col6a
1Ccl8
Cas
p3
Sta
t5b
-4
-2
0
2
4 Micro arrayRT-qPCR
Lo
g2
FC
S-PT genes (156): GO-BP clusters
0.0
0.5
1.0
1.5
Defense response
Immune response
Wound healing
Enrichment Score
0 1 2 3 4
Gap juction
Focal adhesion
Long-term depression
Chemokine signaling pathway
Leukocyte transendothelial mig
- Log (p value)
S-PT genes (156): KEGG pathways
0.0
0.5
1.0
1.5
Bone development
Protein processing
ECM organization
Regulation of growth
Enrichment Score
S-ESM genes (165): GO-BP clusters
0.0 0.5 1.0 1.5
ECM-receptor interaction
- Log (p value)
S-ESM genes (165): KEGG pathways
Figure 3
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A C
D F
H G
4T1-
SCR
4T1-
C5
4T1-
C18
0.0
0.5
1.0
1.5
CO
X-2
mR
NA
re
lativ
e
exp
ressio
n le
vels
in 3
D c
ultu
re
MCF7-
GFP
MCF7-
SPARC
0.0
0.5
1.0
1.5
2.0 hSPARC
hCOX-2
mR
NA
re
lativ
e
exp
ressio
n le
vels
0 20 40 600
500
1000
1500
2000 4T1-SCR
4T1-C18(E)
4T1-C18(COX2-C7)
4T1-C18(COX2-C15)
***
Days after injection
Tum
or
volu
me
(m
m3)
4T1-
SCR
4T1-
C18
(E)
4T1-
C18
(COX2-
C7)
4T1-
C18
(COX2-
C15
)0
5
10
15
20
25***
******
nsns
Num
be
r o
f sp
onta
ne
ous
lung
macro
me
tasta
se
s
- + - +
4T1-SCR 4T1-C18
CO
X-2
N
ucle
i
hSPARC
E
4T1-
SCR
4T1-
C18
(E)
4T1-
C18
(COX2-
C7)
0
1
2
3
4*
*
% A
rea K
i-6
7 s
tain
ing
I
4T
1-S
CR
4T
1-S
CR
4T
1-C
18
4T
1-C
18
0
2
4
6
8
ns
***
ns
- + - + hSPARC
2g/mL
Lo
g1
0C
TS
FB
4T1-
SCR
4T1-
C5
4T1-
C18
0.0
0.5
1.0
1.5
2.0
CO
X-2
mR
NA
re
lativ
e
exp
ressio
n le
vels
in 2
D c
ultu
re
4T1-
SCR
4T1-
C18
(E)
4T1-
C18
(COX2-
C7)
0
10
20
30
40
50 *
ns
***
Cle
ave
d-C
asp
3+ c
ells
pe
r 2
0x fie
ld
Figure 4
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64.5%
0 102 103 104 105
0
102
103
104
105
70.3%
0 102 103 104 105
0
102
103
104
105 45.3%
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CI
4T1-SCR
4T1-WT
4T1-C18
CD11b
Gr-
1
Bone Marrow
A B C D
E F G H
I J K
25.9%
0 102 103 104 105
0
102
103
104
105
28.1%
0 102 103 104 105
0
102
103
104
105 4.9%
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CI
4T1-SCR
4T1-WT
4T1-C18
CD11b
Gr-
1
Spleen
CD11b
Gr-
1
5.0%
0 102 103 104 105
0
102
103
104
105
5.2%
0 102 103 104 105
0
102
103
104
105 1.0%
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CI
4T1-SCR
4T1-WT
4T1-C18
Lung
4T1-
WT
4T1-
SCR
4T1-
C18
0
10
20
30
40 *****ns
%C
D11b
+G
r-1
+ce
lls
in s
ple
en
4T1-
WT
4T1-
SCR
4T1-
C18
0
2
4
6
8
10ns ***
*
%C
D11b
+G
r-1
+ce
lls
in lu
ng
4T1-
WT
4T1-
SCR
4T1-
C18
0
20
40
60
80 nsns
ns
%C
D11b
+G
r-1
+ce
lls
in b
one m
arr
ow
4T1-
SCR
4T1-
C18
(E)
4T1-
C18
(COX2-
C7)
4T1-
C18
(COX2-
C15
)0
20
40
60
80 ***ns**
*****
% C
D11b
+G
r-1
+ce
lls
in s
ple
en
4T1-
SCR
4T1-
C18
0.0
0.5
1.0
1.5
*
Tum
or
infiltr
ating
CD
4+cells
(%
)
4T1-
SCR
4T1-
C18
0
2
4
6
8
10 *
Tum
or-
infiltr
ating
CD
8+cells
(%
)
0 10 20 30 40 500
500
1000
1500
2000 4T1-SCR (CI)
4T1-C18 (CI)
4T1-C18 (AntiCD4+AntiCD8)
*
*
**
**4T1-SCR (AntiCD4+AntiCD8)
Days after injection
Tum
or
volu
me
(m
m3)
4T
1-S
CR
4T
1-S
CR
4T
1-C
18
4T
1-C
18
0
20
40
60
80
ns
CI
CD4 + CD8
+ - - -
+ +- -
Num
be
r o
f sp
onta
ne
ous
lung
macro
me
tasta
se
s
Figure 5
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A B
C D
4T1-
SCR
4T1-
C1
4T1-
C5
4T1-
C18
0
50
100
150
******
Inva
siv
e c
ells
(% o
f th
e c
ontr
ol)
7 140
20
40
60
80
100
4T1-C18
4T1-SCR
******
Days after injection
Num
be
r o
f G
41
8
resis
tent co
lonie
s p
er
fie
ld
4T1-
SCR
4T1-
C1
4T1-
C5
4T1-
C18
0
50
100
150 ******
***
Num
be
r o
f e
xpe
rim
enta
l
lung
macro
me
tasta
se
s
4T1-
SCR
4T1-
C18
0.0
0.5
1.0
1.5
2.0
2.5 *
Lung
ave
rag
e r
ad
iance
(sp
lee
n r
ela
tive
leve
ls)
4T1-
SCR
4T1-
C18
0
500
1000
1500
*A
dhe
rent ce
lls o
n
fib
rone
ctin
pe
r fie
ld
E
Figure 6
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A B
C D
Figure 7
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Published OnlineFirst December 28, 2016.Mol Cancer Res Leandro N. Guttlein, Lorena G. Benedetti, Cristobal Fresno, et al. and metastasis signatures driven by SPARCPredictive outcomes for HER2-enriched cancer using growth
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