Predictive outcomes for HER2-enriched cancer using growth ... · 12/28/2016 · 1 1 Predictive...

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Predictive outcomes for HER2-enriched cancer using growth and metastasis 1

signatures driven by SPARC 2

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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

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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

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* These authors contributed equally to this work 23

** Both should be considered senior authors 24

on January 23, 2021. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 28, 2016; DOI: 10.1158/1541-7786.MCR-16-0243-T

http://mcr.aacrjournals.org/

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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




4

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




16

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




18

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




19

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




20

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




21

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28

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




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




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




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




PAM50 - Her2-Enriched

0.00

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A

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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




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




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)

***


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




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)


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




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

******


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




A B

C D

Figure 7




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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