1
SUPPLEMENTARY INFORMATION
2
3
p63/MT1-MMP axis is required for in situ to invasive transition in basal-like
4
breast cancer
5
Catalina Lodillinsky, Elvira Infante, Alan Guichard, Ronan Chaligné, Laetitia
6
Fuhrmann, Joanna Cyrta, Marie Irondelle, Emilie Lagoutte, Sophie Vacher, Hélène
7
Bonsang-Kitzis, Marina Glukhova, Fabien Reyal, Ivan Bièche, Anne Vincent-
8
Salomon and Philippe Chavrier
9
10
Table of Content
page
11
SUPPLEMENTARY METHODS
2-7
12
SUPPLEMENTARY REFERENCES
8-11
13
SUPPLEMENTARY TABLES
12-
14
Supplementary Tables S1
12-13
15
Supplementary Tables S2
14-15
16
Supplementary Tables S3
16-17
17
Supplementary Tables S4
18
18
SUPPLEMENTARY FIGURE LEGENDS
19-
19
Supplementary Figure S1
19
20
Supplementary Figure S2
19-20
21
Supplementary Figure S3
20-21
22
Supplementary Figure S4
21
23
1
24
SUPPLEMENTARY METHODS
25
RNA extraction and RT-qPCR analysis. Samples of 458 primary unilateral invasive
26
primary breast tumors excised from women at the Institut Curie/René Huguenin
27
Hospital (Saint-Cloud, France) from 1978 to 2008 have been analyzed. Samples
28
were included if the proportion of tumor cells was more than 70%. Tumors were
29
divided into four groups according to hormone-receptor (ER and PR) and HER2
30
status as described 1 (see Table S1).
31
Conditions for total RNA extraction, cDNA synthesis and PCR reaction have been
32
described elsewhere
33
extraction procedure was performed on ice using RNeasy Mini kit (Qiagen) according
34
to the manufacturer’s instructions. Quantitative values were obtained from the cycle
35
number (Ct value) using ABI Prism 7900HT Sequence Detection System and PE
36
Biosystems analysis software according to the manufacturer’s instruction (Perkin-
37
Elmer Applied Biosystems). Each sample was normalized to the level of TATA box-
38
binding protein (TBP) transcripts content and results, expressed as N-fold differences
39
in target gene expression relative to the TBP gene, termed ‘Ntarget’, were
40
determined by the formula: Ntarget = 2Ctsample, where Ct was determined by
41
subtracting average Ct value of target gene from average Ct value of TBP gene.
42
Primers for TBP (upper primer, 5′-TGCACAGGAGCCAAGAGTGAA-3′; lower primer,
43
5′-CACATCACAGCTCCCCACCA-3′),
44
CAACATTGGAGGAGACACCCACT-3′;
45
CCAGGAAGATGTCATTTCCATTCA-3′) were selected with Oligo 6.0 (National
46
Biosciences).
47
For quantification of MT1-MMP mRNA levels in tumor samples, ten specimens of
48
adjacent normal breast tissue from breast cancer patients or normal breast tissue
1.
For RT-qPCR analysis of gene expression, the RNA
MT1-MMP
lower
(upper
primer,
primer,
5′5′-
2
49
from women undergoing cosmetic breast surgery were used as sources of normal
50
RNA. Ntarget values were subsequently normalized such that the median of ten
51
normal breast tissue Ntarget values was 1.
52
53
RT-qPCR analysis of MT1-MMP and p63 expression in breast tumor-derived
54
cell lines. Breast tissue derived cell lines were obtained from ATCC and the German
55
Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) and were
56
cultured in conditions recommended by the providers. Procedures for mRNA
57
extraction and RT-qPCR have been previously described 2. Primers for ∆Np63
58
(upper
59
TGTTCAGGAGCCCCAGGTTC-3’)
60
5′AGATTAGCATGGACTGTATCCGCA-3’;
61
GAGCCCCAGGTTCGTGTACTGT-3’) were selected with Oligo 6.0. RT-qPCR-based
62
analysis of estrogen-receptor expression in these cell lines has been previously
63
described 2.
primer,
5′-GGAAAACAATGCCCAGACTCAAT-3’;
and
Tap63
lower
lower
primer,
(upper
primer,
5′
primer,
5′-
64
65
Immunoblotting analysis. Cells were lysed and proteins were eluted in SDS sample
66
buffer, separated by SDS-PAGE, and detected by immunoblotting analysis with
67
indicated antibodies. Bound antibodies were detected with ECL Western Blotting
68
Detection Reagents (GE Healthcare Life Sciences).
69
70
Chromatin Immunoprecipitation and RT-qPCR: Cells were fixed with 1% para-
71
formaldehyde during 10 minutes at room temperature. Cross-linking was stop by
72
glycine (0.125M) for 5 min. Chromatin from fixed cells was fragmented by sonication
73
(30 min 30’’ on / 30’’ off on Bioruptor®, Diagenode, Liège, Belgium) and
3
74
immunoprecipitated in incubation buffer (10mM TrisHCl pH8, 1mM EDTA, 1% Triton,
75
0.1% Na-deoxycholate) complemented with protease inhibitor cocktail (Roche cat#
76
11873580001). 20 µg of sonicated chromatin was immunoprecipitated with 15 µl
77
beads (Mix of Protein G and protein A (Dynabeads®) pre-incubated with BSA 0.5
78
mg/mL and antibodies). After overnight chromatin immunoprecipitation in presence of
79
the corresponding pre-incubated beads, 7 washes with ice-cold RIPA (3’ each time)
80
and 2 washes with 1xTE (supplemented with 50 mM NaCl) were performed before
81
chromatin elution at 65°C (30 min in elution buffer: 50mM TrisHCl pH8, 10mM EDTA,
82
1% SDS). The immunoprecipitated chromatin was decrosslinked overnight (65°C in
83
elution buffer), the remaining proteins were removed by proteinase K treatment
84
(Roche; cat# 03115852001) and phenol-chloroform extraction. The purified DNA was
85
then evaluated by quantitative real-time PCR (qPCR, Roche LightCycler 480 II
86
device; Roche LightCycler 480 SYBR Green I Master mix reagents). Chromatin
87
immunprecipitation assays were performed with an antibody directed against p63
88
(ab53039, Abcam). A non-relevant IgG was used as negative control (ab171870).
89
Here are the sequence of primer used for the ChIP-qPCR : MT1-MMP #1 :
90
GCACCACAAAAAGGCAACTT and TGGGGACGTGGTTGTTTTAG; MT1-MMP #2 :
91
AACGACTCCAGAGGGGATTT and GGGGAGAAGACAGAACGACA; MT1-MMP #3
92
: GCCTTCCAGCGTCAGTAGAC and TTTTGCCCCTAGCATACCTG; MT1-MMP #4:
93
GCTAGGTGGTGCTAGGGTTG and GCAACATGGTTCTGGGAAGT; MT1-MMP #5:
94
AAGGGGAAAGAGGTGGAAGA and CCTGAAATTCTCTCCGCTTG; MT1-MMP #6:
95
ATCACAAGTTCCCGCTGAGT and GTCTTTCGGAAGCCACAGAG. Finally, 3’ MT1-
96
MMP located 20kb away from MT1-MMP has been used as negative control for p63
97
enrichment: ATCAAAAATCCCTGGCTTCC and TTCTTCCACTTGGACCTTGG.
98
4
99
Multicellular spheroid invasion assay and quantification of pericellular
100
collagenolysis. Stably or transiently knocked down cells were used. For transient
101
silencing, we used a double-round siRNA treatment for prolonged (up to 6 days)
102
silencing; cells were first electroporated with 100 nM siRNA using Amaxa kit V and
103
Nucleofector; after overnight incubation, cells were transfected with lullaby reagent
104
(OZ Bioscience) with 100 nM siRNA. Six hrs after treatment, multicellular spheroids
105
were prepared using 3x103 cells in 20 l of complete medium for 3 days using the
106
hanging droplet method as previously described 3. Spheroids were then embedded in
107
2.2 mg/ml acid extracted rat tail type I collagen (BD Biosciences), fixed immediately
108
(T0) or after 2 days at 37°C (T2) and then stained with Alexa546-phalloidin, anti-KI-
109
67 antibody and DAPI. Images were taken with a LSM 510 Meta confocal
110
microscope (Zeiss) with a 5X dry objective, collecting stack of optical sections along
111
the Z axis with 10µm interval. Quantification of invasion was done with ImageJ
112
software by estimating the diameter of spheroids at T0 and T2 as described 3. Values
113
were averaged and used to calculate the mean invasion area (πr 2). Mean invasion
114
area at T2 was normalized to mean invasion area at T0. Quantification of pericellular
115
collagenolysis using anti-Col1-3/4C antibodies was performed as previously described
116
4.
117
118
Analysis of MT1-MMP (MMP14) and TP63 expression in TNBC subtypes
119
according to Lehmann classification. We collected 21 publicly available datasets
120
that contained raw gene expression micro-array data (Affymetrix GeneChip Human
121
Genome HG-U133A and HG-U133Plus2) of 3247 primary human breast cancer
122
samples. Raw data were downloaded from NCBI’s Gene Expression Omnibus or
123
ArrayExpress with the following identifiers: GSE1456 5, GSE1561 and GSE2034 6,
5
124
GSE2603 7, GSE2990 8, GSE3494 9, GSE5327 and GSE5847
125
GSE11121
126
GSE16446, GSE18864 and GSE19615
127
16.
128
(http://cran.rproject.org).
129
For each dataset, we identified TNBC samples using a bimodal mixture of two
130
Gaussian distribution for ER and HER2 gene expression, and the median value for
131
PR expression. ER, PR and HER2 expression was represented using Affymetrix
132
probes 205225_at, 208305_at and 216836_s_at, respectively
133
filtered before normalization, then normalized by RMA method (R EMA package)
134
(Affymetrix GeneChip Human Genome HG-U133A and HG-U133Plus2, separately)
135
18.
136
20tool
137
TNBCs in BL1, BL2, IM, M, MSL, LAR subgroups was according to 21.
138
Affymetrix probes 202828_s_at and 209863_s_at were chosen to represent MT1-
139
MMP and TP63 expression, respectively. Density plot of MT1-MMP and TP63 gene
140
expression in the 550 TNBC samples did not show multimodal distribution. MT1-
141
MMP and TP63 expression was described using a 3-classe variable based on
142
quartiles (low, < first quartile ; high, > third quartile and moderate). The distribution of
143
these 2 qualitative variables (expression level of MT1-MMP and TP63) was
144
described in each TNBC Lehmann’s classification subtype using X2 test. A
145
combined-class variable of MT1-MMP and TP63 expression was built as followed:
146
correlated-high when both expression level of MT1-MMP and TP63 were high,
147
correlated-low when both expression level of MT1-MMP and TP63 were low,
148
correlated-moderate, expression levels of MT1-MMP and TP63 were both moderate,
12,
GSE20194, MDA133, GSE2109, GSE7904
15,
10,
13,
GSE7390
GSE12276
11,
14,
GSE22513, GSE28796 and GSE28821
All statistical analyzes were performed using R software version 2.13.2
17.
TNBC outliers were
Data were merged together and corrected for batch effect using ComBat 19. JetSet
was used to select the optimal probe set for each gene
20.
Classification of
6
149
and dissociated in other cases. Description of the distribution of this variable and
150
graphic representation were done as above
7
151
SUPPLEMENTARY REFERENCES
152
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breast cancer by qRT-PCR: Evidence that PKCiota/lambda overexpression is
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2
Karam, M. et al. Protein kinase D1 regulates ERalpha-positive breast cancer
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cell growth response to 17beta-estradiol and contributes to poor prognosis in
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patients. J Cell Mol Med 18, 2536-2552, doi:10.1111/jcmm.12322 (2014).
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3
Rey, M., Irondelle, M., Waharte, F., Lizarraga, F. & Chavrier, P. HDAC6 is
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required for invadopodia activity and invasion by breast tumor cells. Eur J Cell
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Biol 90, 128-135, doi:10.1016/j.ejcb.2010.09.004 (2011).
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4
Monteiro, P. et al. Endosomal WASH and exocyst complexes control
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exocytosis of MT1-MMP at invadopodia. J Cell Biol 203, 1063-1079,
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doi:10.1083/jcb.201306162 (2013).
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5
Pawitan, Y. et al. Gene expression profiling spares early breast cancer
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patients from adjuvant therapy: derived and validated in two population-based
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cohorts. Breast Cancer Res 7, R953-964, doi:10.1186/bcr1325 (2005).
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6
Wang, Y. et al. Gene-expression profiles to predict distant metastasis of
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lymph-node-negative
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doi:10.1016/S0140-6736(05)17947-1 (2005).
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Minn, A. J. et al. Genes that mediate breast cancer metastasis to lung. Nature
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Sotiriou, C. et al. Gene expression profiling in breast cancer: understanding
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Inst 98, 262-272, doi:10.1093/jnci/djj052 (2006).
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Miller, L. D. et al. An expression signature for p53 status in human breast
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cancer predicts mutation status, transcriptional effects, and patient survival.
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Boersma, B. J. et al. A stromal gene signature associated with inflammatory
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Desmedt, C. et al. Strong time dependence of the 76-gene prognostic
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signature for node-negative breast cancer patients in the TRANSBIG
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multicenter independent validation series. Clin Cancer Res 13, 3207-3214,
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Schmidt, M. et al. The humoral immune system has a key prognostic impact in
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breast cancer. Cancer Cell 9, 121-132, doi:10.1016/j.ccr.2006.01.013 (2006).
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Richardson, A. L. et al. X chromosomal abnormalities in basal-like human
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Juul, N. et al. Assessment of an RNA interference screen-derived mitotic and
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ceramide pathway metagene as a predictor of response to neoadjuvant
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Bauer, J. A. et al. Identification of markers of taxane sensitivity using
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Karn, T. et al. Homogeneous datasets of triple negative breast cancers enable
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Lehmann, B. D. et al. Identification of human triple-negative breast cancer
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Karn, T. et al. Data-driven derivation of cutoffs from a pool of 3,030 Affymetrix
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Singletary, S. E. et al. Staging system for breast cancer: revisions for the 6th
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239
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240
241
242
11
243
SUPPLEMENTARY TABLES
244
Supplementary Table 1 (related to Figure 1 and Table 1). Correlation of MT1-
245
MMP transcript levels with clinicopathological parameters of 458 breast cancer
246
cases analysed by RT-qPCR.
Number of patients (%)
pa
Total population (%)
MT1-MMP mRNA
expression relative to
normal
99 (21.6)
359 (78.4)
1.78 (0.17-17.36)
1.63 (0.01-39.17)
0.55 (NS)
58 (12.9)
230 (51.2)
161 (35.9)
1.58 (0.05-17.36)
1.58 (0.00-10.14)
1.79 (0.05-39.17)
0.045
120 (26.2)
237 (51.9)
100 (21.9)
1.63 (0.01-17.36)
1.70 (0.05-39.17)
1.78 (0.03-9.37)
0.93 (NS)
223 (49.6)
227 (50.4)
1.76 (0.05-17.36)
1.62 (0.01-39.17)
0.83 (NS)
ER status
Negative
Positive
119 (26.0)
339 (74.0)
1.95 (0.05-39.17)
1.54 (0.01-17.36)
PR status
Negative
Positive
195 (42.6)
263 (57.4)
1.86 (0.03-39.17)
1.55 (0.01-17.36)
HER2 status
Negative
Positive
360 (78.6)
98 (21.4)
1.63 (0.01-39.17)
1.84 (0.05-17.36)
Molecular subtypes
RH- HER2- (TNBC)
RH- HER2+ (HER2)
RH+ (Luminal A+B)
69 (15.1)
45 (9.8)
344 (75.1)
1.99 (0.05-39.17)
2.07 (0.34-8.30)
1.53 (0.01-17.4)
Age
50
>50
SBR histological grade
I
II
III
Lymph node status
0
1-3
>3
b,c
d
Macroscopic tumor size
25mm
>25mm
e
0.00024
0.014
0.038
0.00040
247
12
248
The table displays median (range) of MT1-MMP mRNA levels; values of the samples
249
were normalized such that the median of 10 normal breast tissue mRNA values was
250
1. Estrogen receptor (ER), progesterone receptor (PR) and HER2 status were
251
determined as described
252
classification. c Information available for 449 patients. d Information available for 457
253
patients. e Information available for 450 patients.
23,24. a
Kruskal Wallis’s H test.
b
Scarff-Bloom-Richardson
254
255
13
256
Supplementary Table 2 (related to Figure 1). Characteristics of primary tumors
257
included in the TMA
258
IDC
N=496
(%)
DCIS
N=101
(%)
Microinvasive
N=50
(%)
I
83 (16.7)
NA
NA
II
154 (30.0)
NA
NA
III
258 (52.0)
NA
NA
1 (0.2)
NA
NA
High
NA
55 (54.5)
40 (80.0)
Non high
NA
46 (45.5)
10 (20.0)
Ductal carcinoma
487 (98.2)
NA
NA
Lobular carcinoma
6 (1.2)
NA
NA
Others
3 (0.6)
NA
NA
Tis
NA
101 (100)
NA
T1mic
NA
NA
50 (100)
T1 (<2)
330 (66.5)
NA
NA
T2 (2 NA 5)
148 (29.8)
NA
NA
T3 (>5)
14 (2.8)
NA
NA
T4
4 (0.8)
NA
NA
N0
272 (54.8)
99 (98.0)
NA
N1
149 (30.0)
2 (2.0)
NA
N2
55 (11.1)
0
NA
N3
17 (3.4)
0
NA
Unknown
3 (0.6)
0
NA
Positive
286 (57.7)
56 (55.4)
23 (46.0)
Negative
210 (42.3)
45 (44.6)
27 (54.0)
Positive
255 (51.4)
59 (58.4)
20 (40.0)
Negative
241 (48.6)
42 (41.6)
30 (60.0)
0 (0.0)
1 (1.0)
0 (0.0)
Positive
93 (18.7)
31 (30.7)
32 (64.0)
Negative
403 (81.3)
69 (68.3)
18 (36.0)
0 (0.0)
1 (1.0)
0
Positive (>20%)
363 (74.0)
24 (23.7)
32 (64.0)
Negative (<20%)
133 (26.0)
75 (74.3)
18 (36.0)
0 (0.0)
2 (2.0)
0 (0.0)
Characteristics
Histological grade a
Unknown
Nuclear grade
b
Histological subtype
Tumour size (cm)
N stage
c
d
ER status
PR status
ND
HER2 status
ND
Ki67
NA
Molecular subtype
14
TNBC
131 (26.4)
14 (13.9)
3 (6%)
HER2
79 (15.9)
19 (18.8)
18 (36)
Luminal A
147 (29.6)
42 (41.6)
9 (18)
Luminal B
139 (28.0)
23 (22.8)
20 (40)
0 (0)
3 (2.9)
0
ND
259
260
Molecular subtypes were based on estrogen receptor (ER), progesterone receptor
261
(PR) and HER2 status as described
262
breast cancers classified based on the Elston-Ellis classification system (grade I-III)
263
27. b
264
grading system 28 or EORTC. c, d Based on TNM staging 29. NA, non applicable.
25,26
(see Experimental procedures).
a
Invasive
Grading of DCIS and microinvasive tumors based on Bloom-Richardson nuclear
265
15
266
Supplementary Table 3 (related to Table 1).
267
(A) Comparison of MT1-MMP expression and grade of breast cancer IDCs (TMA
268
cohort). Comparisons were made with X2 test (one-sided).
269
Histological
grade
Total
N=448
MT1-MMP low MT1-MMP high
(%)
(%)
I
68
54 (79)
14 (21) a, b
II
141
98 (70)
43 (30) c
III
239
125 (52)
114 (48)
270
271
a
Grade I vs. grade II, NS; b grade I vs. grade III, p < 0.0001; c grade II vs. grade III, p = 0.0005.
272
273
(B) p values of X2 test (one-sided) corresponding to comparison of plasma
274
membrane MT1-MMP intensity levels in the different subgroups of DCIS and
275
IDC cases shown in Table 1.
276
DCIS
Normal
Normal
LUM
HER2
TNBC
-
0.0037
0.01
0.0001
-
NS
NS
-
NS
LUM
HER2
IDC
TNBC
Normal
LUM
-
0.0258
0.0001
0.0001
-
0.0001
0.0001
16
HER2
TNBC
-
0.0009
-
277
278
279
280
281
282
17
283
Supplementary Table 4 (related to Figure 6). Distribution of MT1-MMP and TP63
284
expression in TNBC subtypes defined by Lehmann.
550 TNBC
MT1-MMP
expression
level
TP63
expression
level
MT1-MMP
/TP63
coexpression
level
High
Moderate
Low
High
Moderate
Low
Correlated
Disssociated
High
Moderate
Low
BL1
99 (0.18)
BL2
54 (0.10)
IM
101
(0.18)
LAR
53 (0.10)
M
113
(0.21)
MSL
45 (0.08)
UNS
85 (0.15)
P value
19 (0.19)
53 (0.54)
27 (0.27)
7 (0.07)
48 (0.48)
44 (0.44)
2 (0.02)
28 (0.28)
11 (0.11)
58 (0.59)
22 (0.41)
24(0.44)
8 (0.15)
34 (0.63)
18 (0.33)
2 (0.04)
15 (0.28)
5 (0.13)
0
32 (0.59)
11 (0.11)
55 (0.54)
35 (0.35)
26 (0.26)
55 (0.54)
20 (0.20)
0
30 (0.30)
6 (0.06)
65 (0.73)
6 (0.11)
34 (0.64)
13 (0.25)
10 (0.19)
29 (0.55)
14 (0.26)
0
15 (0.28)
2 (0.04)
36 (0.68)
40 (0.35)
50 (0.44)
23 (0.20)
24 (0.21)
58 (0.51)
31 (0.27)
11 (0.10)
23 (0.20)
5 (0.04)
74 (0.65)
15 (0.33)
20 (0.44)
10 (0.22)
18 (0.40)
17 (0.38)
10 (0.22)
5 (0.11)
7 (0.16)
3 (0.07)
30 (0.66)
25 (0.29)
38 (0.45)
22 (0.26)
19 (0.22)
49 (0.58)
17 (0.20)
7 (0.08)
24 (0.28)
6 (0.07)
48 (0.56)
2.00 10-4
1.34 10-12
2.18 10-7
285
286
Distribution of 550 TNBC samples classified using Lehmann’s subtypes according to
287
three MT1-MMP or TP63 expression classes (low, < first quartile ; high, > third
288
quartile and moderate) or four MT1-MMP and TP63 combined-classes (correlated,
289
MT1-MMP and TP63 low, MT1-MMP and TP63 high, MT1-MMP and TP63 moderate
290
or dissociated). BL1, basal-like 1; BL2, basal-like 2; IM, immunomodulatory; M,
291
mesenchymal; MSL, mesenchymal stem-like; LAR, luminal androgen receptor; UNS,
292
unstable. Description of the distribution of these variables using X2 (single
293
quantitative variables) or Fisher exact test (combined quantitative variables).
294
295
296
297
18
298
SUPPLEMENTARY FIGURE LEGENDS
299
Supplementary Figure 1 (accompanying Fig. 2). Progression of DCIS.com
300
xenograft tumors. (A, C) Whole-mount carmine-stained glands analyzed 5 or 10
301
weeks after intraductal injection of DCIS.com cells. Scale bars, 1 mm. (B, D)
302
Hematoxylin and Eosin staining of corresponding paraffin-embedded tissue sections.
303
Scale bars, 100 m. (E-G) Reorganization of collagen fibers during DCIS.com tumor
304
progression from in situ (5 weeks p.i.i.), to microinvasive (7 weeks p.i.i.) and invasive
305
(10 weeks p.i.i.) stages. Second-harmonic generation imaging of collagen fibres
306
(Magenta) at the tumor/ECM boundary of primary tumors from xenografts of
307
DCIS.com cells (green) transduced with YFP-expressing lentivirus. Scale bars, 40
308
m.
309
310
Supplementary Figure 2 (accompanying Fig. 3). Control experiments with
311
DCIS.com cells stably knocked down for MT1-MMP expression. (A) Proliferation
312
curve of shNT- and shMT1-MMP-expressing DCIS.com cells. No statistically
313
significant difference was found between the two cell populations. (B) Multicellular
314
spheroids of DCIS.com cells expressing shNT or shMT1-MMP without or with rescue
315
expression of shRNA-resistant MT1-MMPmCherry were embedded in 3D type I
316
collagen matrix and fixed immediately (T0) or after 2 days of invasion (T2). Data are
317
mean invasion area at T2 normalized to the mean invasion area at T0 ±S.E.M. with n
318
= 3 (***p<0.001, 2 way ANOVA, Bonferroni post test). (C, D) Immunofluorescence
319
320
generated by intraductal injection of shNT or shMT1-MMP-expressing DCIS.com
321
cells. shNT and shMT1-MMP xenografts were analyzed 5 and 10 weeks p.i.i.,
322
respectively. Percentage of Ki-67positive nuclei was determined from 3 different
19
323
fields from 2-3 independent tumors. No statistically significant difference was found
324
between
325
overexpressing MT1-MMPmcherry were generated by lentiviral transduction and
326
analyzed by immunoblotting with anti-MT1-MMP. ß-actin was used as a loading
327
control. (F) Phenotypic analysis of primary tumors from intraductal xenografts of
328
DCIS.com cells overexpressing MT1-MMPmCherry or not. Analysis was based on
329
whole-mount staining at 5-weeks p.i.i. (X2 tests, one-sided; MT1-MMPmCh vs. CTRL,
330
p=0.0178).
shNT
and
shMT1-MMP
xenografts.
(E)
DCIS.com
cells
stably
331
332
Supplementary Figure 3 (accompanying Fig. 3). MT1-MMP is required for
333
formation of invasive tumors by MDA-MB-231 cells in the intraductal xenograft
334
model. (A) Immunoblotting analysis of MT1-MMP expression in MDA-MB-231 cells
335
expressing the indicated MT1-MMP shRNAs. Total ERK was used as a loading
336
control. (B) Proliferation curve of parental and shNT- and shMT1-MMP-expressing
337
MDA-MB-231 cells. No statistically significant difference was found between the
338
different cell populations. (C) Multicellular spheroids of MDA-MB-231 cells expressing
339
shNT or shMT1-MMP were embedded in 3D type I collagen matrix and fixed
340
immediately (T0) or after 2 days of invasion (T2). Data are mean invasion area at T2
341
normalized to the mean invasion area at T0 ±S.E.M. with N = 3 (***p<0.001, 2 way
342
ANOVA, Bonferroni post test). (D) Whole mount carmine staining of a mammary
343
gland injected with MDA-MB-231 cells expressing shNT. Tumor foci are observed 4
344
weeks p.i.i. LN, lymph node. (E) Hematoxylin and Eosin staining of a corresponding
345
paraffin-embedded tissue section. (F) IHC analysis of a tumor foci generated by
346
intraductal injection of MDA-MB-231 cells with anti-MT1-MMP antibody. (G) Whole
347
mount staining of a mammary gland 4 weeks after intraductal injection of MDA-MB-
20
348
231 cells knocked down for MT1-MMP. (H) Phenotypic analysis of primary tumors
349
from intraductal xenografts of MDA-MB-231 cells expressing control Non-Targeting
350
shRNA (shNT) or depleted for MT1-MMP by expression of three independent shMT1-
351
MMP shRNAs (X2 tests, one-sided; shNT vs. shMT1-MMP#2 or #4, p=0.0451; shNT
352
vs. shMT1-MMP#3, p=0.0119). Scale bars, 1 mm (D, G), 50 m (E, F).
353
354
Supplementary Figure 4 (accompanying Fig. 6 and 7). Modification of p63
355
levels does not affect proliferation of DCIS.com cells in vitro and in vivo. (A)
356
Proliferation curve of shNT- and shp63-expressing DCIS.com cells cultured in vitro.
357
No statistically significant difference was found between the cell populations. (B)
358
DCIS.com tumor xenografts generated by intraductal injection of shNT or shp63-
359
expressing DCIS.com cells were analyzed by immunofluorescence with anti-
360
antibody. Percentage of Ki-67positive nuclei was determined from 8 different fields
361
from 4 independent tumors expressing shp63#1 or #4. No statistically significant
362
difference was found between shNT and shp63 DCIS.com cells in tumor xenografts.
21