Breast Cancer Res Treat (2014) 148:19–31 DOI 10.1007/s10549-014-3142-0

PRECLINICAL STUDY

Growth and metastatic behavior of molecularly wellcharacterized human breast cancer cell lines in mice Muhammad Riaz • Buddy Setyono-Han • Mieke A. Timmermans • Anita M. Trapman • Joan Bolt-de Vries • Antoinette Hollestelle • Roel C. Janssens Maxime P. Look • Mieke Schutte • John A. Foekens • John W. M. Martens

•

Received: 26 June 2014 / Accepted: 17 September 2014 / Published online: 30 September 2014 Ó Springer Science+Business Media New York 2014

Abstract Breast cancer (BC) is a disease with intra- and inter-tumor heterogeneity, and models representing the complete variety of clinical BC phenotypes are not available. We explored the tumor growth potential and metastatic behavior of human BC cell lines and determined whether these cell lines can recapitulate subtype-related biological characteristics of tumors. Eighteen human BC cell lines were implanted under the mammary fat pad of nude mice. Subtype-specific differences in tumor growth, metastatic ability to distant sites, and tumor-related survival of mice were

The author Buddy Setyono-Han was deceased.

Electronic supplementary material The online version of this article (doi:10.1007/s10549-014-3142-0) contains supplementary material, which is available to authorized users. M. Riaz (&)
B. Setyono-Han
M. A. Timmermans
A. M. Trapman
J. Bolt-de Vries
A. Hollestelle
M. P. Look
J. A. Foekens
J. W. M. Martens Department of Medical Oncology and Cancer Genomics Centre, Erasmus University Medical Center – Daniel den Hoed Cancer Center, 3000 CA Rotterdam, The Netherlands e-mail: [email protected] J. W. M. Martens e-mail: [email protected] R. C. Janssens Department of Genetics, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands M. Schutte The Lorentz Center, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands J. W. M. Martens Department of Medical Oncology, Erasmus University Medical Center – Daniel den Hoed Cancer Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands

recorded. Eighty-nine percent of the cell lines gave rise to xenografts of which 56 % showed metastasis to distant sites. A clear difference was observed in growth of xenografts from cell lines of different molecular subtypes (P = 0.001; Kruskal–Wallis test). Mice bearing the basal-like and the normal-like xenografts showed poor tumor-related survival (HR: 10.50; P = 0.002 and HR: 9.89; P = 0.003, respectively) compared with those bearing the ERBB2-positive xenografts, which had the longest survival. Subtype-specific metastasis to distant sites between xenografts was not however observed. Comparable to clinical behavior in humans, we observed that the basal-like and the normal-like cell lines grew more aggressively in mice than the cell lines of other molecular subtypes. However, in contrast to clinical findings, we observed no relationships between intrinsic subtype and preferences for site of relapse. Importantly, we have established xenograft models from 16 phenotypically and molecularly diverse human BC cell lines, which can be exploited as useful tools to perform functional studies and screening of interfering drugs. Keywords Breast cancer cell lines
Molecular subtypes
In vivo growth
Metastasis
Xenografts Abbreviations APC Adenomatous polyposis coli ARRIVE Animal Research Reporting In Vivo Experiments BC Breast cancer CI Confidence interval Cag-Luc CMV early enhancer/chicken beta actin geneluciferase DEC Dier Experimenten Commissie ERBB2 Erythroblastic leukemia viral oncogene homolog 2 ER Estrogen receptor

123

20

EGFR EMC FBS HR P/S PgR STR TP53

Breast Cancer Res Treat (2014) 148:19–31

Epidermal growth factor receptor Erasmus medical center Fetal bovine serum Hazard ratio Penicillin and streptomycin Progesterone receptor Short tandem repeat Tumor protein 53

Introduction Breast cancer is not a single disease but rather a group of multiple distinct diseases with an extensive intra- and intertumor heterogeneity. Heterogeneity of breast tumors has been captured histologically decades ago by pathologists and by clinicians during clinical examinations. Based on such differences, breast tumors were classified into various histological subtypes with a different prognosis [1]. The classical pathology-based classification has been refined over the time and replaced by modern molecular classifications, which have the potential to combine disease mechanisms with clinical outcome measures [2]. Molecular classification is an on-going process, which is currently mainly driven by differences in the expression of several thousands of genes detectable in the human breast tissues, and such changes are generally measured using gene expression microarray technology [3]. Using cDNA microarrays, five main molecular subtypes have initially been identified [3]. These subtypes include two luminal ones: luminal A and B, an ERBB2-positive, a basal-like, and a less well-defined normal-like subtype. These five subtypes in breast cancer are very stable and have been observed and validated by a number of subsequent studies in independent series of tumors from breast cancer patients with different ethnic background [4]. These subtypes not only display distinct gene expression patterns, but also show a significant difference in disease-free as well as overall survival [5]. Additionally, these subtypes show distinct preferences for organ-specific metastases [6, 7]. For instance, the luminal subtypes of breast cancer preferentially metastasize to bone, and the basal-like subtype metastasizes more frequently to lungs and brain [6]. Furthermore, response to chemotherapy as well as observed genomic instability of these subtypes is also diverse [8, 9]. Grouping of breast tumors based on gene expression analysis has allowed the identification of several subtypespecific molecular drivers, which have been successfully targeted therapeutically using human BC cell lines as model systems [10–12]. Recently, a large collection of cell

123

lines including BC cell lines has been used as in vitro preclinical model systems to uncover new biomarkers of sensitivity to the currently applied therapeutic drugs [13, 14]. Furthermore, accumulating evidence suggests that the currently available BC cell lines mirror the molecular portraits of primary breast tumors. For instance, BC cell lines cluster into luminal, ERBB2-positive, basal-like, and normal-like molecular subtypes, as do primary tumors [15]. Additionally, the cell lines exhibit a comparable degree of heterogeneity in copy number and expression abnormalities as observed in primary tumors, and they carry many of the genetic abnormalities commonly found in primary breast tumors [15, 16]. The interpretation and extrapolation of cell line-related data, however, require an understanding of how uniformly and stably these cell lines mimic primary breast tumors at both in vitro and in vivo levels. Previous studies have suggested that the cell lines, despite of considerable similarities, also show some discrepancies with clinical breast tumors. These discrepancies include higher frequency of TP53 gene mutation and over representation of ER-negative cell lines compared with human clinical breast tumors. In the current study, we developed a large series of luciferase-tagged breast cancer xenografts derived from breast cancer cell lines from different molecular subtype. By doing this, we explored in vivo growth potential and metastatic behavior of the cell lines to generate useful models for experimentation and to determine whether these cell lines in mice recapitulate their subtype-related clinical behaviors such as aggressiveness and capacity to metastasize to specific distant sites.

Materials and methods Human breast cancer cell lines The 18 human breast cancer cell lines used in this study are listed in online resource 1, supplementary Table 1 and are described elsewhere [17]. All cell lines were unique and monoclonal as determined by STR analysis. All cell lines were maintained in RPMI-1640 medium supplemented with 10 % FBS (GibcoÒ) and P/S (100/80). For implantation, at least three cell lines were selected from each of four well-known molecular subtypes. Molecular subtypes of the cell lines were determined using intrinsic gene-set published by [3] as described previously [18]. Transduction and clone selection Breast cancer cell lines were seeded in T-162 tissue culture flasks and grown until 70–80 % confluency. The cells were

Luciferaselabeled clones

36

25

25

T47D-CL-10D8*

T47D-CL-9E11

T47D-CL-10G7

225

144

UACC893-CL-4C11

UACC893-CL-4F8

225

100

225

225 225

225

225

225

MDA-MB-468-CL-9A10*

MDA-MB-468-CL-6B4

MDA-MB-468-CL-6D2

SUM229PE-CL-8C5 SUM229PE-CL-9C7

SUM229PE-CL-8E11

SUM149PT-Luc-5D5

SUM149PT-Luc-5E4

Basal-like cell lines

225

UACC893-CL-4B5

0

MDA-MB-453-CL-10D2

36

0

MDA-MB-453-CL-9B1

SK-BR-3-CL-6C11*

0

MDA-MB-453-CL-10A7

36

0

EVSA-T-CL-10H12

SK-BR-3-CL-5C11

0 49

EVSA-T-CL-10D11 EVSA-T-CL-10H8

ERBB2-positive cell lines

100

SUM52PE-CL-8F10

4

MDA-MB-175VII-CL-4D3

25

64

MDA-MB-175VII-CL-4B8

25

225

MCF-7-CL-5D10

SUM52PE-CL-6F5

225

MDA-MB-175VII-CL-4G5

225

MCF-7-CL-5D8

Maximum PXT size (mm2)b

1.6

1.6

8.7

2.2 5.5

10

NR

9

NR

10

11

NR

NR

NR

NR

NR

NR

NR NR

NR

NR

NR

NR

NR

NR

NR

NR

5

8

1.1

Time of PXT removal

9

9

8.7

2.2 5.5

10

10.4

9

12

12

12

12

12

12

12

12

12

12 12

3

3

8.3

11

2.4

12

2

8

8.6

8

1.1

Maximum follow-up time

Xenograft size and follow-up period (months)

MCF-7-CL-4D4

Luminal-type cell lines

Molecular subtypea

SAT

SAT

SAT

SAT SAT

SAT

SOT

SAT

EPR

EPR

EPR

EPR

EPR

EPR

EPR

EPR

EPR

EPR EPR

SBWL

SBWL

SBWL

SBWL

SBWL

EPR

SBWL

SBWL

SAT

SAT

SAT

Remarks

Table 1 Xenograft growth and sites of relapse of human breast cancer cell line in nude mice

-

-

?

?

?

-

?

?

?

?

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

?

?

?

Lymph nodes

-

-

-

-

?

-

?

-

-

?

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

?

?

?

Lungs

-

-

-

-

?

-

-

-

-

?

-

-

-

-

-

--

-

-

-

-

-

-

-

-

-

-

-

-

Liver

Sites of relapse in mice body

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Spleen

-

-

-

-

?

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

?

?

-

Uterus/ Ovaries

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Kidneys

-

-

-

-

-

-

-

-

-

?

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Brain

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Pancreas

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Bone

Breast Cancer Res Treat (2014) 148:19–31 21

123

123

Luciferaselabeled clones

225

225

225

225

225

225 0 4

MDA-MB-436-CL-6C12

MDA-MB-436-CL-6C12

SK-BR-7-CL-7E3

SK-BR-7-CL-7F9

SK-BR-7-CL-7G3

SUM159PT-CL-8A4

SUM159PT-CL-7B3

SUM159PT-CL-7C6

12

12

12

12

3.6

3.8

4

4

6.1

8

5.8

3.7

3.4

6.5

12

5.2 12

12

12

12

9

Maximum follow-up time

EPR

EPR

EPR

EPR

SAT

SAT

SAT

SAT

SAT

SAT

SAT

SAT

SAT

As

EPR

SAT EPR

EPR

EPR

EPR

SAT

Remarks

-

-

-

-

?

-

-

-

?

?

?

?

?

-

-

? -

-

-

-

?

Lymph nodes

-

-

-

-

?

-

-

-

?

?

?

?

?

-

-

-

-

-

-

-

Lungs

Molecular subtyping of the cell lines was done using intrinsic gene-set published by [3] as described previously [18]

0.47

0.47

NR

NR

3.3

3.8

4

4

0.8

2

1.4

3.6

3.4

6.5

NR

2.5 NR

NR

NR

NR

0.8

Time of PXT removal

-

-

-

-

-

-

-

-

?

?

?

?

?

-

-

-

-

-

-

-

Liver

Sites of relapse in mice body

-

-

-

-

-

-

-

-

-

?

-

-

-

-

-

-

-

-

-

-

Spleen

-

-

-

-

?

-

-

-

-

?

?

-

-

-

-

-

-

-

-

-

Uterus/ Ovaries

-

-

-

-

?

-

-

-

-

?

?

?

?

-

-

-

-

-

-

-

Kidneys

-

-

-

-

-

-

-

-

-

-

-

-

?

-

-

-

-

-

-

-

Brain

-

-

-

-

?

-

-

-

-

-

-

?

-

-

-

-

-

-

-

-

Pancreas

-

-

-

-

-

-

-

–

?

-

-

-

-

-

-

-

-

-

-

-

Bone

Only two luciferase-labeled clones were implanted for three cell lines: DU4475, SK-BR-3, and SUM52PE

Due to ethical reasons, primary xenografts reaching the size of 225 mm2 were removed leaving a tumor portion of 36 mm2 on the primary site to induce organ metastasis. CL cag-Luc, PXT primary xenograft tumor, NR not removed, SBWL sacrificed due to body weight loss, EPR end point reached, SAT sacrificed due to aggressive tumor, SOT sacrificed due to open tumor, As ascites, ? metastasis (observed through bioluminescence), - no metastasis

b

a

225

225

MDA-MB-436-CL-5C3

DU4475-CL-8A8

225

MDA-MB-231-CL-5D11

225

225

MDA-MB-231-CL-5D6

Other cell lines DU4475-CL-6A8

81

225 81

Hs578T-CL-4A8 Hs578T-CL-5D4

192

0

BT549-CL-8G12

MDA-MB-231-CL-8C1*

0

Hs578T-CL-5D8

0

BT549-CL-10B3

225

Maximum PXT size (mm2)b

Xenograft size and follow-up period (months)

BT549-CL-9A12

Normal-like cell lines

SUM149PT-Luc-5H4

Molecular subtypea

Table 1 continued

22 Breast Cancer Res Treat (2014) 148:19–31

Breast Cancer Res Treat (2014) 148:19–31

then seeded in 24-well tissue culture plates in 0.5 mL culture medium and grown until 50–60 % confluency. Concentrated viral particles containing luciferase Cag-Luc construct, which were produced as described by van der Wegen [19], were added to the cell line (50 lL to each well) and incubated for 6–8 h. The cells were then trypsinized and subjected to the serial dilution to select monoclonal population of the transduced cells. Multiple clones with similar luciferase expression were further propagated for morphological characterization, which was done through microscopic inspection of the clones, comparing them with each other and with the corresponding parental lines. Three clones with similar morphological characteristics as the parental cell line (online resource 2, supplementary Fig. 1) were propagated further for implantation experiments. A flow chart of the methodology is presented in online resource 2, supplementary Fig. 2. To ensure clonal and their corresponding xenografts identity, we performed STR analysis on clones and xenografts and found them to be matching with the original lines they were derived from. For four clones marked with asterisk in Table 1, the identity at xenograft levels could not be confirmed due to suboptimal storage of the xenografts. Cytospin staining and Immunohistochemistry Immunohistochemistry was performed on cytospin preparations or on tissue sections, using standard procedures [20]. ER expression analysis (clone 1D5, M7047 at 1:40) was developed using EnvisionTM Plus system (mouse, K4006; rabbit, K4008 for cytospins and the animal research kit horse reddish peroxidise (K3954) for tissue sections). PgR expression (A0098 at 1:50) was developed using EnvisionTM Plus system for both cytospins and tissue sections. ERBB2 and EGFR (D38B1 at 1:100) staining was performed using the HercepTestTM kit and EGFR pharmDxTM kit, respectively. All reagents were supplied by Dako, Glostrup, Denmark except for D38B1, which was supplied by Cell Signaling, BZ Leiden, The Netherland. Mouse strain and animal care The NMRI/Nu–Nu strain of immune-compromised female mice (Mus musculus), aged 6–8 weeks (Charles River Laboratories, Wilmington, Massachusetts, USA), was used in this study. The animals were housed in the individually ventilated cages with sterile bedding, water, rodent chew feed, and air. Experiments were carried out in accordance with the ARRIVE guidelines [21], and the animal research protocol [EMC 2541 (136-11-03)] was approved by the Institutional Animal Ethical Committee (DEC), Erasmus University Medical Center, Rotterdam, the Netherlands.

23

Cell line implantation and tumor size determination Three mice were used one for each clone from each cell line. Approximately, 12 million cells were implanted orthotopically under mammary fat pad of mice under anesthesia (2 % isoflurane, Pharmachemie BV, Haarlem, The Netherlands). The xenografts viability was assessed by bioluminescence as explained below, and their size was measured twice a week using a vernier calliper [xenograft size (mm2) = length 9 width]. To allow growth of ERpositive luminal cell lines, 1.5 mg b-estradiol pellet with 90 days release time (Innovative Research of America, Sarasota, Florida, USA) was implanted subcutaneously on the dorsal side of mouse’s neck two days before cell implantation [22]. The mice were followed for a total period of 12 months. Due to ethical reasons, primary xenografts reaching the size of 225 mm2 before the end of the follow-up period were surgically removed while leaving an approximately 16 mm2 portion on the site to facilitate invasion and metastasis. After the follow-up period, the intact animals were subjected for whole body luminescence to visualize distant organ metastasis. Subsequently, mice were sacrificed, and body organs were excised, which were re-imaged to assess micrometastases. The xenografts and body organs were preserved in 10 % formalin solution (Sigma-Aldrich, St. Louis, Missouri, USA) for subsequent histological analyses. Bioluminescence imaging Bioluminescence imaging was performed using IVIS Imaging System (Xenogen product from Caliper Life Sciences, Hopkinton, Massachusetts, USA) according to manufacturer’s instructions. Briefly, for whole body bioluminescence, the substrate 200 lL of D-luciferin (10 lg/ gram of mouse weight) was injected intraperitoneally about 10–15 min before imaging. Mice were then anesthetized with a continuous flow of 2 % isoflurane, and bioluminescence images were acquired with an acquisition time ranging from 0.1 s to 5 min depending upon the luminescence signals from the implanted cells. For ex vivo imaging of cells and organs, 150 and 300 lg/mL of D-luciferin in culture medium, respectively, were used. Imaging and quantification of signals were controlled by acquisition and analysis software Living Imaging 3.0 (Xenogen, Caliper Life Sciences, Hopkinton, Massachusetts, USA) and wherever necessary data were expressed as total number of photon/sec/cm2. Data analyses, study end point, and event Cox regression analysis was used to reveal subtype-specific tumor-related survival in the mice. Kruskal–Wallis and

123

24

Breast Cancer Res Treat (2014) 148:19–31

Mann–Whitney tests were used to evaluate significant differences in growth rate of the cell lines across the intrinsic subtypes. A P value of\0.05 was considered to be statistically significant. Twelve months follow-up time after the implantation of cell lines was considered as the study end point for determination of organ-related micrometastases. For tumor-related survival analysis, mice in which primary tumor reached the maximal allowed size (i.e., 225 mm2) within the 12 months follow-up time were considered as event harboring mice at this point and time. Mice showing serious health problems beyond the ethically permissible limits were sacrificed and taken out from the study. The data from these mice were censored for tumorrelated survival analysis.

like cell lines were successfully established. Among normal-like cell lines, only one cell line (BT549) did not establish xenografts in this strain of mice, whereas other 5 cell lines gave rise to xenografts of 225 mm2 size in a short time span (range 24 days–6.5 months) (Fig. 1; Table 1). Additionally, two clones of the DU4475 cell line also showed an extremely aggressive growth behavior in the mice, and the primary xenografts reached 225 mm2 size in only 2 weeks (Fig. 1; Table 1). Overall, in this nude mice model, basal-like and normal-like cell lines gave rise to xenograft of maximum size more quickly compared to the luminal-type and ERBB2-positive cell lines.

Results

The primary aim of the study was to determine whether the xenograft growth rate and tumor-related survival in the mice were associated with the molecular subtype of the implanted cell lines. For this, we first compared the growth rates of xenografts from all cell lines across 4 molecular subtypes and then analyzed subtype-specific survival in the mice using the Cox regression model. Our analyses revealed that the growth rate of the cell lines accordingly to molecular subtypes was significantly different (Kruskal– Wallis Test, P = 0.001; Fig. 2a). Importantly, cell lines of the basal-like and the normal-like molecular subtypes combined showed a significantly faster growth when compared with those of the luminal and the ERBB2-positive cell lines (Mann–Whitney Test P \ 0.001; Fig. 2b). Additionally, mice bearing xenografts from the basal-like and the normal-like cell lines showed worse tumor-related survival compared with the mice bearing xenografts from the ERBB2-positive molecular subtype, which had the best prognosis (HR: 10.50; 95 % CI: 3.18–50.57; P = 0.002 and HR: 9.89; 95 % CI: 2.15–45.48; P = 0.003, respectively) (Fig. 2c). However, mice bearing xenografts from luminal cell lines showed intermediate tumor-related survival (HR: 4.24; 95 % CI: 0.76–23.60; P = 0.1) (Fig. 2c). Overall, the xenografts of basal-like and normal-like cell lines showed more aggressive behaviors compared to the xenografts of cell lines of other subtypes.

Subtype-specific growth characteristics of BC cell line xenografts To determine the intrinsic molecular subtype-specific growth characteristics of BC cell lines in vivo, we selected a panel of 18 cell lines, which included 4 cell lines from the luminal-type, 4 from the ERBB2-positive, 3 from the basal-like, and 6 from the normal-like subtype. DU4475 cell line was also selected, which according to microarray gene expression analysis does not cluster to either of the known intrinsic subtypes [18]. The cell lines were labeled with luciferase reporter gene, and three clones from each cell line were implanted in mammary fat pat of mice. Detail of luciferase-labeled clones corresponding to each cell line is given in Table 1. Among 19 cell lines, 16 (84 %) of lines gave rise to xenografts in mice albeit with variable primary xenograft size in the set follow-up time of 1 year. Based on corresponding primary xenograft size, we grouped clones of all cell lines into four arbitrary groups: group I through group IV, where group I included clones producing no xenograft and group IV included clones producing xenografts of maximum size (225 mm2) (Fig. 1). Among the luminal-type cell lines, all clones showed growth in mice however, in majority of cases, a dramatic loss in body weight of the mice was observed within 1 month of post cell implantation (Table 1). These mice were therefore taken out from the study and censored at this time point in survival analyses. Interestingly, all three clones of the MCF-7 cell line showed aggressive growth and gave rise to primary xenografts of the maximal allowed size (225 mm2) in 1, 5, and 8 months, respectively (Fig. 1; Table 1). Among ERBB2-positive cell lines, except MDA-MB-453 clones from all cell lines gave rise to xenografts though with variable size (xenografts size range: 36–225 mm2). Similarly, xenografts from all basal-

123

Subtype-specific cell line primary xenograft growth rate and tumor-related survival

Subtype-specific metastatic potential and specificity for distant sites of relapse Besides variable growth patterns associated with the cell lines, we determined the propensity of these cell lines to colonize common distant sites in mice and determined whether this behavior was molecular subtype related. For this, we determined the frequent metastatic sites in mice by performing whole body bioluminescence as well as by imaging the inner body organs after sacrificing them. As

Breast Cancer Res Treat (2014) 148:19–31

25

Fig. 1 Subtype-specific growth characteristics of BC cell line xenografts. Based on their corresponding primary xenograft size in mice, luciferases-labeled clones from all cell lines are grouped into four groups; group I through group IV

expected, the most common sites of relapse in mice bearing 225 mm2 xenograft tumors were the adjacent lymph nodes (68 %). Second, in line to the lymph nodes were the lungs (48 %). Liver and uterus/ovaries (28 and 24 %, respectively) were, respectively, the third and fourth most frequently colonized distant organs by the cell lines (Table 1 and Fig. 3). Relapses to spleen, bone, and brain were also observed though less frequently compared with those to lymph nodes and lungs. Additionally, occasional relapses to some uncommon sites with regard to breast cancer, such as to the kidney and pancreas, were also observed in the mice (Table 1). Interestingly, within same follow-up time, the isogenic clones of SUM149, SUM159, and UACC893, respectively, showed differences in metastatic ability and preferences for distant relapse sites (Table 1). It should however be noted that the majority of these micrometastases were only detectable by the bioluminescence and could not be confirmed histologically except for the metastases to lymph nodes and lungs (Fig. 4). Although majority of cell lines were able to give rise to xenografts in mice, but unlike clinical tumors, they did not demonstrate subtype-specific preferences for sites of relapses in mice body. Breast cancer cell line xenografts as preclinical models Next, we investigated whether this panel of BC cell lines can be used as a xenograft preclinical mouse model. For this, we assessed the robustness of the growth rate of three representative luciferase-labeled cell lines (MDA-MB-468CL-9A10, MDA-MB-231-CL-5D6, SUM159PT-CL-8A4). Slices of 2 mm3 from primary xenografts were serially implanted into three additional recipient mice. As expected, the take rate of the daughter xenografts was 100 % (online resource 2, supplementary Fig. 3). Importantly, the growth rate of three serially implanted xenografts from

each cell line was uniform as concluded from the small error bars and showed rapid occurrence compared with their corresponding parental xenografts generated directly from cultured cells of the selected clones (Fig. 5).

Discussion In this study, we explored the molecular subtype-related growth and metastatic potential of an in vitro propagated molecularly diverse collection of human BC cell lines in nude mice. We determined whether human breast cancer cell lines as a model system can recapitulate the clinical and biological behavior of tumors in patients. We found that the majority of breast cancer cell lines from the four main molecular subtypes are able to give rise to xenografts in mice. This suggests that despite of being propagated for years in vitro, most of these cell lines retain their innate ability to exploit the mouse microenvironment and give rise to full blown primary tumors. However, most mice bearing xenografts from luminal cell lines suffered from loss of body weight. The cause of such weight loss remains unknown as pathological and microbiological analyses of these mice did not reveal any peculiar cell line-related concerns. It is however important to mention that all these cell lines are ER-positive [15] and therefore required a suitable concentration of estrogen for their steady growth under in vivo conditions [22]. To provide sufficient estrogen for optimal growth of these cell lines in mice, 1.5 mg b-estradiol pellet with 90 days release time was implanted subcutaneously in each mouse 2 days before implantation of luminal cells. As this weight loss was not observed in mice not receiving the b-estradiol pellet, a surplus of released b-estradiol above the physiological range in mice bearing the luminal xenografts could have caused the unexpected decline in body weight. This appears to be in

123

26

Breast Cancer Res Treat (2014) 148:19–31

Fig. 2 In vivo subtype-specific growth rate and tumor-related survival of human breast cancer cell lines. a Comparison of growth rate of primary xenograft tumors from the cell lines according to the molecular subtypes. b Comparison between growth rate of primary xenograft tumors of basal-like and normal-like cell lines combined and luminal and ERBB2-positive cell lines. In a, Kruskal–Wallis test revealed a significant difference in the growth rate across four molecular subtypes. In b, further exploration of the differences between growth rates of the cell lines showed that both the basal-like

and normal-like cell lines combined showed faster growth rates than the luminal-type and ERBB2-positive cell lines combined. c Xenograft tumor-related survival analysis of the cell lines across four molecular subtypes. Hazard ratio, 95 % confidence interval, and P values for each subtype are indicated in the figure. The ERBB2positive group, which showed the best tumor-related survival, was used as reference with HR set at one. The censored animals from each subtype are also indicated in the survival curves. Number of mice at risk is indicated below the x-axis

line with a previous study by Kang et al. showing that bestradiol causes renal damage and bladder stone formation in mice in a dose-dependent manner, where mice supplemented with higher dose of b-estradiol (1.7 mg/pellet) showed poor survival compared to mice supplemented with the lowest dose (0.18 mg/pellet), which showed better survival [23]. For future studies, we suggest that for each ER-positive line, the lowest possible effective dose of bestradiol should be determined. Our findings that the basal-like and normal-like cell lines showed rapid growth is in line with the currently published in vitro growth behavior of these cell lines with doubling times for basal-like and normal-like cell lines being shorter when compared with cell lines of the two

other subtypes [24]. Interestingly, these results also confirm the clinical findings showing basal-like and normal-like tumors to be associated with a worse prognosis [4]. Additionally, similar to clinical specimens, the normal-like cell lines do not exhibit ER, PgR, or ERBB2 expression (triple-negative) [25] and rather they exhibit a high expression of stem cell and mesenchymal markers such as CD44high/CD24low and vimentin, N-cadherin, and fibronectin, respectively [11, 26, 27]. In contrast to clinical observations, however, none of the ERBB2-positive cell lines showed aggressive growth and poor survival in mice (Fig. 1a and c) [28, 29]. This discrepancy may, on the one hand, be just a matter of bias due to the fact that only few number of ERBB2-positive cell lines were successfully

123

Breast Cancer Res Treat (2014) 148:19–31

27

Fig. 3 Bioluminescence imaging of the intact animals as well as organs showing micrometastases of representative cell line xenografts from four molecular subtypes. Primary xenograft tumor and metastatic lesions on distant sites from a MCF-7-Cag-Luc-5D8 luciferaselabeled clone, a representative picture of the luminal subtype of cell lines, b UACC893-Cag-Luc-4F8 luciferase-labeled clone, a representative picture of the ERBB2-positive subtype of cell lines, c MDA-

MB-468-Cag-Luc-9A10 luciferase-labeled clone, a representative picture of the basal-like subtype of cell lines, and d SUM159PTCag-Luc-8A4 luciferase-labeled clone, a representative picture of the normal-like subtype of cell lines. LN lymph nodes, LU lungs, LI liver, PA pancreas, KD kidneys, AG adrenal glands, SP spleen, OV ovaries, UT uterus, BR brain, HR heart

implanted in the mice. On the other hand, it is in line with in vitro behavior of ERBB2-positive cell lines where they show an overall lower proliferation rate (as assessed by Ki67 staining) compared with the basal-like and normal-like cell lines [15]. Another explanation could be an incompatibility of murine ERBB2 ligands to recognize human ERBB2 receptors and as a result xenograft tumor from ERBB2-positive human cell line lack aggressiveness in mice compared to the clinical presentation of ERBB2 tumor in human. Whether this is true requires more research as current literature is limited to support this argument. Interestingly, the Du4475 cell line, which has APC mutation and was previously suggested to have stem cell-like phenotype [15], demonstrated high growth rate but poor metastatic capacity in this strain of mice. However, more research is needed to elucidate whether the DU4475 cells indeed possess stem cell-like properties. It has been previously observed that the intrinsic molecular subtypes show a preference to relapse to specific distant sites [6, 7] and also when intravenously injected breast cancer cells show organ-specific relapse [30–32]. Unfortunately, in this strain of mice, subtype-specific preferences of the cell lines of 4 molecular subtypes could

not be reproduced (Table 1). One explanation of not reproducing this clinical behavior of breast tumors could be that our panel of cell lines is small and therefore lacks sufficient power to observe subtype-specific sites of relapse in the mice. Particularly, xenograft tumors from the luminal cell lines were under-represented as the mice bearing these xenografts had to be sacrificed due to technical reasons before they could generate xenograft tumors reaching a size that would allow distant organ metastases. This could in part explain the almost complete absence of bone metastasis in our study as it is luminal cancer that particularly relapses to bone [6, 7]. Another explanation would be that although these cells are able to disseminate, they may not have the ability to produce histologically detectable micrometastases in the distant organs in mice. This also appears to be in line with a previous observation that metastasizing mouse mammary cancer cells harbor different potential to give rise to detectable micrometastases in the target organs [33]. It may therefore be arguable to improve the power of the study by expanding the panel of cell lines to better understand the relevance of cell lines model with regard to subtype-specific preferences for sites of relapse in breast cancer. However, it is important to

123

28

Fig. 4 A representative figure showing hematoxylin and eosin staining of the primary xenografts and metastases to lymph nodes and lungs. Staining of four representative luciferase-labeled clones

Fig. 5 Comparison between growth rates of the xenograft tumors generated from clonally selected cells and from serially implanted 2 mm3 tumor cubes of the primary xenografts from the same clone

mention that in our study irrespective of cell types, mice generally harbor mostly lungs metastasis, which may pose a challenge to the efficacious use of mouse as a model to properly study organ-specific metastases of human tumors. Our observation that isogeneic clones of some cell lines

123

Breast Cancer Res Treat (2014) 148:19–31

from four molecular subtypes is shown. Arrows in the inserts indicate metastatic tumor cells in the tissue at 10 times higher magnification

showed different metastatic ability and preferences for distant relapse sites is also interesting. These differences may likely be the intrinsic properties of these isogenic clones as such isogenic clones of MDA-MB-231 with different dissemination ability have been previously reported [30–32]. Additionally, for the SUM159 line, which was successfully shown to give rise xenografts in the NOD-SCID mice by others [34, 35], but in our study, a more heterogeneous picture of this line seems to emerge with one clone showing aggressive growth and metastatic behaviors, while other two clones behaving rather benign. These differences between different isogenic clones are likely to be intrinsic in nature and may not be related to the growth conditions. Our findings that serially implanted xenografts from the donor mice to the recipient mice show fast and robust growth compared with the xenografts raised from cell lines indicate that the BC cell line xenograft model could potentially be an attractive preclinical model for drug screening. Importantly, these xenografts grow in a practically feasible time frame and are accompanied with mouse stroma, which has been suggested to have a critical effect on the efficacy of the tested drugs [36, 37]. Moreover, as suggested previously [16], the group of cell lines with the

Breast Cancer Res Treat (2014) 148:19–31

same molecular subtype can be used as a system rather than individually for better and more robust screening of the drugs.

Conclusion In summary, we show that human breast cancer cell lines, which have been propagated on plastic for years, have retained their innate abilities to exploit the (orthotopic) microenvironment of the implantation site and can give rise to xenografts when implanted under the mammary fat pad of the mice. The most common sites colonized by all types of cell lines in mice are lymph nodes, lungs, liver, and ovaries/uterus, whereas some other sites of relapse such as kidneys, bone, brain, and pancreas were infrequently observed. Importantly, xenografts from breast cancer cell lines of the basal-like and normal-like subtypes showed a more aggressive growth behavior and worse tumor-related survival of the mice compared with xenografts of cell lines from the other molecular subtypes. Finally, serially generated xenografts in the recipient mice grew reproducibly and robustly and can serve as diverse and practically feasible preclinical models to screen breast cancer interfering drugs in vivo in the presence of mouse stroma and also to do functional studies on clinically important genes. Acknowledgments We acknowledge Professor B. van der Horst from Genetics Department of Erasmus University Medical Center for his generous donation of HEK293T cells and Dr. Ali Imam from Cell Biology Department of Erasmus University Medical Center for his gift of luciferase containing vector. We also appreciated the help provided by personnel from the animal facility. This study was supported financially by the Netherlands Genomics Initiative (NGI)/ Netherlands Organization for Scientific Research (NWO), and MR was funded by the Higher Education Commission (HEC) of the Governments of Pakistan (Ref: HEC.07/170). Conflict of interest All authors declared that they have no competing interest.

References 1. Weigelt B, Reis-Filho JS (2009) Histological and molecular types of breast cancer: is there a unifying taxonomy? Nat Rev Clin Oncol 6(12):718–730. doi:10.1038/nrclinonc.2009.166 2. Weigelt B, Horlings HM, Kreike B, Hayes MM, Hauptmann M, Wessels LF, de Jong D, Van de Vijver MJ, Van’t Veer LJ, Peterse JL (2008) Refinement of breast cancer classification by molecular characterization of histological special types. J Pathol 216(2):141–150. doi:10.1002/path.2407 3. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406(6797):747–752. doi:10. 1038/35021093

29 4. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100(14):8418–8423. doi:10.1073/pnas.09326921000932692100 5. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Lonning PE, BorresenDale AL (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98(19):10869–10874. doi:10.1073/pnas. 19136709898/19/10869 6. Smid M, Wang Y, Zhang Y, Sieuwerts AM, Yu J, Klijn JG, Foekens JA, Martens JW (2008) Subtypes of breast cancer show preferential site of relapse. Cancer Res 68(9):3108–3114. doi:10. 1158/0008-5472.CAN-07-5644 7. Kennecke H, Yerushalmi R, Woods R, Cheang MC, Voduc D, Speers CH, Nielsen TO, Gelmon K (2010) Metastatic behavior of breast cancer subtypes. J Clin Oncol 28(20):3271–3277. doi:10. 1200/JCO.2009.25.9820 8. Smid M, Hoes M, Sieuwerts AM, Sleijfer S, Zhang Y, Wang Y, Foekens JA, Martens JW (2011) Patterns and incidence of chromosomal instability and their prognostic relevance in breast cancer subtypes. Breast Cancer Res Treat 128(1):23–30. doi:10. 1007/s10549-010-1026-5 9. Rouzier R, Perou CM, Symmans WF, Ibrahim N, Cristofanilli M, Anderson K, Hess KR, Stec J, Ayers M, Wagner P, Morandi P, Fan C, Rabiul I, Ross JS, Hortobagyi GN, Pusztai L (2005) Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 11(16):5678–5685. doi:10.1158/ 1078-0432.CCR-04-2421 10. Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D, Macgrogan G, Bergh J, Cameron D, Goldstein D, Duss S, Nicoulaz AL, Brisken C, Fiche M, Delorenzi M, Iggo R (2005) Identification of molecular apocrine breast tumours by microarray analysis. Oncogene 24(29):4660–4671. doi:10.1038/ sj.onc.1208561 11. Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI, He X, Perou CM (2010) Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12(5):R68. doi:10.1186/bcr2635 12. Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121(7):2750–2767. doi:10. 1172/JCI45014 13. Garnett MJ, Edelman EJ, Heidorn SJ, Greenman CD, Dastur A, Lau KW, Greninger P, Thompson IR, Luo X, Soares J, Liu Q, Iorio F, Surdez D, Chen L, Milano RJ, Bignell GR, Tam AT, Davies H, Stevenson JA, Barthorpe S, Lutz SR, Kogera F, Lawrence K, McLaren-Douglas A, Mitropoulos X, Mironenko T, Thi H, Richardson L, Zhou W, Jewitt F, Zhang T, O’Brien P, Boisvert JL, Price S, Hur W, Yang W, Deng X, Butler A, Choi HG, Chang JW, Baselga J, Stamenkovic I, Engelman JA, Sharma SV, Delattre O, Saez-Rodriguez J, Gray NS, Settleman J, Futreal PA, Haber DA, Stratton MR, Ramaswamy S, McDermott U, Benes CH (2012) Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature 483(7391):570–575. doi:10.1038/nature11005 14. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, Wilson CJ, Lehar J, Kryukov GV, Sonkin D, Reddy A, Liu M, Murray L, Berger MF, Monahan JE, Morais P, Meltzer J, Korejwa A, Jane-Valbuena J, Mapa FA, Thibault J, Bric-Furlong E, Raman P, Shipway A, Engels IH, Cheng J, Yu GK, Yu J, Aspesi P Jr, de Silva M, Jagtap K, Jones MD, Wang L, Hatton C,

123

30

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Breast Cancer Res Treat (2014) 148:19–31 Palescandolo E, Gupta S, Mahan S, Sougnez C, Onofrio RC, Liefeld T, MacConaill L, Winckler W, Reich M, Li N, Mesirov JP, Gabriel SB, Getz G, Ardlie K, Chan V, Myer VE, Weber BL, Porter J, Warmuth M, Finan P, Harris JL, Meyerson M, Golub TR, Morrissey MP, Sellers WR, Schlegel R, Garraway LA (2012) The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483(7391):603–607. doi:10. 1038/nature11003 Hollestelle A, Nagel JH, Smid M, Lam S, Elstrodt F, Wasielewski M, Ng SS, French PJ, Peeters JK, Rozendaal MJ, Riaz M, Koopman DG, Ten Hagen TL, de Leeuw BH, Zwarthoff EC, Teunisse A, van der Spek PJ, Klijn JG, Dinjens WN, Ethier SP, Clevers H, Jochemsen AG, den Bakker MA, Foekens JA, Martens JW, Schutte M (2010) Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines. Breast Cancer Res Treat 121(1):53–64. doi:10.1007/s10549-009-0460-8 Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10(6):515–527. doi:10.1016/j.ccr. 2006.10.008 Hollestelle A, Elstrodt F, Timmermans M, Sieuwerts AM, Klijn JG, Foekens JA, den Bakker MA, Schutte M (2010) Four human breast cancer cell lines with biallelic inactivating alpha-catenin gene mutations. Breast Cancer Res Treat 122(1):125–133. doi:10. 1007/s10549-009-0545-4 Riaz M, van Jaarsveld MT, Hollestelle A, Prager-van der Smissen WJ, Heine AA, Boersma AW, Liu J, Helmijr J, Ozturk B, Smid M, Wiemer EA, Foekens JA, Martens JW (2013) MicroRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs. Breast Cancer Res 15(2):R33. doi:10.1186/bcr3415 van der Wegen P, Louwen R, Imam AM, Buijs-Offerman RM, Sinaasappel M, Grosveld F, Scholte BJ (2006) Successful treatment of UGT1A1 deficiency in a rat model of Crigler-Najjar disease by intravenous administration of a liver-specific lentiviral vector. Molecul Ther 13(2):374–381. doi:10.1016/j.ymthe.2005. 09.022 Kuenen-Boumeester V, Timmermans AM, De Bruijn EM, Henzen-Logmans SC (1999) Immunocytochemical detection of prognostic markers in breast cancer; technical considerations. Cytopathology 10(5):308–316 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8(6):e1000412. doi:10. 1371/journal.pbio.1000412 Osborne CK, Coronado-Heinsohn EB, Hilsenbeck SG, McCue BL, Wakeling AE, McClelland RA, Manning DL, Nicholson RI (1995) Comparison of the effects of a pure steroidal antiestrogen with those of tamoxifen in a model of human breast cancer. J Natl Cancer Inst 87(10):746–750 Kang JS, Kang MR, Han SB, Yoon WK, Kim JH, Lee TC, Lee CW, Lee KH, Lee K, Park SK, Kim HM (2009) Low dose estrogen supplementation reduces mortality of mice in estrogendependent human tumor xenograft model. Biol Pharma Bull 32(1):150–152 Heiser LM, Sadanandam A, Kuo WL, Benz SC, Goldstein TC, Ng S, Gibb WJ, Wang NJ, Ziyad S, Tong F, Bayani N, Hu Z, Billig JI, Dueregger A, Lewis S, Jakkula L, Korkola JE, Durinck S, Pepin F, Guan Y, Purdom E, Neuvial P, Bengtsson H, Wood KW, Smith PG, Vassilev LT, Hennessy BT, Greshock J, Bachman KE, Hardwicke MA, Park JW, Marton LJ, Wolf DM, Collisson EA, Neve RM, Mills GB, Speed TP, Feiler HS, Wooster

123

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

RF, Haussler D, Stuart JM, Gray JW, Spellman PT (2012) Subtype and pathway specific responses to anticancer compounds in breast cancer. Proc Natl Acad Sci USA 109(8):2724–2729. doi:10.1073/pnas.1018854108 Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K, Gilcrease MZ, Krishnamurthy S, Lee JS, Fridlyand J, Sahin A, Agarwal R, Joy C, Liu W, Stivers D, Baggerly K, Carey M, Lluch A, Monteagudo C, He X, Weigman V, Fan C, Palazzo J, Hortobagyi GN, Nolden LK, Wang NJ, Valero V, Gray JW, Perou CM, Mills GB (2009) Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 69(10):4116–4124. doi:10.1158/ 0008-5472.CAN-08-3441 Sieuwerts AM, Kraan J, Bolt J, van der Spoel P, Elstrodt F, Schutte M, Martens JW, Gratama JW, Sleijfer S, Foekens JA (2009) Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst 101(1):61–66. doi:10.1093/jnci/djn419 Hollestelle A, Peeters JK, Smid M, Timmermans M, Verhoog LC, Westenend PJ, Heine AA, Chan A, Sieuwerts AM, Wiemer EA, Klijn JG, van der Spek PJ, Foekens JA, Schutte M, den Bakker MA, Martens JW (2013) Loss of E-cadherin is not a necessity for epithelial to mesenchymal transition in human breast cancer. Breast Cancer Res Treat. doi:10.1007/s10549-0132415-3 Buzdar AU, Ibrahim NK, Francis D, Booser DJ, Thomas ES, Theriault RL, Pusztai L, Green MC, Arun BK, Giordano SH, Cristofanilli M, Frye DK, Smith TL, Hunt KK, Singletary SE, Sahin AA, Ewer MS, Buchholz TA, Berry D, Hortobagyi GN (2005) Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer. J Clin Oncol 23(16):3676–3685. doi:10.1200/JCO.2005. 07.032 Gonzalez-Angulo AM, Litton JK, Broglio KR, Meric-Bernstam F, Rakkhit R, Cardoso F, Peintinger F, Hanrahan EO, Sahin A, Guray M, Larsimont D, Feoli F, Stranzl H, Buchholz TA, Valero V, Theriault R, Piccart-Gebhart M, Ravdin PM, Berry DA, Hortobagyi GN (2009) High risk of recurrence for patients with breast cancer who have human epidermal growth factor receptor 2-positive, node-negative tumors 1 cm or smaller. J Clin Oncol 27(34):5700–5706. doi:10.1200/JCO.2009.23.2025 Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX, Minn AJ, van de Vijver MJ, Gerald WL, Foekens JA, Massague J (2009) Genes that mediate breast cancer metastasis to the brain. Nature 459(7249):1005–1009. doi:10.1038/nature08021 Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J (2005) Genes that mediate breast cancer metastasis to lung. Nature 436(7050):518–524. doi:10.1038/nature03799 Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, CordonCardo C, Guise TA, Massague J (2003) A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3(6):537–549 Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117(7):927–939. doi:10. 1016/j.cell.2004.06.006S0092867404005768 Korkaya H, Paulson A, Charafe-Jauffret E, Ginestier C, Brown M, Dutcher J, Clouthier SG, Wicha MS (2009) Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling. PLoS Biol 7(6):e1000121. doi:10.1371/journal.pbio.1000121 Conley SJ, Gheordunescu E, Kakarala P, Newman B, Korkaya H, Heath AN, Clouthier SG, Wicha MS (2012) Antiangiogenic

Breast Cancer Res Treat (2014) 148:19–31 agents increase breast cancer stem cells via the generation of tumor hypoxia. Proc Natl Acad Sci USA 109(8):2784–2789. doi:10.1073/pnas.1018866109 36. Dittmer A, Fuchs A, Oerlecke I, Leyh B, Kaiser S, Martens JW, Lutzkendorf J, Muller L, Dittmer J (2011) Mesenchymal stem cells and carcinoma-associated fibroblasts sensitize breast cancer cells in 3D cultures to kinase inhibitors. Int J Oncol 39(3):689–696. doi:10.3892/ijo.2011.1073

31 37. Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J, Davis A, Mongare MM, Gould J, Frederick DT, Cooper ZA, Chapman PB, Solit DB, Ribas A, Lo RS, Flaherty KT, Ogino S, Wargo JA, Golub TR (2012) Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487(7408):500–504. doi:10.1038/nature11183

123