Electronic Supplementary Materials
Intraductally administered pegylated liposomal doxorubicin reduces mammary stem cell function
in the mammary gland but in the long term, induces malignant tumors
Yong Soon Chun1#$, Takahiro Yoshida1#, Tsuyoshi Mori1$, David L Huso2, Zhe Zhang1, Vered Stearns1,
Brandy Perkins1, Richard J Jones1, Saraswati Sukumar1*
Affiliations: Departments of 1Oncology and 2Molecular and Comparative Pathobiology, Johns Hopkins
University School of Medicine, Baltimore, Maryland, U.S.A.
#Equal
contribution
address:
Yong Soon Chun: Department of Surgery, Gachon University Graduate School of Medicine,
1198 Guwol-dong, Namdong-gu, Incheon 405-760, Korea
Tsuyoshi Mori: Department of Surgery, Shiga University of Medical Science,
Seta, Tsukinowa-cho, Otsu Shiga, 520-2191 Japan
$Current
Yong Soon Chun:
Takahiro Yoshida:
Tsuyoshi Mori:
David L Huso:
Zhe Zhang:
Vered Stearns:
Brandy Perkins:
Richard J Jones:
Saraswati Sukumar:
chunysmd@gachon.ac.kr
tyoshid9@jhmi.edu
t252m@belle.shiga-med.ac.jp
dhuso@jhmi.edu
zzhang16@jhmi.edu
vstearn1@jhmi.edu
bperkin6@jhmi.edu
rjjones@jhmi.edu
saras@jhmi.edu
*Corresponding author
Saraswati Sukumar, Ph.D.
Breast Cancer Program
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
CRB1, Room 143, 1650 Orleans Street, Baltimore, MD 21231, U.S.A.
Phone: 410-614-2479; Fax: 410-614-4073; E-mail: saras@jhmi.edu
Isolated and enriched mammary epithelial cells
Mammary glands were digested for 8 h at 37ºC in serum-free breast tissue digestion medium [1]
containing collagenase (80mg/50ml) and hyaluronidase (12.5mg/50ml). Organoids obtained after
vortexing were subjected to red blood cell lysis in red blood cell lysing buffer (Sigma). Further dissociation
was done using 0.25% trypsin for 2 min, 5mg/ml dispase (StemCell Technologies) with 0.1mg/ml DNase I
(StemCell Technologies) for 2 min, and the cell suspension was filtered through a 40um mesh to obtain
single cells. Enriched mammary epithelial cells, Lin- (CD45-, Ter119-, CD31-) cells were collected using
immunomagnetic bead separation with the EasySep mouse epithelial cell enrichment kit (StemCell
Technologies, #19758). Mammary epithelial cells were thus enriched and used for transplantation studies.
1
In vivo limiting dilution assay
Cells were resuspended in a 10ul volume containing 25% Matrigel (BD Biosciences) and 75% Hank’s
balanced salt solution (StemCell Technologies) plus 2% FBS. Cells (10 6, 105, 104, 103, 102, and 10) were
injected into bilateral 4th cleared mammary fat pads of FVB/N mice. The frequency of mammary
repopulating units (MRUs) in single cell suspensions was calculated using complementary log-log
generalized linear model. Two-sided 95% Wald confidence intervals were computed, except in the case of
zero outgrowths, when one-sided 95% Clopper-Pearson intervals were used instead. The single-hit
assumption was tested as recommended [2] and was not rejected for any dilution series.
Whole mount analyses of morphological characteristics of mammary glands in parous Her2/neu
transgenic mice
Six whole mount specimens of the 4th mammary gland harvested from 6 different mice each group were
evaluated based on presence of entire mammary ducts and appropriate red carmine staining. Images of
whole mount slides were visualized by a Nikon SMZ 1500 with HR plan Apo 0.5X WD136 lens and 0.75X.
The distal mammary gland (from the central lymph node) was divided into 4 equal parts. Field 1 to 4
(5mmX5mm each field) were set along the midline of each mammary gland as shown in Figure 1d. The
end buds IN the each field were counted on the JPEG file saved as 3840 X 3073 pixels and 150 dpi. The
primary ductal dilatation was examined by measuring the diameter of the largest primary duct adjacent to
the central lymph node based on the JPEG image files containing a 100μm black scale bar captured by a
Nikon Digital Camera DXM 1200, objective lens X5 and synophthalmia lens X10 (saved as 2560 X 2048
pixels and 150 dpi). The external diameter was measured in increments of 5 um for each picture.
Histopathological analyses of mammary glands in i.duc PLD-treated FVB/N mice
Histopathological characteristics in mammary glands were examined by H&E. Features assessed
included 1) lymphocytic and plasmacytic infiltration 2) neutrophil and/or macrophage infiltration; and 3)
fibrosis in FVB/N wild type mice treated by 40ug PLD (n=38 slides from 38 mice) or untreated (n=10
slides from 10 mice).
Fibrotic changes were examined by H&E staining in terms of proliferation of fibroblasts and stromal cells
around terminal ductal lobular units as healing with scoring in FVB/N mice for stem cell analysis. Scores
(0, 1, 2 or 3) were determined for cell infiltration and fibrosis based on criteria described in the table
below. Distribution of foci was considered. Representative images of lymphocytic infiltration and fibrosis
are shown.
Criteria
Cell infiltration
Within normal limits
Mild
Moderate
Severe
Fibrosis
Within normal limits
Few fibroblasts and limited collagen
Moderate fibroblasts and collagen
Proliferative fibroblasts, heavy
collagen bundles
Score
0
1
2
3
2
Distribution
Within normal limits
1 focus or less/hpf, occasional foci
2-4 foci/hpf, several foci but also
normal units
>4 foci/hpf, almost all units
affected, rare or no normal
Microscopic findings (X200, bar 20um)
Lymphocytic infiltration
Fibrosis
Score
0
1
2
3
Supplementary Table 1. Tumor development in transplanted mammary fat pads*
No. of cells injected per mfp
No. of tumor outgrowths/No. of mfps injected
106
2/2
105
4/8
104
9/18
103
4/13
*Mammary glands from five i.duc PLD-treated mice were subjected to in vivo limiting dilution assay 33
weeks after the second PLD injection. Enriched mammary epithelial cells (103 to 106) were transplanted
into the 4th cleared mfps of recipient females. Recipient mice developed solid tumors in their fat pads by
8 weeks after transplantation. Tumors developed in 19/41 mfps injected.
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Supplementary Table 2. Malignant mammary tumors developing in I.duc PLD treated mice*
i.duc PLD
(ug)/Tag#
Reasons of
euthanasia
Histological diagnosis
40 / #176
Tumor latency
(months after 2nd
i.duc)
9
sick
40 / #177
8
mammary tumor
carcinomas
(non-palpable tumors)
carcinoma
40 / #178
7
mammary tumor
carcinoma
-
40 / #179
9
mammary tumor
carcinosarcoma
+
40 / #181
9
termination of expt.
carcinoma
-
40 / #184
8
mammary tumor
carcinoma
-
20 / #153
9
termination of expt.
-
20 / #155
4
mammary tumor
carcinoma
(non-palpable tumor)
carcinoma
+
20 / #157
8
mammary tumor
carcinoma
+
10 / #127
5
mammary tumor
undifferentiated sarcoma
+
10 / #128
5
mammary tumor
carcinoma
-
5
sick
carcinoma
(non-palpable tumor in LN)
-
@10
/ #129
EMT
findings
(+/-)
-
*Three groups of multi-parous FVB/N mice received i.duc PLD at doses of 10, 20 and 40 ug/duct
respectively, one group received vehicle, and one group was left untreated. Palpable tumors appeared
starting at 16 weeks after the 2nd injection in PLD-treated groups during the 42 weeks of observation, but
not in the two control groups. Histopathology was performed on three tumors per group.
@One case of microscopic epithelial tumor in the lymph node (LN) without visible or additional
microscopic lesions in the mammary gland sections examined (same case as **LN tumor in Table 2).
Supplementary references
[1] Stampfer M, Hallowes RC, Hackett AJ (1980) Growth of normal human mammary cells in culture. In
Vitro 16:415-425
[2] Bonnefoix, T, Bonnefoix, P, Verdiel, P & Sotto, JJ (1996) Fitting limiting dilution experiments with
generalized linear models results in a test of the single-hit Poisson assumption. J Immunol Methods
194:113-119
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