Mutations in Bone Marrow-Derived Stromal Stem Cells
Unmask Latent Malignancy
Houghton, Jeanmarie et al. “Mutations in Bone Marrow-Derived
Stromal Stem Cells Unmask Latent Malignancy.” Stem Cells and
Development 19.8 (2010) : 1153-1166. Copyright © 2010, Mary
Ann Liebert, Inc.
Mary Ann Liebert
Final published version
Thu Oct 13 23:20:33 EDT 2011
Article is made available in accordance with the publisher's policy
and may be subject to US copyright law. Please refer to the
STEM CELLS AND DEVELOPMENT
Volume 19, Number 8, 2010
© Mary Ann Liebert, Inc.
Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple
risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to ma-
lignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse
clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk
tissue. To address this question, Apc Min/+ and Apc Min/+Rag2−/− mice, which have a predilection to mammary car-
cinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC)
transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the mar-
row cavity where they could be recovered up to a year later without apparent effect on the health of the mouse.
No mammary tumors were found. However, in mice carrying the Apc Min/+ mutation, p53MSC homed to mam-
mary tissue and signifi cantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-
α-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent
disease. The increased cancer phenotype was completely preventable with neutralization of TNF-α or by transfer
of CD4 + regulatory T cells from immune competent donors, demonstrating that immune competency to regulate
infl ammation was suffi cient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and
stromal mutations. The signifi cant synergy between host immunity and mesenchymal cells identifi ed here may
restructure treatments to restore an anticancer microenvironment.
dence of clinically apparent tumors is strikingly lower [ 1 ].
Autopsy studies reveal a profound discrepancy between the
presence of subclinical malignancy and clinically apparent
cancer [ 2–4 ]. Multiple factors may affect this switch from
dormant to overt disease, including additional gene muta-
tions within tumor cells, changes in the systemic immune
response, and/or alterations in the microenvironment and
stromal cells proximate to the cancer cells.
Bone marrow-derived cells (BMDC) invade and re-
side within infl ammed tissues. Within these tissues, cells
y midlife, the majority of people have microscopic foci
of malignant cells throughout their body, yet the inci-
derived from the marrow can differentiate as myofi broblasts
and fi broblasts and contribute substantially to the signaling
environment surrounding regenerating tissue [ 5 ], and they
are instrumental in directing epithelial cell regeneration
and wound repair. Within established tumors, BMDC are
recruited as cancer-associated fi broblasts (CAF), which have
a phenotype similar to that of activated fi broblasts thus
sharing a “wound-healing” repertoire of chemokine and
cytokine [ 6 ] production. Mesenchymal stem cells (MSC),
which can be found in the marrow, also localize to areas
of established carcinoma and infi ltrate into the tumor as
tumor stroma [ 7 ]. MSC promote breast cancer metastasis
through effects on local tumor growth and migration, and
Mutations in Bone Marrow-Derived Stromal Stem Cells
Unmask Latent Malignancy
JeanMarie Houghton , 1,2 Hanchen Li , 1 Xueli Fan , 1 Yingwang Liu , 1 Jian Hua Liu , 1 Varada P. Rao , 3
Theofi los Poutahidis , 3,4 Christie L. Taylor , 5 Erin A. Jackson , 3 Christine Hewes , 3 Stephen Lyle , 2 Anna Cerny , 6
Glennice Bowen , 6 Jan Cerny , 7 Nathan Moore , 5 Evelyn A. Kurt-Jones , 6 and Susan E. Erdman 3
1 Department of Medicine, Division of Gastroenterology, 2 Department of Cancer Biology, 6 Department of Medicine, Division of
Infectious Disease, and 7 Department of Medicine, Division of Hematology/Oncology, University of Massachusetts Medical School,
3 Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts.
4 Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece.
5 University of Massachusetts GSBS, Worcester, Massachusetts.
HOUGHTON ET AL. 1154
School under IACUC approval. Female C57BL/6 Apc Min/+
(B6Min), Apc Min/+ Rag2 −/− (B6RagMin), and wt (B6) littermates
were genotyped prior to experiments, and at euthanasia.
Experiments were conducted using separate trials with
5–10 mice each and results combined for statistical anal-
yses. C57BL/6 Apc Min/+ mice are at risk for mammary tumors
and 1% of female mice develop tumors by 26 weeks of age.
Apc Min/+ Rag2 −/− mice are at a higher risk of mammary carci-
noma and 10% of female mice develop mammary tumors by
26 weeks of age.
Wild-type bone marrow transplantation
and MSC infusion
Bone marrow transplantation was performed as previ-
ously described [ 12 ] using enhanced green fl uorescent
protein (eGFP)-labeled total bone marrow from C57BL/6J-
β-actin-EGFP mice (Jackson laboratories, Bar Harbor, ME)
or eGFP-MSC plus 3–5 million unmarked support marrow
cells transplanted into irradiated (900 rads) recipients via
tail vein injection. Recipient mice were 6–8 weeks of age.
Donor mice were 8–12 weeks of age. Freshly cultured cells
were used at fi rst passage. Cultured wtMSC or p53MSC
were age-matched for experiments and used at Passages
Isolation, culture, and infusion of p53MSC
Wild-type bone marrow was isolated and cultured as
previously described [ 13 ]. p53wt (wt) and MSC with a CAF
phenotype and carrying p53 mutation (p53MSC) were iden-
tifi ed, characterized, and transfected with a monomeric
DS-red containing plasmid as described in Supplemental
T REG transfer and neutralization of TNF-α by anti-TNF-α
antibody is described in Supplemental Methods.
Primary tumor, tumor transplantation, and histological
evaluation of tissues
p53MSC-induced B6Min mammary tumors were excised.
One half was processed for histology and Ki-67 expression,
and ½ was minced, digested, and cell suspensions injected
into the mammary fat pad of recipient female B6Min mice.
All mammary tissue and tumors were evaluated for his-
tology and epithelial cell proliferation as described in
Supplemental Methods. For ex vivo proliferation studies,
cells were placed in tissue culture with Dulbecco’s modifi ed
Eagle’s medium (DMEM) 10% fetal calf serum (FCS) and
grown as a monolayer.
Migration by monolayer wound-healing assay
MCF-7 cells were grown to 90% confl uence and the
cell monolayer was then wounded by a plastic tip (1 mm).
Monolayers were washed, photographed, and media
replaced with control medium or p53MSC-conditioned me-
dium ± chemokines and healing measured as described in
via priming of distant sites to become conducive docking
sites for tumor cells [ 7 ].
Cancer-associated fi broblasts have been shown to carry
genetic and epigenetic mutations, which are thought to
impact their clinical behavior [ 8–10 ]. Recently, it has been
suggested that CAF may play as much a role in cancer for-
mation as do the epithelial-derived tumor cells themselves.
Mutations in stromal p53 have been demonstrated in CAF
associated with multiple tumor types. Mammary tumors
containing p53-defi cient stromal fi broblasts developed
faster and are more aggressive than tumors containing
wild-type (wt) stromal fi broblasts [ 11 ]. Though it is clear
that stromal cells have dramatic impact on established
tumors, it is less clear what effect these cells have on the
earliest stages of malignancy or what role, if any, they may
have in converting microscopic malignant foci within at-
risk tissue to frank malignant disease. Interestingly, it has
been suggested that mammary stroma may be a critical
target for chemical-induced transformation. Studies using
NMU as a transforming agent demonstrate that the mam-
mary stroma can be a direct target of chemical transfor-
mation and precedes the transformation of rat mammary
epithelial cells in a rat model of chemical-induced mam-
mary carcinoma [ 8 ].
Here we show that mutant p53MSC home to mammary
tissue under the infl uence of dysregulated infl ammation.
Once within at-risk tissue, p53MSC accelerated neoplastic
cell growth. Homing and proliferation of p53MSC within
the mammary epithelium resulted in a dramatic increase
in disease incidence and tumor size, and a decrease in
the time to clinically apparent disease in mice at-risk for
mammary carcinoma. The p53MSC themselves did not
promote mammary tumors in wt mice that were not at
risk for the disease. Further, the increased cancer pheno-
type was dependent upon tumor necrosis factor (TNF)-α,
and accelerated tumor formation was completely reversed
with neutralization of TNF-α via neutralizing antibody or
by transfer of competent T-regulatory cells (T REG ). Altered
MSC signaling converted quiescent in situ carcinoma to
clinically apparent disease in a tissue-specifi c fashion.
The signifi cant contribution of immune dysregulation to
procancer MSC activity provides additional mechanisms
for the clinical stratifi cation of breast cancer and may re-
structure treatment targets toward restoring an anticancer
Materials and Methods
Please see Supplemental Methods for additional details.
Human breast cancer and normal breast tissue sam-
ples from de-identifi ed individuals were obtained from
the UMass Cancer Center Tissue and Tumor Bank http://
with IRB approval and evaluated for p53 expression (see
Animal work was performed at Massachusetts Institute
of Technology or at the University of Massachusetts Medical
MUTATED STROMA UNMASKS LATENT MALIGNANCY
non-neoplastic mammary glands and associated connective
tissue for p53 and α-SMA expression. Small glands were
devoid of α-SMA expression. Myoepithelial cells along lob-
ular ductules stain strongly positive for α-SMA. Fibroblasts
within the stroma were devoid of α-SMA signal. We did not
detect any p53 staining within any cell of benign mammary
tissue ( Fig. 1H and 1I ). These fi ndings confi rm the published
observation of rare (1%–5%) p53 + CAF cells within breast
cancer stroma [ 11 , 15 , 17–19 ].
Wild-type MSC are not associated with an increase
risk of mammary carcinoma
We hypothesized that p53 mutations in stromal cells
could drive the transition of occult malignant cells to frank
mammary carcinoma in at-risk tissue. As a fi rst step in eval-
uating the p53MSC promotion of mammary carcinoma, we
characterized the in vivo activity of wtMSC in wtC57BL/6
mice (B6) not prone to mammary carcinoma compared
with wtMSC given to C57BL/6 Apc Min/+ (B6Min) mice that
have a low but signifi cant spontaneous rate of developing
mammary carcinoma, and found that wtMSC did not con-
tribute to a malignant phenotype in either mouse model (see
Supplemental Table 1 ; Supplementary materials are avail-
able online at http://www.liebertonline.com/scd).
Infused p53MSC-CAF are not associated
with mammary carcinoma in B6 mice
We next investigated the fate of MSC hemizygous for p53
307 Thr–Ile mutation [ 13 ] (p53MSC) (which coincides with
position 312 in the DNA-binding region of human p53, a
mutation found in human cancers [ 20 ]) in the B6 mice. This
Mammary tumor incidence was compared using Fisher
exact t -test. Ki-67 index and assessment of stroma was com-
pared between groups using 2-tailed t -test. In vitro data
were compared using Student’s t -test using a minimum of 3
replicates per experimental point, and experiments repeated
2–3 times for reproducibility.
Cancer-associated fi broblasts from human breast
cancer contain a population of p53-mutated
Stromal cells with p53 mutations have been identifi ed as-
sociated with breast [ 14,15 ], prostate [ 16 ], and other epithelial
cancer cells and are predictive of aggressive tumor behavior
and metastasis. While there is controversy with regard to
the specifi c mutation and frequency of stromal p53 muta-
tions in human breast cancer specimens [ 15 , 17–19 ], there
is a growing literature that demonstrates a role for nonau-
tonomous p53 effects between stroma and mammary epi-
thelial cells [ 11 ]. We fi rst assessed p53 expression in human
breast cancer stroma. Specifi c p53 immunohistochemistry
(IHC) of 15 consecutive breast cancer specimens archived
by the UMass tumor bank was done and our data support
the notion that overexpression of p53, which can be asso-
ciated with clinically relevant mutations, occurs commonly
within a population of CAF ( Table 1 ). p53 + stromal cells were
found in 67% of breast cancer specimens ( Fig. 1A–1C ) and
expressed α-smooth muscle actin (α-SMA) indicative of an
activated fi broblast phenotype ( Fig. 1D–1G ). p53 + cells were
found as isolated cells within activated stroma. We evaluated
T able 1. H uman B reast C ancer —C linical I nformation
Sample number Age Cancer typeGradepTpN pM ER/PR/Her2
Grade: 1, well-differentiated; 2, moderately differentiated; and 3, poorly differentiated.
pT: tumor size and extent.
pN nodes: a ≤ 0.2 cm; b ≥ 0.2 cm; mi, micrometastasis <0.2 cm.
pM metastasis: +/−.
ER/PR/Her2: estrogen receptor, progesterone receptor, and Her2 status of tumor sample.
p53 status for epithelium and stroma: Negative (−) if there is no staining, (+) if any cell in the appropriate
compartment stains for nuclear p53.
HOUGHTON ET AL. 1156
were verifi ed by fl uorescence-activated cell sorting (FACS)
to lack hematopoietic stem cell markers and lineage-spe-
cifi c markers (negative for CD45, CD3, CD4, CD8, CD11b,
CD11c, CD19, CD25, Gr1, F4/80 NK1.1, and CD31) and were
positive for CD105, CD73, and CD44. All MSC populations
used maintained trilineage differentiation (fat, cartilage,
and bone) capacity in culture. wtMSC and p53MSC main-
tained similar phenotypes and growth characteristics
mutation is a gain-of-function mutation (see Supplemental
Fig. 1; Supplementary materials are available online at
http://www.liebertonline.com/scd). wtMSC cultured under
the same conditions, and at the same age, do not have de-
monstrable p53 protein by immunofl uorescence (IF) or by
western blot analysis, while p53MSC stain heavily by p53
IF, and have abundant protein by western blot analysis
[ 13 ]. Freshly isolated MSC and cultured MSC populations
Human Breast Cancer
p53 Positive IHC/IF
Within Human Breast
p53 Positive IHC/IF
Within Benign Human
Human Non-Neoplastic Mammary Tissue
FIG. 1. Human breast cancer stroma contains nuclear p53. Specifi c anti-p53 IHC of 3 different representative human breast
cancer specimens containing ( A ) abundant stroma ( B ) moderate amount of stroma, and ( C ) little stroma. Arrows point out
p53 + nuclei of stromal cells (brown nuclear staining). Frozen tissue was used to colocalize ( D ) anti-α-smooth muscle actin
(SMA) (green), ( E ) p53 (red, arrows), ( F ) nuclei (blue). ( G ) Merged image shows p53 colocalizing with dapi (purple, arrows)
within α-SMA + cells. ( H ) Anti-p53 (brown staining) and ( I ) anti-α-SMA (brown staining) immunohistochemistry of con-
secutive sections of non-neoplastic mammary tissue containing mammary epithelial and stromal structures. Boxed areas
shown in higher power as indicated. The chart summarizes the number of p53 + cells within the epithelial and stromal com-
partments of the human tumors and within benign mammary tissue.
MUTATED STROMA UNMASKS LATENT MALIGNANCY
wtMSC but, in the absence of infl ammation or a predisposi-
tion to tumors, do not themselves initiate epithelial malig-
nancy (see Supplemental Methods for further details).
p53MSC and mammary carcinoma in the Apc Min/+
cancer prone mouse
To address the effects of stroma on the growth of dormant
or premalignant mammary cells, we chose a mouse model
that develops mammary carcinoma at a low but reproducible
rate. Less than 1% of B6Min mice housed at MIT or UMass
develop mammary carcinoma as they age (personal obser-
vation). Tumor incidence is directly linked to infl ammatory
triggers [ 21–23 ] as tumor formation occurs with chronic in-
fection [ 23 ]. Congenital or acquired immunodefi ciency is
strongly associated with an increased incidence of tumors in
both human and mice. Immune cells are thought to prevent
tumor outgrowth by either direct elimination of tumor cells
or by maintaining an equilibrium state where occult tumors
are kept in check. These processes are termed immunosur-
veillance and immunoediting [ 24–26 ]. Rag2-defi cient mice
lacking T, B, and NK-T immune cells are at an increased
risk for chemically induced tumors attributed to genetic
with doubling times of 26 and 28 h, respectively. B6 mice
were infused with 1 million p53MSC or age-matched cul-
tured wtMSC labeled with red fl uorescent protein (RFP)
as control. Infused cultured wtMSC were not maintained
at detectable levels long term in host tissue, and were not
found after 3, 6, 9, or 12 months postinfusion (Supplemental
Fig. 2; Supplementary materials are available online at
http://www.liebertonline.com/scd). In contrast to wtMSC,
p53MSC remained detectable in the blood for many months
postinfusion and were recovered at all time points tested
( Fig. 2A and 2B and Supplemental Fig. 2). There were no
gross or microscopic tumors in any tissue from B6 mice.
The spleen and the marrow cavity contained a few scattered
RFP + cells ( Fig. 2D ). To confi rm the presence of p53MSC in
the bone marrow, we cultured the bone marrow and recov-
ered 35 ± 5 RFP-p53MSC colony-forming units per mouse
( Fig. 2C ), which we calculated was found to be ~3% of the
stroma. These MSC lacked specifi c hematopoietic or lineage
markers (CD3, CD4, CD8, CD11b, CD11c, CD19, CD25, Gr1,
F4/80, and NK1.1), and retained CD44 positivity and the
ability to differentiate into bone fat and cartilage, confi rm-
ing they were MSC. Further, these data demonstrate that
p53-mutated MSC have a survival advantage in vivo over
FIG. 2. Analysis of red fl uorescent protein (RFP)-p53MSC recovered in the mature wild-type mouse after IV injection. RFP-
labeled p53MSC injected IV into wild-type C57BL/6 mice are identifi ed within the circulation and bone marrow cavity. ( A )
Fluorescence-activated cell sorting (FACS) analysis of peripheral blood 9 months after injection of 1 × 10 6 RFP p53MSC. ( B )
FACS analysis of peripheral blood 9 months after injection of PBS. Boxed area indicates the number of RFP + cells detected.
( C ) Bone marrow fl ushed from the right femur of mouse shown in Panel A was grown in culture and RFP-p53MSC recov-
ered. RFP + cells in culture are seen under the fl uorescent inverted tissue culture microscope. ( D ) RFP-specifi c IHC of the
marrow cavity of the left femur of the same mouse. Arrows highlight brown-colored positive signal in RFP + cells (DAB
chromogen and hematoxylin counterstain 10×).
HOUGHTON ET AL. 1158
were evident. Tumors in B6Min mice had a larger propor-
tion of keratinized glands compared with the tumors in the
B6MinRag mice. The amount of supporting stroma varied
from small to moderate amounts within a group, and did
not differ between groups as a whole; p53MSC did not alter
the amount of stroma in tumors (B6MinRag spontaneous
tumor 8.39% stroma vs. B6MinRag+p53MSC 8.41% stroma).
Neutrophilic infi ltrate was noted in all tumors and ter-
minal ductular (alveolar) differentiation was an occasional
p53MSC-induced mammary tumors are
transplantable into a second host
We next assessed p53MSC-induced tumors for malignant
characteristics. Cell suspensions were prepared from B6Min
p53MSC tumors and injected into the mammary fat pad of
secondary B6Min hosts. Tumors formed by 8 days after injec-
tion in 1/3 B6Min mice (1.6 × 0.9 × 0.8 cm). Hematoxylin and
eosin (H&E) staining revealed adenosquamous carcinoma
with interspersed fat and stroma, similar in characteristics to
the original parent tumor as well as to the histology of spon-
taneous mammary tumors arising in aged B6Min mice ( Fig.
3E and 3F [transplanted tumor] compared with Figure 3A and
3B [parent tumor]). Marked nuclear atypia was noted within
disorganized glands ( Fig. 3F ), and a large number of cells
were proliferative by Ki-67 staining ( Fig. 3G ). Transplanted
tumors contained 1% p53MSC within the stroma, which
were Ki-67 − . Recovered tumor from the secondary host con-
tained ~1% RFP-p53MSC within a predominantly p53 − (by
IHC) stroma. Cells from the secondary tumor grew readily
in tissue culture, and maintained an epithelial morphology
in monolayer ( Fig. 3H ). We did not isolate any p53MSC in
mutations and escape of immune-mediated surveillance [ 21 ]
and for microbially induced tumors attributed to inability
to down-regulate host infl ammatory response [ 27 ]. Placing
the Apc Min/+ mutation on the Rag2 background increases the
incidence of spontaneous mammary carcinoma to ~10% by 5
months of age. Tumors are usually solitary.
p53MSC are recruited to mammary tissue
of the B6Min and B6MinRag mouse, surround ducts,
and dramatically increase tumor incidence
We tested our hypothesis that circulating p53MSC con-
tribute to carcinoma progression. One million p53MSC,
wtMSC, or vehicles were injected via the tail vein into fe-
male B6, B6Min, or B6MinRag mice at 8 weeks of age. Mice
were followed for an additional 4–6 weeks. Fifty percent of
the B6Min mice and 80% of the B6MinRag mice that received
p53MSC developed single mammary tumors ranging from
0.3 to 2.0 cm in size by 4–6 weeks postinjection ( Table 2 ). In
marked contrast, B6, B6Min, or B6MinRag mice that received
either vehicle or wtMSC developed no tumors over 6 weeks
p53MSC-associated mammary tumors in the B6Min and
the B6MinRag mouse had histological features similar to
spontaneous tumors that are found in the aged (>26-week-
old) B6Min and B6MinRag mice ( Fig. 3A–3D , higher-power
images of B6MinRag p53MSC tumor in Supplemental Fig. 3;
Supplementary materials are available online at http://www
.liebertonline.com/scd). In p53MSC recipients, mammary
adenosquamous carcinoma was characterized by highly
irregular infi ltrative growth pattern with both keratinized
and nonkeratinized gland units forming small nests, cords,
and trabeculae. Cellular atypia and increased proliferation
T able 2. M ammary T umor F requency in M ouse M odels
Group Mammary tumor incidence
B6+BM+marked wtMSC (transplant)
B6+BM+marked wtMSC (transplant)
B6MinRag+p53MSC+isotype control IgG
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
50% ( n = 10)*
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
0 ( n = 10)
80% ( n = 10)**
0 ( n = 10)
0 ( n = 10)
80% ( n = 10)**
Mice 6–8 weeks of age at the start. Tumor frequency determined up to 6
weeks. Several mice removed from study at 4 weeks due to large tumor size. All
groups with 0 tumor frequency observed for at least 6 weeks. * P = 0.0325, ** P =
Tumor incidence between the C57BL/6Apc Min+/− (B6Min)+MSC and
C57BL/6Apc Min+/− /Rag2 − / − (B6MinRag)+MSC groups was not statistically
signifi cant; P = 0.35.
MUTATED STROMA UNMASKS LATENT MALIGNANCY
surrounded by a small population of RFP-p53MSC. We con-
fi rmed p53 nuclear expression (indicative of mutant protein
expression) within a subset of the stromal cells by anti-p53 IF
staining, consistent with continued in vivo expression of the
mutant protein ( Fig. 4E ). The majority of stromal cells within
the tumor were RFP − , and therefore of host origin ( Fig. 4C
and 4D and Supplemental Table 1 ). This pattern closely re-
sembled the fi ndings in our human samples, where p53 +
stromal cells comprised 1%–5% of the tumor stroma, while
95%–99% of the cells failed to stain for p53.
Peripheral blood of the B6Min and B6MinRag mice that
had developed mammary carcinoma contained <1% cir-
culating RFP-expressing p53MSC-derived cells, similar to
wt mice. The spleen contained between 1% and 5% RFP-
expressing cells. The p53MSC in the spleen maintained CD44
expression, while lacking surface markers of differentiation:
CD45, CD3, CD4, CD8, CD11b, CD11c, CD19, CD25, Gr1, F4/80,
CD31, and NK1.1 by FACS analysis. Bone marrow contained
few RFP-p53MSC. The p53MSC recovered from B6min or
B6MinRag mice with tumors had the same distribution and
culture; however, the quantity of tissue for this purpose was
p53MSC home to at-risk mammary epithelium
p53MSC were detected in the mammary tissue of mice fol-
lowing injection with RFP-labeled p53MSC, but the level of
engraftment differed substantially between recipient mouse
strains. B6 mice had rare single MSC juxtaposed to mam-
mary ducts ( Supplemental Table 1 ). B6Min (data not shown)
and B6MinRag mice had foci of abundant p53MSC-derived
cells surrounding several ducts ( Fig. 4A and 4B ), while other
ducts within the same mouse had few to no detectable RFP-
p53MSC (summarized in Supplemental Table 1 ) even in the
absence of tumors. This patchy engraftment was consistent
between B6Min and B6MinRag mice. In areas of tumor,
p53MSC were identifi ed both at the periphery of tumors,
and within the infi ltrating stroma, and accounted for 1%–5%
of cells within tumors ( Fig. 4C and 4D and Supplemental
Table 1 ). Carcinomas were consistently infi ltrated with, and
Adenosquamous Mammary Carcinoma Transplanted into
a B6Min Recipient.
FIG. 3. p53MSC induce mammary adenosquamous carci-
noma in the B6Min and B6MinRag mouse. Comparison of
histology of spontaneous mammary carcinoma and p53MSC-
induced mammary tumor in the mouse model. Tumor tissue
from aged (>26-week-old) untreated B6Min and B6MinRag
mice from previously published studies was used for com-
parison purposes, as no spontaneous tumors arose during
our study period. Tumors from ( A ) aged B6Min, ( B ) 8-week-
old B6Min injected with p53MSC and evaluated 4–6 weeks
later, ( C ) aged B6MinRag, ( D ) 8-week-old B6MinRag injected
with p53MSC and evaluated 4–6 weeks later demonstrate
striking similarities when compared with each other in-
cluding highly irregular infi ltrative growth pattern with
nonkeratinized and keratinized glands arranged in variable
sized nests and cords, cellular atypia, and nuclear pleomor-
phism of neoplastic cells. p53MSC-induced mammary tumor
(such as the one shown in Panel B ) was transplanted as a cell
suspension into the mammary fat pad of secondary recip-
ient B6Min mice. ( E ) Low-power and ( F ) high-power images
of transplanted tumor reveal suspended tumor cells have
been organized into abnormal neoplastic glands. Increased
mitotic fi gures, including 2 abnormal ones (arrows) typify
active neoplastic cell proliferation. ( G ) Ki-67-specifi c immu-
nostaining of transplanted mammary carcinoma highlights
increased proliferative activity consistent with neoplasia. ( H )
Cells from the tumor shown in E and F grow readily in tissue
culture as an epithelial monolayer. 40× inverted microscope
image. Images ( A–F ), hematoxylin and eosin staining. ( G ),
Ki-67 IHC, hematoxylin counter stain. Original magnifi cation
( A–E , G ) 10×, ( E ) 20×, ( F , H ) 40×.
HOUGHTON ET AL. 1160
tumors, and behaved as previously described for primary
p53MSC. These cells lacked the surface markers: CD45, CD3,
CD4, CD8, CD11b, CD11c, CD19, CD25, Gr1, F4/80, CD31, and
behavior (when placed in culture or injected back into the
wt mouse) as those isolated from wt nontumor-bearing mice.
Specifi cally, reinjected p53MSC into B6 mice did not form
Ki67 Staining of Mammary Ductal Epithelium of B6MlnRag Mice
P < 0.05
P < 0.001
Ki-67 Labeling Index
(% Ki-67 Positive Cells)
B6MinRag + MSC
Isotype Control IgG
B6MinRag + MSC
7.78 ± 1.4617.3 ± 3.24 3.00 ± 0.55
No TreatmentMSC +
Isotype Control lgG
FIG. 4. p53MSC in mammary epithe-
lium of the B6MinRag increase mam-
mary duct epithelium proliferation via
tumor necrosis factor (TNF) signaling.
( A ) 10× and ( B ) 40× (boxed image
from ( A ) red fl uorescent protein (RFP)-
immunohistochemistry (IHC) (brown
cytoplasmic staining) showing clusters
of p53MSC closely opposed to mam-
mary duct epithelium. ( C ) Direct fl uo-
rescence imaging of tissue surrounding
mammary carcinoma. Abundant RFP +
(top image black and white fl uores-
cent image, below; pseudocolored
image). Asterisk denotes a duct em-
bedded within RFP + stroma. ( D ) RFP −
IHC reveals RFP + MSC at the edge of
mammary tumor and within stroma
(arrows). ( E ) p53 IF staining (green) of
stroma surrounding mammary car-
cinoma. Green nuclei of p53 + stromal
cells are highlighted by arrows. ( F )
Ki-67-specifi c IHC of mammary epi-
thelium of B6MinRag mouse receiving
no intervention (no treatment—fi rst
image), p53MSC and isotype control
IgG (second image), or p53MSC plus
anti-TNF-α IgG (third image). Ki-67
staining is confi ned to epithelial cells.
The stromal cells were predominantly
nonproliferative. We did not detect any
Ki-67 staining in p53MSC. Tumor inci-
dence and Ki-67 proliferation index is
shown below histology as a chart form
and as a bow-and-whisker plot for Ki-67
MUTATED STROMA UNMASKS LATENT MALIGNANCY
tissue ( Supplemental Table 1 ) verifying that the anti-TNF-α
treatment did not affect p53MSC viability or engraftment
ability, but dramatically impacted the ability of p53MSC to
induce tumor in at-risk tissue similar to our fi ndings with
T REG infusion.
Proliferation of malignant cells in at-risk tissue
p53MSC are detected juxtaposed to normal duct epi-
thelium in the B6Min and B6MinRag mice, and rarely in
mammary tissue of B6 mice that do not develop mam-
mary carcinoma. We examined Ki-67 expression in these
nonmalignant mammary tissues to gauge the level of cell
proliferation within the ductal epithelium prior to onset of
overt neoplasia. Baseline proliferation of mammary ductal
cells in both the B6Min (data not shown) and B6MinRag
( Fig. 4E and 4F ) mice were higher than control B6 mice
(data not shown). Proliferation within the B6MinRag tis-
sue increased dramatically with the addition of p53MSC,
and was unaffected by the addition of control Ig Ab treat-
ment ( Fig. 4E and 4F ). Addition of T REG cells or anti-TNF-α
antibody to B6Min (data not shown) and B6MinRag mice
( Fig. 4E and 4F ) with p53MSC decreased the proliferation
rate below baseline levels for these strains to proliferative
levels that were indistinguishable from levels found in
wt non-Min mice. These data indicated that at baseline,
at-risk mammary epithelium in Min mice had a higher
proliferation rate, which was dramatically augmented
by p53MSC. Anti-TNF-α therapy prevented the p53MSC-
mediated increase in proliferation, and restored epithelial
proliferation rates to levels found in normal risk non-Min
p53MSC increased proliferation and migration
of human breast carcinoma cells in vitro and in vivo
To tease out the mechanisms involved in immune reg-
ulation of mammary carcinoma, and establish relevance to
human disease, we utilized a combination of in vitro cell cul-
ture systems and human breast cancer xenograph models.
We fi rst evaluated the effects of soluble factors produced by
p53MSC on the MCF-7 breast cancer cell line. p53MSC had a
phenotype consistent with the one that is reported for CAF.
p53MSC expressed higher levels of MMP3, MMP9, fgf1, and
VEGF [ 13 ] and secreted high levels of MCP-1 and RANTES
when compared with wtMSC and had low but detectable
levels of IL-6, TNF-α, and IFN-γ ( Supplemental Table 2 ;
Supplementary materials are available online at http://www
.liebertonline.com/scd) . Conditioned cell-free media from
24-h p53MSC cultures (but not wtMSC cultures) increased
proliferation of the MCF-7 human breast cancer cell line in
a consistent and sustained fashion ( Fig. 5A ) and increased
the mobility of MCF-7 cells in a wound-healing assay ( Fig.
5B ). Direct addition of TNF-α to MCF-7 wounded monolay-
ers did not signifi cantly increase the motility of the breast
cancer cells (data not shown). In contrast, p53MSC cell-free
conditioned media by itself (which contain on average 153
pg/mL of secreted TNF-α) stimulated motility and migra-
tion of MCF-7 cells signifi cantly over control conditions
( Fig. 5C ). The addition of exogenous TNF-α to p53MSC
cultures prior to harvesting media increased motility fur-
ther, allowing 60% healing by 24 h, and complete healing
by 48 h. Adding neutralizing anti-TNF-α during p53MSC
culture resulted in conditioned media that did not support
NK1.1, retained CD44 positivity, and retained the ability to
differentiate into bone fat and cartilage, confi rming they
were MSC. Further, these data suggest that our results do not
refl ect phagocytosis of p53MSC by macrophages. These data
also suggest that the p53MSC did not gain in vivo growth
characteristics as a result of passage through the mouse.
The phenotype of p53MSC recovered from B6, B6Min, and
B6MinRag was similar with regard to the number in circula-
tion, the number recovered from the marrow cavity (~3% of
the stromal cells of the marrow), and surface marker expres-
sion. Thus these results support our hypothesis that tissue
environment of at-risk tissue itself orchestrates engraftment,
proliferation, and proneoplastic effects of p53MSC in the
cancer prone model, but that outside of the local environ-
ment of the tumor, p53MSC behave normally.
Wild-type CD25 + regulatory T cells abolish p53MSC
augmentation of tumor formation in B6Min
and B6MinRag mice
CD4 + CD25 + regulatory (T REG ) cells signifi cantly con-
tribute to immune homeostasis in autoimmune diseases
[ 27 ], chronic infl ammatory diseases [ 29 ], and cancer
[ 27 , 30 , 31 ] through down-regulation of destructive infl am-
matory responses arising from CD4 + T cells as well as cells
of innate immunity. In our present system, infusion of wt
T REG cells completely inhibited the cancer phenotype. While
50% of the B6Min and 80% of the B6MinRag mice injected
with p53MSC developed clinically relevant mammary car-
cinoma, none of the B6Min or B6MinRag mice injected with
T REG cells along with the p53MSC developed mammary
tumors ( Table 2 ). This fi nding was not the result of T REG elim-
ination of p53MSC, as we were able to identify p53MSC in
the peripheral blood and bone marrow of these mice. The
pattern, number, and distribution of p53MSC in the blood
and marrow were similar between mice receiving T REG and
vehicle. Examination of the mammary tissue of T REG recipi-
ents revealed several important differences compared with
controls. Few, single p53MSC were found in the mammary
epithelium of B6MinRag mice receiving p53MSC and T REG
( Supplemental Table 1 ). These cells were juxtaposed to mam-
mary epithelium suggesting that, in the presence of T REG
cells, p53MSC remain viable and retain their ability to home
to the mammary tissue at risk. However, despite their local-
ization to mammary tissue, no tumor formed with T REG cells
present. Thus, immune dysregulation appears to be required
for p53MSC-orchestrated tumor initiation and growth in the
p53MSC-induced mammary tumor formation in
B6Min and B6MinRag mice was TNF-α-dependent
Previous studies from our group show that intestinal and
mammary tumor development in Apc Min/+ mice depends
upon overexpression of TNF-α [ 22 , 23 , 32 ]. In these models,
T REG cells modulate the immune response, in part by re-
storing elevated TNF-α levels to normal. In our present
study, anti TNF-α, but not isotype control Ig, effectively pre-
vented tumor formation in both the B6Min and B6MinRag
mice receiving p53MSC, indicating that p53MSC modulated
cancer via a TNF-α-dependent signaling pathway ( Table
2 ). Anti-TNF-α therapy did not alter peripheral circulating
levels of p53MSC and p53MSC were present in mammary
HOUGHTON ET AL. 1162
responsible for increased motility of breast cancer cells. The
effect of TNF-α is on MSC themselves rather than a direct
effect of TNF-α on the breast cancer cells.
Next we assessed the interaction between p53MSC and
MCF-7 cells in vivo. NOD-SCID mice received 1 million
MCF-7 cell wound healing ( Fig. 5C ). However, neutralizing
the TNF-α in the conditioned media just prior to adding it
to the MCF-7 monolayers did not have a signifi cant effect
on healing kinetics. These data suggest that TNF-α stimu-
lates the p53MSC in an autocrine manner to secrete factors
FIG. 5. Factors elaborated by p53-mutated mesenchymal stem cells (MSC) increase growth and motility of human breast
cancer in vitro and allow homing to breast cancer in vivo. ( A ) Human breast cancer cell line MCF-7 was grown in control
media or cell-free p53MSC-conditioned media. Cell counts were determined at the indicated times. ( B ) Confl uent MCF-7
wounded monolayers were grown in MCF-7 media (open circle) or p53MSC-conditioned media (black circle) for 48 h and
the wound remaining measured and recorded as a percent of the original wound. ( C ) MCF-7-wounded monolayers were
grown in media as indicated and percent wound closure recorded at 24 h. Cell counts and wound-healing experiments
were performed using triplicate plates, repeated a minimum of one additional time, and reported as the mean ± 1 SD. For
A–C; * P < 0.05 unless otherwise noted. ( D ) Red fl uorescent protein (RFP)-p53MSC surround GFP-labeled MCF-7 cells in the
NOD-SCID mouse. ( E ) wtMSC labeled with RFP do not migrate toward GFP-labeled MCF-7 cells. ( F ) Cartoon depicting the
distribution of p53MSC within human breast cancer stroma and within surrounding muscle.
Percent Wound Remaining
*P = 0.0054**P = 0.00002
0 h24 h48 h
162440 486472 h
Wound Healing Assay
Migration of MCF-7 Cells
RFP-MSC Surrounding Tumor Under Skin
And Within Muscle
RFP-MSC in Surrounding Muscle Trafficking to Tumor
MCF-7 MediaMSC Media MSC + TNF
MSC + TNF
% Wound Closure at 24 h
Growth of MCF-7 Cells
1 × 106 Cells
MUTATED STROMA UNMASKS LATENT MALIGNANCY
of the stroma, or vice versa, that is, if the stroma forces a
neoplastic phenotype on the epithelial cell, thus creating
the classical chicken and egg dilemma. Our studies using
p53MSC and wtMSC in the Apc Min+/− mouse demonstrate
that stromal cell mutations accelerate tumor progression
in at-risk tissue.
For the most part, it has been assumed that transformed
cells within an organ proliferate and orchestrate stroma
recruitment and organization via infl uence on local fi bro-
blasts [ 35–37 ], or through epithelial–mesenchymal transi-
tion of normal or transformed epithelial cells [ 38 ] or by
recruiting bone marrow-derived mesenchymal [ 39–41 ] and
other progenitor cells [ 42 ]. Factors elaborated by stromal
cells create a unique milieu that has profound effects on
cancer cell proliferation, invasion, and migration [ 43–45 ]
similar to the contribution of activated stromal cells to
wound healing. This viewpoint of stromal cell–cancer
cell interaction presupposes that the tumor cells orches-
trate the complex interaction with the stromal cells, and
these stromal cells likely undergo gene mutations as a re-
sult of the hyperproliferative environment of the tumor.
This view maintains the stromal cells are under the in-
fl uence and direction of the cancer cell. Our data support
an alternate view that stromal cells may initiate epithelial
transformation via cytokine-mediated signaling of at-risk
tissue and cause the transition of preneoplastic tumor
foci to overt malignant tumors by promoting tumor cell
Though controversial, there are data suggesting that
p53 mutations can be found selectively within the stromal
component of breast cancer and may be associated with a
more aggressive phenotype [ 14 , 15 , 17–19 ]. The vast majority
(95%–99%) of stromal cells within the B6Min+p53MSC
and B6MinRag+p53MSC mammary tumors were host-
derived, and thus wt for p53. Although rare, p53MSC
were found both within tumors, and at the very edge of
the epithelial cancer cells interface. Thus, sampling at any
distance from the tumor itself would lead to a negative re-
sult for mutant p53, and sampling within the tumor could
yield negative results due to sampling error based on the
p53MSC being a small minority of cells within the stroma.
Immunohistochemistry and IF analysis of our archived
human breast cancer samples verifi es that in 67% of cases
examined, overexpression of p53 was found suggesting a
mutant protein, but p53 + stromal CAF were only 1%–5% of
the total stromal cells. Thus, p53CAF are <5% of the total
stroma, but may have a major impact on the biology of the
Despite this controversy, our human data and compel-
ling data from other systems warrants investigation of the
association between p53MSC and breast cancer. MCF-7
mammary carcinoma cells grow faster and demonstrate
a more aggressive phenotype when mixed with p53-defi -
cient fi broblasts compared with wt fi broblasts suggesting
a nonautonomous mechanism of p53-mutated stroma and
transformed mammary epithelial cells [ 11 ]. In these stud-
ies, p53-defi cient fi broblasts are derived from p53 null mice.
Signaling differs between p53-deleted animal models and
clinically relevant p53 point mutations that occur in human
disease. Our model addresses the function of clinically rel-
evant p53 mutations [ 20 ] in mesenchymal cells and their
ability to initiate cancer in a tissue-specifi c fashion in an
at-risk mammary carcinoma model. Our results in both
p53MSC, or wtMSC as a single intracapsular injection, fol-
lowed 1 week later by 1 × 10 6 GFP-labeled MCF-7 cells or
mouse embryonic fi broblast (MEF) cells injected onto the
fl ank. At 4 weeks, p53MSC were detected in the surround-
ing skin and muscle and encasing small well-demarcated
GFP-MCF-7 tumors ( Fig. 5D , and cartoon in Fig. 5F ). p53MSC
did not migrate to normal fl ank nor to normal mammary
fat pads, or in response to the primary GFP-MEF cell cul-
ture (data not shown). In contrast to p53MSC, wtMSC could
not be recovered in MCF-7 tumors or peripheral tissues in-
cluding mammary fat at times up to 4 weeks ( Fig. 5E ). These
fi ndings confi rm that p53MSC migrate toward human breast
cancer cells, providing relevance to human disease.
Adults have in situ malignancy in multiple organs by
midlife [ 1–4 ], but remarkably few people manifest clinical
disease. We sought to understand factors involved in allow-
ing subclinical cancers to progress to overt clinical disease.
We chose our Apc Min+/− mouse model because of the low in-
cidence of mammary tumors in a controlled environment,
and the association of clinically apparent tumor formation
with increasing systemic infl ammation. This pattern follows
the known association of chronic infl ammation with many
types of human tumors. Apc mutations can also be found
in human breast cancer [ 33 ] and breast cancer cell lines [ 34 ].
Thus the Apc Min+/− mouse represents a model whereby the
mammary tissue is at-risk for malignancy, but depending
upon the local environment, cancer potential may or may
not be realized.
Our experiments show that genetic changes including
p53 mutations in MSC are well-tolerated in genetically
normal tissues, and are not associated with an increase in
cancer incidence and that p53MSC have a homing advan-
tage over wtMSC to “at-risk” peripheral tissue by an as of yet
unclear mechanism. Alone, p53MSC are unable to induce
disease; however, in combination with systemic immune
dysregulation, they allow neoplastic growth in at-risk mam-
mary tissue. Restoration of immune homeostasis prevents
disease despite unchanged genetic mutations within both
the stromal and epithelial compartments strongly support-
ing the notion that immune modulation may be an effective
means to prevent cancer in high-risk individuals.
Epigenetic and genetic changes within tumors, which
drive proliferation and aggressive clinical behavior, have
historically been assessed at the level of the cancer cell it-
self, with less attention given to gene alterations within
the supporting stroma. Animal models of cancer typically
focus on gene mutations within the epithelial cell com-
partment, and in many cases multiple gene alterations
are required to initiate tumors. The behavior of resulting
tumors differs from those found in human and rarely is
the spectrum of disease caused by epithelial mutations
alone as diverse as the diseases seen in humans. One way
to view these discrepancies is to recognize that genes
targeted to specifi c cell populations alter the behavior of
“tumor” cells, but do not take into account the contribu-
tion of stromal cells. Several recent reports demonstrate
the importance of mutations within tumor stroma and the
dramatic infl uence these cells have on the behavior of the
tumor as a whole [ 8 , 14 , 15 ]. It is not clear, however, if the
neoplastic epithelial cells infl uence and dictate behavior
HOUGHTON ET AL. 1164
that new targets for preventative therapy for at-risk human
populations can be developed. One can also envision the
possibility that quiescent metastatic foci, which may be-
come active many years after seemingly curative cancer
therapy, could be kept in check by therapy targeted at dis-
rupting infl ammation–stroma–cancer cell interactions.
We thank Kathy Cormier and Chakib Boussahmain for
help with histology and IHC, Glenn Paradis and Michele
Perry for assistance with cell sorting, Chung-Wei Lee for help
with fi gures, and Dario Altieri for assistance with the MCF-7
cell culture. This work is supported by RO1CA119061 (JH)
Pythagoras II Grant 80860 (TP), R01 AI51405 (EAKJ), and DOD
Contract W81XWH-05-01-0460 and RO1CA108854 (SEE).
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Received for publication November 5, 2009 Download full-text
Accepted after revision March 11, 2010
Prepublished on Liebert Instant Online March 11, 2010
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