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Carcinogenesis |
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The contribution of stroma to early events in carcinogenesis has recently begun to be appreciated. Whereas it is well-recognized that tumors can recruit cells (e.g., endothelial cells) and induce changes in the stroma that are conducive to their growth (4 , 5) , it has also been suggested that disruption of stromal/epithelial interactions may provide a stimulus for initiated cells to move further down the neoplastic pathway (6 , 7) . Specialized microenvironments, composed of insoluble ECM3 and soluble growth factors, mediate epithelial-stromal interactions and play a pivotal role in normal tissue development and function (8 , 9) . Such interactions can efficiently suppress the expression of the neoplastic phenotype (3 , 10 , 11) . Conversely, abnormal stromal/epithelial interactions have been shown to enhance the ability of cells to express the neoplastic phenotype (12 , 13) . Transgenic manipulation of the microenvironment, rather than the target cell, can also stimulate tumorigenesis (14) . Recent studies in chronic myelogenous leukemia and in human mammary cancer cells indicate that reestablishing appropriate interactions with the ECM can reverse tumorigenesis and neoplastic behavior, even in the presence of grossly abnormal genetic damage (15 , 16) .
Ionizing radiation is a complete carcinogen, able both to initiate and promote neoplastic progression (17) , and is a known carcinogen of human breast (18, 19, 20, 21) and rodent mammary glands (22, 23, 24) . Our previous studies have demonstrated that radiation exposure elicits rapid and persistent global remodeling of the mammary gland ECM (25) . Because cell-ECM interactions are pivotal in mammary differentiation and growth control (26 , 27) , we postulated that the radiation-induced mammary microenvironment may contribute to radiation carcinogenesis by disrupting cell interactions in a manner that is conducive to expression of neoplastic potential (28) .
To test the above hypothesis, we transplanted epithelial-free mammary stroma with the functionally normal COMMA-D mammary epithelial cell line. The COMMA-D mammary epithelial cell line arose spontaneously from a primary culture of epithelial cells from mammary glands of pregnant BALB/c mice and is nontumorigenic at early passages (29) . It was shown subsequently to contain two mutations in p53 characterized as substitution of Trp for Cys at codon 138 and deletion of the first 21 nucleotides of exon 5 (30) , which result in nuclear accumulation (31) . The mutations in p53 in these cells suggest that the population may be genomically unstable and susceptible to neoplastic transformation (30) .
We show here that COMMA-D cells transplanted to cleared mammary glands in irradiated hosts gave rise to significantly more tumors that arose more quickly and grew larger than in unirradiated animals. Hemibody irradiation supports the conclusion that radiation effects on mammary microenvironment is critical because tumors arose only when COMMA-D cells were transplanted to the irradiated side of animals. These data demonstrate that radiation effects on stroma facilitate expression of neoplastic potential in the absence of exogenously induced mutagenic events in mammary epithelial cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture
COMMA-D mouse mammary epithelial cells originating from midpregnant
BALB/c mice (29)
were obtained at passage 11 from Dr.
Peggy Neville (University of Colorado, Denver, CO) and were expanded
and frozen at passage 13. Cells were grown routinely in 1:1 F12:DMEM
supplemented with 2% fetal bovine serum, epidermal growth factor (5
ng/ml), insulin, transferrin, and selenium on tissue culture plastic in
a 5% CO2 humidified incubator at 37°C and
passaged twice weekly at <90% confluency. Cells were used between
passages 15 and 24. Differential trypsinization was used to enrich for
morphological variants at passage 22 by collecting cells released in
the first 3 min of trypsinization (CD-3T), adding trypsin for an
additional 2 min (discarded), followed by an additional 5 min of
trypsinization to release the remaining cells (CD-5R). Each
subpopulation was expanded one passage, and aliquots were frozen for
subsequent characterization. Cells were trypsinized, counted, and
resuspended in serum-free medium or PBS immediately before
transplantation.
Transplantation Assay
The female mammary gland is unique among all glands in that the
epithelium develops postnatally from a rudiment that can be removed
from the inguinal glands at 
3 weeks of age (32)
.
Surgical removal of the parenchyma results in a gland-free mammary fat
pad, referred to as a CFP, suitable for receiving donor tissue at the
time of clearing or later (32)
. Transplantation of normal
mammary epithelial cells produces ductal outgrowths that fill the fat
pad and are nearly indistinguishable in whole mounts or histologically
from intact gland (32)
. Anesthetized 3-week-old mice were
opened s.c. at the anterior midline and along each flank to reveal the
inguinal mammary gland. The epithelium was destroyed by cauterizing the
connection to the 5th gland, and removing the tissue from the nipple to
just above the lymph node, leaving approximately two-thirds of the fat
pad. The procedure was repeated on the opposite side, and the skin was
closed with wound clips.
Mammary glands were cleared of epithelia at 3 weeks of age. The mice were irradiated and transplanted at 10–12 weeks of age. The CFPs were exposed from the rostral midline to avoid the scar tissue formed by clearing. Known numbers of COMMA-D cells were injected using a Hamilton syringe into the fat pad in a volume of 10 µl. Animals were monitored weekly for gross changes and sacrificed at 6–10 weeks as indicated, and the inguinal glands were removed for analysis. Glands were fixed in 100% ethanol:glacial acetic acid (3:1) for 24 h, followed by alum carmine, according to published methods (33) . Tumor sizes were determined by caliper measurements of width, thickness, and length in the whole mounts, and the approximate tumor volume was calculated as the product of these. Selected tissues were fresh frozen in OCT (Miles Associates, St. Louis, MO).
Irradiation
Unanesthetized mice were whole-body irradiated in perforated Plexiglas
tubes using 60Co 
-radiation at a dose rate of
0.32 Gy/min to a total dose of 4 Gy. Dosimetry was determined using an
Victoreen ionization chamber. Anesthetized animals were used for
hemibody irradiation (4 Gy) by shielding the right lateral half with
lead collimators. Control animals were sham irradiated with or without
anesthesia, as indicated.
Immunofluorescence
Cells were seeded in 24-well plates in 1 ml of 0.5% serum-containing
medium. After reaching confluence, cells were washed with PBS and fixed
with 500 µl of methanol for 10 min at -20°C for vimentin and
keratin staining and in methanol overnight at 4°C for p53
immunostaining. Fixed cells were stored in PBS at 4°C before
immunostaining. Cryosections (5 µm) were fixed with 2% buffered
paraformaldehyde for 20 min for p53 immunostaining and 4% buffered
paraformaldehyde for 10 min for vimentin and keratin immunostaining.
Polyclonal rabbit antibodies against p53 protein (CM5; Vector Laboratories, Burlingame, CA) were used at 1:500 (0.5 µg/ml). Monoclonal mouse antibodies against vimentin (clone VIM 13.2; Sigma) were used at 1:200 dilution (10 µg/ml), and polyclonal guinea pig antibodies to keratin (Sigma) were used at 1:100 dilution.
Nonspecific reactivity was blocked with the supernatant of 0.5% casein stirred for 1 h in PBS. Specimens were incubated with 50 µl of primary antibody overnight (18–20 h) at 4°C. Vimentin and pan-keratin primary antibodies were incubated together, followed by sequential incubation with fluorochrome-labeled secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI at 0.5 µg/ml (Sigma) during the last wash. Specimens were mounted with Vectashield mounting medium (Vector Laboratories) and stored at -20°C prior to viewing. Whole mounts were embedded in paraffin and sectioned at 5 µm for standard histology staining with H&E. Sections from whole mounts were incubated with p53 antibodies as above and detected with alkaline phosphatase-linked secondary antibody.
Image Acquisition and Processing
Fluorescence microscopy of double or triple fluorescence-stained
sections were imaged using a Zeiss Axiovert equipped with
epifluorescence and multiband pass filter and differential wavelength
filter wheel. Images were acquired using a scientific-grade 12-bit
charged coupled device (KAF-1400, 1317 x 1035 6.8-µm
square pixels) camera (Xillix, Vancouver, Canada). The images were
captured so that intensities for a given experiment fell within the
12-bit linear range. Relative intensity was maintained when
constructing figures by scaling the data set to a common 8-bit scale
using Scilimage (TNO, Delft, the Netherlands).
Statistical Analysis
Tumor incidence was evaluated using 95% confidence intervals and the
z test of proportion for data derived from a single
experiment or two-tailed t test for data from multiple
experiments (SigmaStat, Santa Rosa, CA). The significance of
differences between tumor size for sham-irradiated control and
irradiated animals were determined using two-tailed Mann-Whitney rank
sum test (34)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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COMMA-D Cells Transplanted to CFPs of Irradiated Hosts Establish
Tumors Rapidly.
Our previous studies indicated that ionizing radiation leads to global
remodeling of the ECM and induces activity of potent modulators of
epithelial behavior (25
, 36
, 37)
. Individually irradiated
mammary glands exhibit the same microenvironment changes as those from
whole-body-irradiated animals, indicating that these effects are
mediated by local
factors.4
CFPs from irradiated mice show similar remodeling of the
microenvironment as that observed in intact mammary glands (not shown).
We tested whether the irradiated mammary microenvironment could
modulate the neoplastic potential of COMMA-D mammary epithelial cells.
Different numbers of unirradiated COMMA-D cells were transplanted to
CFPs in adult mice that were sham-irradiated or that received 4 Gy
60Co -radiation 3 days prior to
transplantation (Fig. 1)
. A single tumor formed in sham-irradiated CFPs transplanted with
2 x 106
cells
(n = 6); no tumors were observed when fewer
cells were injected. However, tumors arose in irradiated CFPs as a
function of cell number, even when transplanted with as few as
2.5 x 105
cells. Every CFP
(100%) from irradiated hosts contained tumors when injected with
2 x 106
unirradiated COMMA-D
cells. In four independent experiments (n = 36 injected CFPs), tumor incidence was significantly
(P < 0.005, two-tailed t test)
greater in irradiated CFPs (30 of 36) versus sham-irradiated
(8 of 36). The percentage of CFP-bearing tumors averaged 19% ± 2 SE in sham-irradiated hosts and 81% ± 12 SE in
hosts irradiated with 4 Gy, 3 days prior to transplantation.
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25%) in adult
mice, but the frequency and character were similar in irradiated and
sham-irradiated CFPs. In addition, we found that the transplantation
take of normal mammary tissue fragments to sham and irradiated hosts
was
equivalent.5
Thus, the ability of cells to survive and/or grow in mammary glands in
both sham and irradiated hosts was similar. Some tumors were found in
association with otherwise normal-appearing ductal outgrowths (Fig. 2A)
, but most were isolated.
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.
Tumors were both vimentin and keratin positive and exhibited nuclear
p53, indicative of their origin from COMMA-D cells (Fig. 2C)
. Note that p53 immunostaining was heterogeneous but more
evident using the more sensitive immunofluorescence detection. This
heterogeneity is also evident in culture (Fig. 4)
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-radiation exposure (Fig. 3)
. All irradiated animals showed a significant (P < 0.05) increase in tumor incidence, ranging from 100% at 3 day
to 58% at 14 days after irradiation compared with 25% in the
sham-irradiated host. The peak occurred when irradiated animals were
transplanted at 3 days after irradiation, at which time tumors arose in
100% of glands in irradiated hosts. Furthermore, the mean size of
tumors from irradiated animals was 243 mm3
± 61 SD compared with 31
mm3
± 9 SD in the few tumors that
arose in sham-irradiated hosts. The mean size of tumors in irradiated
animals at all times after irradiation were significantly
(P < 0.05; Mann-Whitney rank sum test)
larger than the size of tumors in sham-irradiated control animals.
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COMMA-D Parental Heterogeneity Gives Rise to Subpopulations That
Are Also Preferentially Tumorigenic in Irradiated Hosts.
Clonal COMMA-D subpopulations have been shown to retain the capacity to
produce ductal outgrowths and to respond to hormone stimulation
(38)
, suggesting that some COMMA-D cells have multipotent
characteristics of putative stem cells. The multipotent nature of these
cells is also suggested by the observation that COMMA-D cell cultures
contain multiple morphologically distinct cells consisting of flat,
polygonal cells that form contact-inhibited monolayer islands and
spindle-shaped cells that form ridges that surround the islands. This
heterogeneity is maintained by routine passaging at 80–90%
confluency. These cells are p53 positive and exhibit various levels of
keratin and vimentin immunoreactivity (Fig. 4A and E)
. Keratin is observed predominantly in
polygonal cells, whereas vimentin expression is less prominent and is
associated with the ridges of spindle cells.
The possibility that these distinct cell types have different
tumorigenic potentials or are preferentially selected during growth
in vivo was addressed by testing their respective behaviors
in CFPs. Morphologically distinct subpopulations were enriched from the
parent population by differential trypsinization. Trypsinization
releases spindle-shaped cells in the first 3 min (designated CD-3T),
whereas predominantly polygonal cells remain after 5 min of
trypsinization (designated CD-5R). Both cell types exhibited p53
immunoreactivity in culture (Fig. 4, E–G)
. The CD-3T
subpopulation was primarily vimentin positive and keratin negative and
had large nuclei (Fig. 4, B and F)
. The CD-5R
subpopulation contained keratin-immunoreactive cells, with little
expression of vimentin, and had small nuclei (Fig. 4, C and G)
.
Both the CD-3T and CD-5R subpopulations were significantly
(P < 0.05) more tumorigenic in irradiated
hosts when transplanted to sham- versus irradiated-hosts
(Fig. 5)
. However, the CD-5R subpopulation produced fewer tumors (4 of 12) than
the parent in irradiated CFPs and did not give rise to any tumors in
the sham-host. The tumors from the CD-5R were also considerably smaller
than either the parent or the CD-3T subpopulation (Fig. 5B)
.
In contrast, the CD-3T subpopulation was more efficient (12 of 12) in
generating tumors than the parent population (8 of 12) in this
experiment. Furthermore, unlike the parent COMMA-D population, the size
of tumors from the 3T-CD subpopulation were similar in the
sham-irradiated CFPs and irradiated CFPs. Thus, the size of the tumors
is influenced by both the nature of the microenvironment and of the
epithelial population.
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-irradiation of anesthetized mice was
used to test whether systemic factors from the irradiated host
contributed to tumor promotion in irradiated CFPs. No tumors were found
in nonirradiated CFPs (n = 8), whereas three
tumors were observed in irradiated CFPs (P = 0.05). To determine whether the low incidence of tumor formation was
attributable to the systemic effects of anesthesia necessary for
hemibody irradiation, we asked whether anesthesia compromised tumor
formation. Anesthetized, whole-body-irradiated mice formed tumors in
two of eight transplanted CFPs, whereas unanesthetized,
whole-body-irradiated mice in this experiment formed seven tumors in
eight CFPs. Thus, the low incidence of tumors in the partial body
protocol compared with whole-body exposures appears to be attributable
to the use of general anesthesia; tumor incidence was restricted to
CFPs on the irradiated side, indicating that local tissue effects were
dominant over the systemic consequences of irradiation. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The studies reported here address the question of whether radiation-induced changes in the composition of the mammary microenvironment promote the expression of neoplastic potential. We showed that COMMA-D mammary epithelial cells, which retain normal developmental capacity, were nontumorigenic by standard assays (lack of tumor formation when injected s.c. or in juvenile CFPs). Small, infrequent (19%) tumors were obtained when COMMA-D cells were transplanted to adult CFPs, suggesting that the physiological status of the host can modulate COMMA-D neoplastic potential. Tumorigenic efficiency increased to 100% when unirradiated COMMA-D cells were transplanted to CFPs in irradiated hosts. Furthermore, tumor size also substantially increased in irradiated CFPs. The effect of radiation on enhanced tumorigenesis persisted for at least 14 days after a single radiation exposure. We concluded that this was attributable to radiation-induced changes in the mammary stroma by comparing the irradiated and nonirradiated CFPs after hemibody irradiation; tumors formed only in the CFPs from the irradiated side. Thus, radiation exposure altered the mammary microenvironment in a manner that promotes the neoplastic behavior of these mammary epithelial cells, which harbor defective p53.
Although clonal, COMMA-D cells exhibit morphological and phenotypic diversity in culture (30 , 38) . We therefore determined some of the characteristics of the tumors arising from COMMA-D cells and found that tumors maintained cytokeratin immunoreactivity whether they arose in normal or irradiated CFPs. We then used selective trypsinization to isolate the CD-3T vimentin-positive versus the CD-5R keratin-positive subpopulations and asked whether one population was selected and/or preferentially influenced by the irradiated microenvironment during growth in vivo. We found that both subpopulations were preferentially tumorigenic in irradiated hosts. Thus, differential selection of these particular phenotypes alone does not appear to be responsible for the preferential effect of the irradiated stroma on promoting COMMA-D neoplastic behavior. The two cell populations may, however, interact because tumors arising from each showed distinctly different characteristics from the parent. CD-5R were less tumorigenic and considerably smaller than the parent cell line. In contrast, the CD-3T subpopulations were somewhat more efficient than the parent cell line in establishing tumors, and unlike the parent, were as large in the sham-irradiated host as in the irradiated host.
Our previous studies showed that irradiated mammary gland undergoes rapid remodeling of the microenvironment characterized by changes in ECM and activation of latent TGF-ß (25 , 36) . We tested whether these events were functionally related by treating animals with TGF-ß neutralizing antibodies prior to irradiation (37) . TGF-ß panspecific neutralizing antibody administered shortly before irradiation inhibited collagen III staining in the adipose stroma at 24 h in an antibody dose-dependent manner and blocked collagen III through 7 days after irradiation. Quantitative image analysis demonstrated that exposure to radiation doses of as little as 0.1 Gy elicited a significant increase in TGF-ß immunoreactivity, which showed a linear dose response after exposure to 0.1–5 Gy without an apparent threshold of the mammary epithelium. Qualitative evaluation of the reciprocal pattern of latency-associated peptide and collagen III immunoreactivity in the adipose stroma, in contrast, demonstrated a threshold of 0.5 Gy. These data provide functional confirmation of the hypothesis that radiation induces TGF-ß activation and implicate TGF-ß as a mediator of tissue response to ionizing radiation.
TGF-ß orchestrates responses of multiple cell types and has emerged as a key coordinator of tissue response to damage during wound healing, inflammation, and development. Aspects of radiation remodeling parallel those associated with dermal wound healing (46) . Ionizing radiation effects on the tissue microenvironment that foster neoplastic behavior may be similar to those observed by other agents that elicit an activated stroma. Experimental animal models have demonstrated that carcinogenesis is enhanced by the activated stroma induced by wounding (12) , overexpression of platelet-derived growth factor (5) , or misregulation of stromelysin (41 , 47) .
Although the particular aspect of the irradiated mammary microenvironment that is stimulating COMMA-D tumorigenic conversion is unknown, the ability of radiation to induce TGF-ß activation may indeed play a role (36 , 48) . We have argued that the role of TGF-ß in cancer appears paradoxical in that normal epithelial cells are profoundly sensitive to TGF-ß growth inhibition, whereas cancer cells are generally TGF-ß resistant (49) because conversion during tumor progression is a critical juncture in establishing malignant behavior of certain epithelia, particularly skin and breast (50) . TGF-ß has been identified as a cancer-promoting agent in wounds (13 , 51 , 52) , and recent studies have implicated cyclosporin-induced TGF-ß in the increased frequency of neoplasms after immunosuppressive therapy (53) . TGF-ß also mediates phenotypic conversion of epithelial cells to vimentin-positive spindle cells (54) . Such a transition is observed in a transgenic keratinocyte model overexpressing TGF-ß activity in which the frequency of benign papillomas is suppressed after chemical carcinogenesis, but progression to more malignant spindle cell carcinomas is stimulated (55) . In the SCp2 cell line, a subclone of late-passage COMMA-D cells (56) , TGF-ß treatment stimulates the transition from nontumorigenic keratin-positive cells to tumorigenic, vimentin-positive cells.6 Studies to determine whether the carcinogenesis-promoting effect of radiation on stroma is attributable to TGF-ß activation are under way.
Our hemibody irradiation data support the conclusion that radiation
alters the local tissue microenvironment in a way that compromises the
restraints imposed by normal stroma on initiated epithelial cells. A
role for stroma early in neoplastic progression has also been suggested
in hematopoietic malignancies, which have been proposed to result from
misregulation of adhesive properties by diseased or genetically
aberrant stroma (57)
. Conversely, the therapeutic benefit
of IFN-
in chronic myeloid leukemia has been shown recently to be
attributable in part to the reestablishment of cell adhesion signals
(15)
. Greenberger et al. (58)
proposed a model of indirect -irradiation leukemogenesis based on
cocultures of heavily irradiated bone marrow stromal cell lines that
selectively bound macrophage-colony-stimulating factor
receptor-positive unirradiated hematopoietic progenitor cells,
resulting in selection of tumorigenic subclones. Additional evidence
that radiation effects on stroma alter the behavior of neoplastic cells
comes from studies of tumor bed effect, in which stroma that is heavily
irradiated prior to tumor transplantation inhibits tumor growth but
fosters metastatic behavior (59)
. Such studies support the
conclusion that radiation has global and persistent consequences in
terms of stromal function, which in turn can influence the expression
of neoplastic potential.
In effect, these experiments demonstrate a bystander-type phenomena in vivo in which the products of irradiated cells can significantly alter the phenotype of unirradiated cells. On the basis of these data, we propose that radiation-induced microenvironments are evidence of an additional class of carcinogenic action, distinct from those leading to mutations or proliferation (60) . Studies in cell culture indicate that the frequency of morphological transformation can be modulated by restrictive conditions that select for preexisting cell variants (61) and that, conversely, normal cells may actively restrain the expression of the transformed cell phenotype (62) . In vivo studies by Cha et al. (63) show that mammary tumors with Hras1 gene mutations from N-nitroso-N-methylurea-treated rats arose from cells with preexisting Hras1 mutations that occurred during early development. Thus, although clearly mutagenic in its own right, N-nitroso-N-methylurea exposure led to the expansion and neoplastic progression of Hras1-mutation containing populations. In our studies, radiation did not directly induce additional mutagenic events because the epithelial cells were unirradiated. We propose that a further action of carcinogens, such as ionizing radiation, is to modify paracrine interactions between the stroma and epithelium in a manner that affects the frequency with which previously initiated cells progress (46) . Carcinogen-induced microenvironments are not necessarily mutagenic or mitogenic per se. Rather, changes in the microenvironment may promote neoplastic behavior by disrupting normal cell functions that are regulated through cell-cell contact, cell-ECM interactions, and growth factor signaling. Thus, if ionizing radiation induces a microenvironment that modifies restrictive interactions, then it may promote malignant phenotype in manner that is functionally equivalent to the acquisition of additional mutations in the initiated cell. Alternatively, the microenvironment elicited by carcinogen exposure could create novel selective pressures that would affect the features of a developing tumor. Disruption of solid tissue interactions is a heretofore unrecognized activity of radiation as a carcinogen and a novel avenue by which to explore new strategies for intervening in the neoplastic process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA-5184, by the United
States Army Medical Research and Materiel Command under
DAMD-17-96-1-6716, and the Office of Health and Environmental Research,
Health Effects Research Division, of the United States Department of
Energy under Contract DE-AC-03-76SF00098. 
2 To whom requests for reprints should be
addressed, at Life Sciences Division, Lawrence Berkeley National
Laboratory, 1 Cyclotron Road, Berkeley, CA 94720. Phone:
(510) 486-6371; Fax: (510) 486-6746; E-mail: mhbarcellos-hoff{at}lbl.gov
3 The abbreviations used are: ECM, extracellular
matrix; CFP, cleared fat pad; TGF-ß, transforming growth factor-ß1;
DAPI, 4',6-diamidino-2-phenylindole.
4 E. J. Ehrhart and M. H. Barcellos-Hoff,
unpublished data.
6 S. Galosy, M. H. Barcellos-Hoff, Z. Werb, and
M. J. Bissell, unpublished observations.
Received 7/19/99. Accepted 12/20/99.
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P. J. Coates, J. K. Rundle, S. A. Lorimore, and E. G. Wright Indirect Macrophage Responses to Ionizing Radiation: Implications for Genotype-Dependent Bystander Signaling Cancer Res., January 15, 2008; 68(2): 450 - 456. [Abstract] [Full Text] [PDF]
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H. Koyama, N. Kobayashi, M. Harada, M. Takeoka, Y. Kawai, K. Sano, M. Fujimori, J. Amano, T. Ohhashi, R. Kannagi, et al. Significance of Tumor-Associated Stroma in Promotion of Intratumoral Lymphangiogenesis: Pivotal Role of a Hyaluronan-Rich Tumor Microenvironment Am. J. Pathol., January 1, 2008; 172(1): 179 - 193. [Abstract] [Full Text] [PDF]
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T. Kaneko, Z. Zhang, M. G. Mantellini, E. Karl, B. Zeitlin, M. Verhaegen, M. S. Soengas, M. Lingen, R. M. Strieter, G. Nunez, et al. Bcl-2 Orchestrates a Cross-talk between Endothelial and Tumor Cells that Promotes Tumor Growth Cancer Res., October 15, 2007; 67(20): 9685 - 9693. [Abstract] [Full Text] [PDF]
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K. L. Andarawewa, A. C. Erickson, W. S. Chou, S. V. Costes, P. Gascard, J. D. Mott, M. J. Bissell, and M. H. Barcellos-Hoff Ionizing Radiation Predisposes Nonmalignant Human Mammary Epithelial Cells to Undergo Transforming Growth Factor {beta} Induced Epithelial to Mesenchymal Transition Cancer Res., September 15, 2007; 67(18): 8662 - 8670. [Abstract] [Full Text] [PDF]
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C.-Q. Zhu, S. N. Popova, E. R. S. Brown, D. Barsyte-Lovejoy, R. Navab, W. Shih, M. Li, M. Lu, I. Jurisica, L. Z. Penn, et al. Integrin {alpha}11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells PNAS, July 10, 2007; 104(28): 11754 - 11759. [Abstract] [Full Text] [PDF]
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 D. Mahadevan and D. D. Von Hoff Tumor-stroma interactions in pancreatic ductal adenocarcinoma Mol. Cancer Ther., April 1, 2007; 6(4): 1186 - 1197. [Abstract] [Full Text] [PDF]
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J.-P. Coppe, K. Kauser, J. Campisi, and C. M. Beausejour Secretion of Vascular Endothelial Growth Factor by Primary Human Fibroblasts at Senescence J. Biol. Chem., October 6, 2006; 281(40): 29568 - 29574. [Abstract] [Full Text] [PDF]
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C. M. Blakely, A. J. Stoddard, G. K. Belka, K. D. Dugan, K. L. Notarfrancesco, S. E. Moody, C. M. D'Cruz, and L. A. Chodosh Hormone-Induced Protection against Mammary Tumorigenesis Is Conserved in Multiple Rat Strains and Identifies a Core Gene Expression Signature Induced by Pregnancy. Cancer Res., June 15, 2006; 66(12): 6421 - 6431. [Abstract] [Full Text] [PDF]
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 A. Y. Khakoo, S. Pati, S. A. Anderson, W. Reid, M. F. Elshal, I. I. Rovira, A. T. Nguyen, D. Malide, C. A. Combs, G. Hall, et al. Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma J. Exp. Med., May 15, 2006; 203(5): 1235 - 1247. [Abstract] [Full Text] [PDF]
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C. Sonnenschein and A. M. Soto Carcinogenesis and Metastasis Now in the Third Dimension--What's in It for Pathologists? Am. J. Pathol., February 1, 2006; 168(2): 363 - 366. [Full Text] [PDF]
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S. M. McDaniel, K. K. Rumer, S. L. Biroc, R. P. Metz, M. Singh, W. Porter, and P. Schedin Remodeling of the Mammary Microenvironment after Lactation Promotes Breast Tumor Cell Metastasis Am. J. Pathol., February 1, 2006; 168(2): 608 - 620. [Abstract] [Full Text] [PDF]
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C. Bavik, I. Coleman, J. P. Dean, B. Knudsen, S. Plymate, and P. S. Nelson The Gene Expression Program of Prostate Fibroblast Senescence Modulates Neoplastic Epithelial Cell Proliferation through Paracrine Mechanisms Cancer Res., January 15, 2006; 66(2): 794 - 802. [Abstract] [Full Text] [PDF]
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M. V. Maffini, J. M. Calabro, A. M. Soto, and C. Sonnenschein Stromal Regulation of Neoplastic Development: Age-Dependent Normalization of Neoplastic Mammary Cells by Mammary Stroma Am. J. Pathol., November 1, 2005; 167(5): 1405 - 1410. [Abstract] [Full Text] [PDF]
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K. K.C. Tsai, E. Y.-Y. Chuang, J. B. Little, and Z.-M. Yuan Cellular Mechanisms for Low-Dose Ionizing Radiation-Induced Perturbation of the Breast Tissue Microenvironment Cancer Res., August 1, 2005; 65(15): 6734 - 6744. [Abstract] [Full Text] [PDF]
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Y. Tu, D. J. Jerry, B. Pazik, and S. Smith Schneider Sensitivity to DNA Damage Is a Common Component of Hormone-Based Strategies for Protection of the Mammary Gland Mol. Cancer Res., August 1, 2005; 3(8): 435 - 442. [Abstract] [Full Text] [PDF]
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N. Jessani, M. Humphrey, W. H. McDonald, S. Niessen, K. Masuda, B. Gangadharan, J. R. Yates III, B. M. Mueller, and B. F. Cravatt Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo PNAS, September 21, 2004; 101(38): 13756 - 13761. [Abstract] [Full Text] [PDF]
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M. V. Maffini, A. M. Soto, J. M. Calabro, A. A. Ucci, and C. Sonnenschein The stroma as a crucial target in rat mammary gland carcinogenesis J. Cell Sci., March 15, 2004; 117(8): 1495 - 1502. [Abstract] [Full Text] [PDF]
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 N. A. Bhowmick, A. Chytil, D. Plieth, A. E. Gorska, N. Dumont, S. Shappell, M. K. Washington, E. G. Neilson, and H. L. Moses TGF-{beta} Signaling in Fibroblasts Modulates the Oncogenic Potential of Adjacent Epithelia Science, February 6, 2004; 303(5659): 848 - 851. [Abstract] [Full Text] [PDF]
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C. d. L. Davies, L. M. Lundstrom, J. Frengen, L. Eikenes, O. S. Bruland, O. Kaalhus, M. H. B. Hjelstuen, and C. Brekken Radiation Improves the Distribution and Uptake of Liposomal Doxorubicin (Caelyx) in Human Osteosarcoma Xenografts Cancer Res., January 15, 2004; 64(2): 547 - 553. [Abstract] [Full Text] [PDF]
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C. C. Park, R. L. Henshall-Powell, A. C. Erickson, R. Talhouk, B. Parvin, M. J. Bissell, and M. H. Barcellos-Hoff Ionizing radiation induces heritable disruption of epithelial cell interactions PNAS, September 16, 2003; 100(19): 10728 - 10733. [Abstract] [Full Text] [PDF]
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G. Zhang, B. He, and G. F. Weber Growth Factor Signaling Induces Metastasis Genes in Transformed Cells: Molecular Connection between Akt Kinase and Osteopontin in Breast Cancer Mol. Cell. Biol., September 15, 2003; 23(18): 6507 - 6519. [Abstract] [Full Text] [PDF]
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K. L. Schmeichel and M. J. Bissell Modeling tissue-specific signaling and organ function in three dimensions J. Cell Sci., June 15, 2003; 116(12): 2377 - 2388. [Abstract] [Full Text] [PDF]
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 J. E. Morgan, J. G. Gross, C. N. Pagel, J. R. Beauchamp, A. Fassati, A. J. Thrasher, J. P. Di Santo, I. B. Fisher, X. Shiwen, D. J. Abraham, et al. Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site J. Cell Biol., May 13, 2002; 157(4): 693 - 702. [Abstract] [Full Text] [PDF]
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H. Rubin Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates Carcinogenesis, December 1, 2001; 22(12): 1903 - 1930. [Abstract] [Full Text] [PDF]
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 C. M. Markey, E. H. Luque, M. Munoz de Toro, C. Sonnenschein, and A. M. Soto In Utero Exposure to Bisphenol A Alters the Development and Tissue Organization of the Mouse Mammary Gland Biol Reprod, October 1, 2001; 65(4): 1215 - 1223. [Abstract] [Full Text] [PDF]
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S. Laconi, P. Pani, S. Pillai, D. Pasciu, D. S. R. Sarma, and E. Laconi A growth-constrained environment drives tumor progression invivo PNAS, June 20, 2001; (2001) 131210498. [Abstract] [Full Text] [PDF]
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H. Rubin Selected Cell and Selective Microenvironment in Neoplastic Development Cancer Res., February 1, 2001; 61(3): 799 - 807. [Abstract] [Full Text]
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B. Elenbaas, L. Spirio, F. Koerner, M. D. Fleming, D. B. Zimonjic, J. L. Donaher, N. C. Popescu, W. C. Hahn, and R. A. Weinberg Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells Genes & Dev., January 1, 2001; 15(1): 50 - 65. [Abstract] [Full Text]
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S. Laconi, P. Pani, S. Pillai, D. Pasciu, D. S. R. Sarma, and E. Laconi A growth-constrained environment drives tumor progression invivo PNAS, July 3, 2001; 98(14): 7806 - 7811. [Abstract] [Full Text] [PDF]
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) or total body irradiated hosts
(
). Animals were irradiated with 4 Gy, 3 days before transplantation
of cells to CFPs. The incidence of tumors was significantly
(P < 0.05) greater in irradiated hosts
transplanted with 106 cells or more.
