| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
University of Colorado Health Sciences Center, Cancer Center, Medical Oncology [L. T. B.], Division of Cancer Prevention and Control [P. S.], Denver, Colorado 80262, and Center for Cancer Causation and Prevention, AMC Cancer Research Center, Denver, Colorado 80214 [P. S.]
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
The stromal component of most organs is relatively stable with respect to composition, structure, and function and does not undergo major remodeling based on endocrine status. In contrast, the stroma of the MG is dramatically modified depending on endocrine status (Refs. 4 and 5 and the references therein). Consequently, the mammary extracellular environment may vary in its ability to support breast tumor cell dormancy and dissemination. To discern whether reproductive status alters the mammary ECM and the ability to influence tumor cell behavior, we evaluated the effects of ECM isolated from nulliparous and postlactational involuting glands on in vitro tumor cell motility and invasion.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Isolation of Stroma.
Isolation of stroma was conducted as described by Hahm and Ip
(6)
, based on the original procedure described by Kleinman
et al. (7)
. Briefly, frozen MG pairs 4–6 (with
lymph nodes removed) were pulverized and extracted with a
high-salt/N-ethylmaleimide solution [3.4
M NaCl, 50 mM Tris-HCl (pH
7.4), 4 mM EDTA-Na2, and 2
mM N-ethylmaleimide] containing a
proteinase inhibitor mixture (100 µg/ml phenylmethylsulfonyl fluoride
and 50 µg/ml aprotinin, leupeptin, and pepstatin) at 4°C.
Homogenates were pelleted and washed three times at
RCFmax 110,000 x g (30 min, 4°C), and supernatants containing soluble
proteins were discarded after each wash. The ECM-enriched pellets were
resuspended in a mid-level salt/urea solution [2
M urea, 0.2 M NaCl, 50
mM Tris-HCl (pH 7.4), 4 mM
EDTA-Na2, and 2 mM
N-ethylmaleimide] with proteinase inhibitor mixture and
extracted overnight at 4°C. Samples were pelleted at
RCFmax 110,000 x g, and the ECM-enriched supernatants were removed and
extensively dialyzed (Mr
12,000–14,000 molecular weight cutoff; Spectrum) against a
low-salt buffer [0.15 M NaCl, 50
mM Tris-HCl (pH 7.4), and 4
mM EDTA-Na2], followed by
dialysis against serum-free DMEM:Ham’s F-12 (Sigma) supplemented with
1 µg/ml gentamicin at 4°C. Matrices were stored on ice at 4°C and
used within 2 weeks of isolation. Six animals were used per stage. For
the Boyden chamber invasion assay, two separate preparations (batches)
of mammary matrix were evaluated.
Cell Line Propagation.
The human breast cancer cell lines used were MCF-7 and derivatives of
MDA-MB-435. The breast tumor cell line MCF-7 (obtained from American
Type Culture Collection) was passaged as described by the supplier in
Eagle’s MEM with nonessential amino acids, 1 mM sodium
pyruvate, 10 µg/ml insulin, and 10% FBS. The highly metastatic
MDA-MB-435 empty vector control (C-100 cells) and MDA-MB-435 cells
transfected with the breast metastasis suppressor gene
nm23-H1 (H1-177 cells) were obtained from Dr. Pat Steeg
(National Cancer Institute, Bethesda, MD) and cultured in DMEM (Sigma)
supplemented with 10% heat-inactivated FBS.
Motility assays were conducted as reported by Kantor et al. (8) . Cells were resuspended in serum-free medium and plated at 125,000 cells/filter (24-well format filters; Falcon). Filters (pore size, 8 µm) were precoated with either 10 µg/ml EHS murine tumor matrix (Matrigel; Collaborative Biomedical) or 10 µg/ml isolated MG matrix. The cells were stimulated to migrate across the filter by providing a chemoattractant (0.5% FBS) in the assay chamber beneath the coated filter to which the cells were added. For FN experiments, filters were coated with 10 µg/ml gelatin control, 10 µg/ml intact FN, or 10 µg/ml FN120, a Mr 120,000 proteolytic fragment containing the RGD cell binding domain (Life Technologies, Inc.). High motility C-100 cells were assayed 4 h after plating, and low motility cells (MCF-7 and H1-177) were assayed 24 h after plating. Cells were fixed in 10% neutral buffered formalin for 5 min and stained in 0.1% crystal violet. The cells, which crossed through the pores toward the chemoattractant, were quantified photographically, as described previously (8) . Assays were performed in triplicate.
Invasion Assays.
To occlude 8-µm pores, transfilters were coated with 200 µl of 200
µg/ml EHS tumor matrix or isolated MG matrix. Cells were suspended in
serum-free medium and plated at 125,000 cells/filter in 24-well plates
using 0.5% FBS as chemoattractant in the lower chamber. The number of
invasive cells, evaluated 24 h after plating, was quantified as
described above. For FN reconstitution experiments, 0, 10, and 20
µg/ml intact FN or FN120 were mixed on ice with 200 µg/ml gelatin,
and filter pores were occluded as described above. For experiments
designed to investigate the effects of inhibition of MMP activity, the
MMP inhibitor GW9471 (British Biotech BB-24) was added to medium at the
time of cell plating to a final concentration of 0.5 µM,
as described previously (9)
. Invasion assays were
performed in triplicate.
Three-Dimensional Culture Model.
C-100 cells were overlaid as single cell suspension onto a thick matrix
substratum in DMEM supplemented with 2% FBS at a concentration of
100,000 cells/well (24-well plate; Falcon). The matrix substratum was
composed of 300 µl of rat MG matrix (isolated based on equal wet
weight of mammary tissue) and mixed 1:1 with EHS control matrix. EHS
matrix was added to the rat MG matrix to facilitate polymerization. The
development of organoids in this three-dimensional culture model is
dependent on the migration of single cells into cell aggregates
(9)
. Organoid development was monitored over 120 h
using an inverted light microscope (Zeiss Axioscope 25) and
photographed with a Polaroid camera at x100. Matrix substratum and
overlying organoids were harvested at 72 and 120 h after plating,
fixed in 10% neutral buffered formalin, embedded in paraffin, and cut
into 5-µm sections before staining with H&E. Cytological sections
were viewed on a Zeiss Axioscope and photographed.
Biochemical Analyses.
Zymography and Western analyses were performed as described previously
(5)
. MG stromal preparations were loaded by either wet
weight isolation or equal protein concentration, as determined by the
Bradford assay, and equal lane loading was confirmed by Ponceau
staining (Sigma). For FN Western analyses, the polyclonal antiserum
used was directed against human plasma FN (Collaborative Biomedical).
RNase Protection Assay.
The rat-specific FN probe was a generous gift from Dr. Pamela Norton
(Thomas Jefferson University, Philadelphia, PA). The FN probe was
sequenced to verify 100% homology to rat (University of Colorado
Health Sciences DNA Sequencing Core Facility). Mammary tissue RNA was
isolated using a modified phenol/chloroform procedure (RNAwiz kit;
Ambion), with six animals/group.
[32P]UTP-labeled antisense FN RNA and
rat-specific actin RNA (Ambion) probes were transcribed with the
Maxiscript kit (Ambion) and gel-purified. The probes, 2 ng of actin
(1.02 x 108 cpm) and 0.5 ng of FN
(2.05 x 105 cpm), were combined
with RNA samples (2.6–4.75 µg; prenormalized for actin),
precipitated, pelleted, and cohybridized overnight at 45°C in RPA III
hybridization buffer (Ambion). Hybridized samples were RNase-digested
at 37°C for 1 h. Denatured samples were resolved on
urea-polyacrylamide gels and exposed to Hyperfilm MP (Amersham).
Semiquantification of the signal was performed on scanned gels (Adobe
Photoshop 4.0; Epson Expression 636 scanner) using the UN-SCAN-IT gel
automated digitizing system.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
. C-100 cells displayed low motility on matrix isolated from
nulliparous animals, whereas motility was enhanced approximately
10-fold on matrix isolated from involuting MGs and on EHS tumor matrix.
In contrast, motility was suppressed on all matrices in MDA-MB-435
cells transfected with nm23-H1 (H1-177 cells), consistent
with previous reports demonstrating the inhibition of migration by
nm23-H1 (8)
.
|
. MCF-7 cells and MDA-MB-435 cells transfected with
nm23-H1 (H1-177 cells) were not observed to be invasive on
nulliparous, involution, or EHS tumor substrata (data not shown).
A second in vitro three-dimensional culture assay, which is
dependent on both cell motility and invasion (9)
, was used
to further evaluate the effects of MG matrix on cancer cell behavior.
In this assay, mammary matrix was evaluated based on wet weight
extractability and was not normalized for protein concentration,
allowing for the detection of quantitative differences between
matrices. The rationale is based on our observation that the
concentration of ECM extracted per mg of tissue is consistently higher
in involuting MG (
2 mg matrix/g tissue, for a final concentration of
1000 µg/ml) than in nulliparous MG (0.4 mg matrix/g tissue, for
a final concentration of 200 µg/ml). The increase in concentration
of ECM proteins in isolated involution matrix may reflect the in
vivo extracellular environment because elevated levels of matrix
proteins have been reported in involuting mammary tissue (5
, 9)
. Using the modified Boyden chamber assays, it is not possible
to test high concentrations of matrix proteins because concentrations
of ECM above 50 µg/ml inhibited motility, and concentrations above
250 µg/ml inhibited
invasion.4
However, in the three-dimensional organoid assay, MG matrix can be
evaluated as isolated. Single cell suspensions of C-100 cells overlaid
onto thick substratum of EHS tumor matrix formed simple sphere-shaped
organoids by 72 h after plating (Fig. 2, a and b)
. More complex organoids resembling
mammary ducts developed when C-100 cells were overlaid onto involution
MG matrix (Fig. 2g)
. In contrast, organoids did not develop
when C-100 cells were plated on nulliparous MG matrix; instead cells
organized into loose two-dimensional sheets (Fig. 2d and e)
. These data suggest that EHS tumor and involution MG
matrices supported the requisite cell migration required for organoid
formation, whereas nulliparous MG matrix did not, observations
consistent with the modified Boyden motility assay results (Fig. 1a)
. Evaluation of 5-µm sections of 120 h organoids
demonstrated that EHS tumor and involution MG matrices also supported
cancer cell dissemination from primary organoids into the surrounding
matrices. Apparent "growth cones" of cells disseminating from the
primary organoid were evident at 120 h in organoids that formed on
EHS tumor and involution MG matrices (Fig. 2, c and i)
, but not on nulliparous MG matrix (Fig. 2f)
,
corroborating the modified Boyden chamber results in which invasion was
observed maximally on involution MG and EHS matrices and was absent or
reduced on nulliparous MG matrix (Fig. 1, b and c)
.
|
97,000 gelatinase activity,
consistent with MMP-9 (5
, 9)
. To determine whether FN
fragments and MMP activity isolate with involution matrix and
contribute to the biological activity of cells plated onto involution
matrix, biochemical characterizations of the matrices were performed.
Matrices isolated from mid-pregnancy and fully regressed MGs are
included for comparison. MMP activity, assayed by a substrate gel
analysis, revealed elevated levels of both a
Mr 97,000 and a
Mr 72,000 gelatinase in involution
MG matrix (Fig. 3a)
. FN integrity was determined by Western analysis. When MG
matrix was analyzed based on wet weight extractability (the conditions
used in the three-dimensional organoid assay), FN fragmentation was
clearly evident in involution matrix (Fig. 3b)
. When FN was
evaluated in matrix samples loaded by equal protein (the conditions
used in the modified Boyden chamber assays), the absolute amount of
intact FN was low in involution matrix (Fig. 3 c and d)
. The reduced level of intact FN in isolated involution
matrix may reflect in vivo MG biology. This argument is
supported by FN RNA levels, which were decreased 70% during
postlactational involution (Fig. 3e)
. Taken together, these
data are consistent with our previous results that indicate that
mammary FN is targeted for degradation during the period of MG
remodeling after weaning in vivo (9)
and
suggest that FN is a candidate ECM component that may modulate tumor
cell motility and invasion.
|
. H1-177 cells, which were not invasive when overlaid onto
involution mammary matrix (Fig. 1a)
, were not induced to be
invasive when plated onto FN120-containing substratum (data not shown).
Evidence that the invasive behavior of C-100 cells is, in part, MMP
dependent is demonstrated by suppression of invasion in the presence of
the MMP inhibitor GW9471 (Fig. 4b)
.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Using C-100 cells, derivatives of the highly metastatic human mammary epithelial cell line MDA-MB-435, in vitro motility and invasion were observed to be low on nulliparous MG matrix, which is consistent with C-100 metastatic potential being down-regulated by stromal interactions provided by nulliparous MG matrix. Intact FN was detected in nulliparous matrix, and, in reconstitution experiments, intact FN was identified as a suppresser of motility and invasion. Whether other ECM components, which can actively suppress in vitro tumor cell motility and invasion, are present in nulliparous MG matrix remains to be determined.
In contrast to results obtained with nulliparous MG matrix, C-100 motility and invasion were significantly elevated on matrix isolated from MGs undergoing postlactational involution and on matrix isolated from EHS murine sarcoma. Importantly, neither of these matrices promoted motility and invasion in the less metastatic cell line, MCF-7, or in C-100 cells transfected with the known tumor suppresser gene nm23-H1 (8) . Taken together, these observations suggest that EHS tumor and involution MG matrices are permissive, but not instructive, for tumor cell motility and invasion. That is, these matrices do not appear to induce motility or invasion in cells that do not already have the ability to express these phenotypes.
MMP activities, consistent with MMP-9 and MMP-2, were significantly increased in involution MG matrix in comparison with nulliparous matrix. This observation demonstrates that MMP activity isolates with the ECM, and is in agreement with a large body of data demonstrating elevated stromal levels of MMPs in mammary tissue after weaning (Ref. 5 and the references therein). In vivo, elevated stromal MMP-9 and MMP-2 activity is highly correlated with increased metastatic potential in most epithelial cancers, including breast cancer (3 , 4 , 10) . MMP-9 and MMP-2 are also present in the EHS tumor matrix used in these studies (13) , further suggesting a causal role between elevated MMP activity and the increased tumor cell motility observed.
Increased fragmentation of FN was detected in mammary stroma isolated from involuting MGs in comparison with nulliparous MG matrix. Experiments aimed at investigating the potential role of FN fragments in modulating cell behavior identified significantly increased C-100 cell migration and invasion on FN120 as compared with intact FN. These observations are in agreement with reports by others demonstrating enhanced cancer cell motility and invasion on fragmented ECM glycoproteins in comparison with intact ECM glycoproteins, including FN (14) and laminin (15) . In addition, cell invasion on FN fragments was found to be inhibited to a greater degree by MMP inhibitors than invasion on gelatin, suggesting that cell invasion on FN fragments may be more MMP-dependent than cell invasion on gelatin. Of potential relevance is the observation that FN120 fragments, but not intact FN, can induce MMP-9 activity in mammary epithelial cells in vitro (9) . In vivo, induction of MMP activity by FN fragments may induce a feedback loop during MG involution, contributing to further matrix degradation, which results in an environment permissive for tumor cell dissemination. However, the magnitude of the enhancement of in vitro motility and invasion on FN fragments was less than that observed on involution MG matrix. In addition, significant fragmentation of FN was not detected in EHS tumor matrix (data not shown), which also supports motility and invasion. These observations suggest that whereas FN fragments may contribute to the enhanced C-100 cell motility observed on involution MG matrix, other ECM components are likely to be involved as well.
The in vitro work reported here raises the question of whether stroma may become permissive for tumor cell dissemination during MG involution in women. Based on our results, we hypothesize that a subset of women with preexisting malignant breast lesions may be at increased risk for tumor progression during postlactational regression of the MG. Our hypothesis that mammary involution might promote tumor progression appears to be in conflict with the demonstrable protective effect of pregnancy. Clearly, early first-term and multiple full-term pregnancies are associated with a lifetime reduction in risk for breast cancer in women, establishing that, overall, pregnancy is protective for breast cancer (16) . However, of potential relevance to our hypothesis are the observations known as the "cross-over" and "dual" effects of pregnancy (see Refs. 16 and 17 , for review). In younger women, breast cancer incidence for parous women is actually greater than that for nulliparous women for 20–30 years after the time of the first birth (16 , 17) . Starting at around age 50 years, a "cross-over" occurs, in which parous women experience a reduction in risk for breast cancer in comparison with aged-matched nulliparous women (16 , 17) . In addition, first full-term pregnancy in older women has been shown to be associated with a permanent increase in breast cancer risk (16) . Because the protective effect of pregnancy is not constant but rather depends on the age of first pregnancy, the effect of pregnancy on breast cancer risk is considered "dual" (16 , 17) . Whereas it is widely recognized that pregnancy may promote breast cancer because of high levels of ovarian hormones, which stimulate tumor cell proliferation (18) , we suggest that, in addition, postpregnancy-associated changes in mammary stroma may contribute to breast cancer promotion. Specifically, we propose that the period of active tissue remodeling that occurs during postlactational involution may be permissive for tumor cell dissemination. Our rationale is based on the observations that tissue remodeling provides a break in the natural stromal barriers that suppress tumor cell motility and invasion. In addition, mammary tissue remodeling may release and/or induce ECM components that can promote tumor cell progression, such as MMPs, transforming growth factor ß, hepatocyte growth factor/scatter factor, and FN fragments (Ref. 5 and the references therein and Ref. 19 ).
In conclusion, we have demonstrated that ECM components of rat MG stroma, which can modulate in vitro measurements of the metastatic phenotype, are altered by reproductive state. The data support a large body of research demonstrating that the "unit of function" of the normal and malignant MG includes both the epithelial cell and its surrounding stromal component (20) . Consequently, it becomes increasingly important to investigate the apparent complex relationships between reproductive status, MG stroma, and modulation of mammary tumor cell dormancy and dissemination.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by American Cancer Society
Grant CB-179 and NIH Grant R29 CA70915-01 (to P. S.). 
2 To whom requests for reprints should be
addressed, at Center for Cancer Causation and Prevention, AMC
Cancer Research Center, 1600 Pierce Street, Denver, CO 80214. Phone:
(303) 239-3482; Fax: (303) 239-3560; E-mail: schedinp{at}amc.org
3 The abbreviations used are: ECM, extracellular
matrix; MG, mammary gland; MMP, matrix metalloproteinase; FN,
fibronectin; FBS, fetal bovine serum; EHS, Engelbreth-Holm Swarm.
4 P. Schedin, unpublished observations.
Received 6/30/99. Accepted 5/15/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. E. Horak, J. H. Lee, A. G. Elkahloun, M. Boissan, S. Dumont, T. K. Maga, S. Arnaud-Dabernat, D. Palmieri, W. G. Stetler-Stevenson, M.-L. Lacombe, et al. Nm23-H1 Suppresses Tumor Cell Motility by Down-regulating the Lysophosphatidic Acid Receptor EDG2 Cancer Res., August 1, 2007; 67(15): 7238 - 7246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 D. Palmieri, D. O. Halverson, T. Ouatas, C. E. Horak, M. Salerno, J. Johnson, W. D. Figg, M. Hollingshead, S. Hursting, D. Berrigan, et al. Medroxyprogesterone Acetate Elevation of Nm23-H1 Metastasis Suppressor Expression in Hormone Receptor-Negative Breast Cancer J Natl Cancer Inst, May 4, 2005; 97(9): 632 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ouatas, D. Halverson, and P. S. Steeg Dexamethasone and Medroxyprogesterone Acetate Elevate Nm23-H1 Metastasis Suppressor Gene Expression in Metastatic Human Breast Carcinoma Cells: New Uses for Old Compounds Clin. Cancer Res., September 1, 2003; 9(10): 3763 - 3772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Su, Y.-Q. Wei, L. Tian, X. Zhao, L. Yang, Q.-M. He, Y. Wang, Y. Lu, Y. Wu, F. Liu, et al. Active Immunogene Therapy of Cancer with Vaccine On the Basis of Chicken Homologous Matrix Metalloproteinase-2 Cancer Res., February 1, 2003; 63(3): 600 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Ellison, T Klinowska, R F R Westwood, E Docter, T French, and J C Fox Further evidence to support the melanocytic origin of MDA-MB-435 Mol. Pathol., October 1, 2002; 55(5): 294 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Binder, Th. Hagemann, B. Husen, M. Schulz, and A. Einspanier Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteases Mol. Hum. Reprod., September 1, 2002; 8(9): 789 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Hartsough, D. K. Morrison, M. Salerno, D. Palmieri, T. Ouatas, M. Mair, J. Patrick, and P. S. Steeg Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway J. Biol. Chem., August 23, 2002; 277(35): 32389 - 32399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Hall, N. J. Maitland, M. Stower, and S. H. Lang Primary Prostate Stromal Cells Modulate the Morphology and Migration of Primary Prostate Epithelial Cells in Type 1 Collagen Gels Cancer Res., January 1, 2002; 62(1): 58 - 62. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Lang, M. Stark, A. Collins, A. B. Paul, M. J. Stower, and N. J. Maitland Experimental Prostate Epithelial Morphogenesis in Response to Stroma and Three-Dimensional Matrigel Culture Cell Growth Differ., December 1, 2001; 12(12): 631 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-K. Park, X. Zeng, and R. I. Glazer Akt1 Induces Extracellular Matrix Invasion and Matrix Metalloproteinase-2 Activity in Mouse Mammary Epithelial Cells Cancer Res., October 1, 2001; 61(20): 7647 - 7653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Hartsough, S. E. Clare, M. Mair, A. G. Elkahloun, D. Sgroi, C. K. Osborne, G. Clark, and P. S. Steeg Elevation of Breast Carcinoma Nm23-H1 Metastasis Suppressor Gene Expression and Reduced Motility by DNA Methylation Inhibition Cancer Res., March 1, 2001; 61(5): 2320 - 2327. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
