Supplementary Figures Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth Kristin M. Nieman 1 , Hilary A. Kenny 1 , Carla V. Penicka 1 , Andras Ladanyi 1 , Rebecca Buell- Gutbrod 2 , Marion R. Zillhardt 1 , Iris L. Romero 1 , Mark S. Carey 3 , Gordon B. Mills 3 , Gökhan S. Hotamisligil 4 , S. Diane Yamada 1 , Marcus E. Peter 5 , Katja Gwin 2 & Ernst Lengyel 1 1 Departments of Obstetrics and Gynecology/Section of Gynecologic Oncology – Center for Integrative Science, and 2 Pathology, University of Chicago, Chicago, Illinois, USA. 3 Department of Systems Biology, MD Anderson Cancer Center, Houston, Texas, USA. 4 Department of Genetics and Complex Diseases and the Broad Institute of Harvard and MIT, Harvard School of Public Health, Boston, MA, USA. 5 Department of Medicine/Division of Hematology and Oncology and the Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois, USA. Correspondence should be addressed to E.L. ([email protected]). 1 Nature Medicine doi:10.1038/nm.2492
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Supplementary Figures Adipocytes promote … promote ovarian cancer metastasis ... Gordon B. Mills3, Gökhan S. Hotamisligil4, S. Diane Yamada1, Marcus E. Peter5, ... MD Anderson Cancer
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Supplementary Figures
Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth
Kristin M. Nieman1, Hilary A. Kenny1, Carla V. Penicka1, Andras Ladanyi1, Rebecca Buell-Gutbrod2, Marion R. Zillhardt1, Iris L. Romero1, Mark S. Carey3, Gordon B. Mills3, Gökhan S. Hotamisligil4, S. Diane Yamada1, Marcus E. Peter5, Katja Gwin2 & Ernst Lengyel1 1Departments of Obstetrics and Gynecology/Section of Gynecologic Oncology – Center for Integrative Science, and 2Pathology, University of Chicago, Chicago, Illinois, USA.
3Department of Systems Biology, MD Anderson Cancer Center, Houston, Texas, USA. 4Department of Genetics and Complex Diseases and the Broad Institute of Harvard and MIT, Harvard School of Public Health, Boston, MA, USA.
5Department of Medicine/Division of Hematology and Oncology and the Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois, USA.
Supplementary Figure 1: Human omental transformation by ovarian cancer.(a) Normal human omentum (extended upward) and hematoxylin and eosin staining ofa section of normal human omentum (inset), showing the omentum consists mostly ofadipocytes covered by a layer of mesothelial cells. (b) Tumor transformed omentum.Ovarian cancer patient undergoing tumor debulking from a midline incision. Thepatient’s head is at the top. The omental tumor is causing a bowel obstruction.
Adipocytes
Transversecolon
Smallbowel
Omentum
2
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Supplementary Figure 2: Adipocytes induce invasion of multiple cancer cell types. (a) Adipocyte isolation. Primary human omental tissues were collected during surgical procedures for benign disease and primary human adipocytes were extracted. Adipocytes were visualized (200x) by (i) phase-contrast, (ii) stained with oil red o to confirm the extraction of mature adipocytes, and (iii) by fluorescence microscopy with Calcein AM to confirm viability. (b) Invasion assay. Primary human omental fibroblasts (HPF), immortalized ovarian surface epithelial (IOSE) cells, human ovarian cancer (OvCa) cells (HeyA8 and SKOV3ip1), mouse OvCa cells (ID8), breast cancer cells (MDA-MB-231 and T47D), colon cancer cells (RKO), and gastric cancer cells (SNU-1) invaded toward primary omental adipocytes. Bars report mean fold change ± s.e.m., as compared to the serum-free medium control.
iiiiii iiiiii
100 µm100 µm
b
aN
umbe
r of
inva
ded
cells
(f
old
chan
ge)
HeyA8HPF IOSE SKOV3ip1 SNU-1ID8
Normal cells Ovarian cancer cells
RKOT47DMDA-MB-231
Breast cancer cells
Colon & gastric cancer cells
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−
b
0.0
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Rel
ativ
e flu
ores
cenc
e un
its
(fol
d ch
ange
)
Goat IgG
TIMP1Mouse IgG
IL-6RCXCR1
P = 0.02P = 0.08
a
e
c P < 0.05
0
2
4
6
8
SKOV3ip1 SKOV3ip1 + Adi
IOSE
CX
CR
1 m
RN
A e
xpre
ssio
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old
chan
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Adi Adi + IgG
Adi + IL-6
Num
ber
of m
igra
ted
cells
(fol
d ch
ange
)
P < 0.05
0.0
0.4
0.8
1.2
Adi + IL-8
P = 0.05
Supplementary Figure 3: Adipokine and receptor inhibition reduce migration, homingand mitogenic signaling. (a) In vitro migration toward primary human omental adipocytes(Adi) after 1 h pretreatment with interleukin (IL)-8 or -6 inhibitory antibodies. Bars report meanfold change ± s.e.m. (b) In vivo mouse homing assay. Fluorescently-labeled SKOV3ip1ovarian cancer cells were pretreated with inhibitory antibodies to the IL-6 receptor (R), IL-8R(CXCR1), or a control mouse IgG. Alternatively, the animals were pre-injected with theinhibitory antibodies, TIMP1 and control goat IgG. SKOV3ip1 cells were injectedintraperitoneally into nude mice, the omentum was excised 20 min later, and fluorescenceintensity measured after digestion. Bars report mean fold change ± s.e.m (c) Quantitative RT-PCR for CXCR1 using RNA from IOSE cells, SKOV3ip1 cells alone, and cocultured withhuman adipocytes. Bars report mean fold change relative to glyceraldehyde 3-phosphatedehydrogenase (GAPDH) expression ± s.e.m. (d) RT-PCR for IL-6R, glycoprotein (gp) 130(IL-6R accessory protein), and GAPDH using RNA from SKOV3ip1 cells alone or coculturedwith human adipocytes, human adipocytes alone, and IOSE cells. (e) SKOV3ip1 cells werepretreated with IL-6R or CXCR1 neutralizing antibodies and cultured with (+) or without (–)adipocytes prior to immunoblotting for the indicated proteins.
d
p-p38
Total p38
AdipocytesIgG antibody
CXCR1 antibodyIL-6R antibody
+−
−−
++
−−
−−
+−+
+−
−−
−+
−+
−+
GAPDH
IL-6R
gp130
251 bp
326 bp
196 bp
SKOV3ip1
43 kDa
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Supplementary Figure 4: Lipid accumulation in human ovarian cancer. Neutral lipid(green) staining in sections of omental metastatic tissue from three ovarian cancer patients(nuclear counterstaining, blue) and visualized by confocal microscopy (right panels). Thecorresponding hematoxylin and eosin section is in the left panels (A, adipocytes; C, cancercells).
3
Bodipy and HoechstHematoxylin and Eosin
1
2
100 µmAA
AA
AA
AA
CC CC
CC CCAA AA
CCCC
50 µm
CCCC
100 µm
Pat
ient
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a
0
5
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0 1 2 3 4
HeyA8HeyA8 + adipocytes
Ave
rage
cel
l cou
nt (
103 )
0
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30
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50
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0 1 2 3 4
MONTY-1MONTY-1 + adipocytes
Ave
rage
cel
l cou
nt (
103 )
**
**
**
**
*P < 0.001 *P < 0.05
c
Supplementary Figure 5: Cocultured ovarian cancer cells accumulate lipids and proliferate.(a) Neutral lipid staining shows lipid accumulation in cancer cells. Coculture of ovarian (HeyA8,MONTY-1), colon (RKO), and breast cancer cells (MDA-MB-231, T47D) with primary human omentaladipocytes (Adi) results in cytoplasmic lipid accumulation (green) as evident by confocal microscopy(nuclear counterstain, blue). The fluorescence intensity is quantified in the lower panel where barsreport mean relative fluorescence ± s.e.m. (* = P < 0.001). (b) Representative images (left) of lipidaccumulation (green) in SKOV3ip1 cells following coculture with an equal number of adipocytesharvested from different anatomic sites (subcutaneous (S); bowel mesentery (M); omental (O);peritoneal (P)). Bars (right) report the mean relative fluorescence ± s.e.m. (c) Adipocytes werecocultured with HeyA8 or MONTY-1 ovarian cancer cells and proliferation was measured over fourdays. The mean number of cells ± s.e.m is reported for each day.
HeyA8ovarian
RKOcolon
MONTY-1 ovarian
MDA-MB-231 breast
T47Dbreast
Cancer cells
alone
Cancer cells +
adipocytes
Rel
ativ
e flu
ores
cenc
eun
its (
fold
cha
nge)
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02468
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Time (d)
b
Time (d)
* ** * *
− + − + − + − + − + Adi
SKOV3ip1 alone
SKOV3ip1 + mesenteric (M)
SKOV3ip1 + subcutaneous (S)
SKOV3ip1 + omental (O)
SKOV3ip1 + peritoneal (P)
50 µm
0
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25
30
alone POS M
Rel
ativ
e flu
ores
cenc
e un
its (
fold
cha
nge)
P < 0.001
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* P < 0.05MONTY-1MONTY-1 + Adi
MONTY-1 + carnitineMONTY-1 + etomoxir
MONTY-1 + Adi + etomoxir
pmol
3H
2O
/mg
prot
ein
Time (h)
0.0
0.4
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1.2
1.6
AdiPer
ilipi
nm
RN
A e
xpre
ssio
n (r
elat
ive
fold
cha
nge)
P < 0.001
d
Adipocytes
(
Ovarian cancer cells
a b
c
Supplementary Figure 6: Cocultivation of ovarian cancer cells with adipocytes activateslipolysis in adipocytes and β–oxidation in cancer cells. (a) Quantitative RT-PCR forperilipin 1 in primary human omental adipocytes (Adi) cultured with and without SKOV3ip1ovarian cancer (OvCa) cells. Bars report mean fold change relative to glyceraldehyde 3-phosphate dehydrogenase expression ± s.e.m. (b) Immunoblot for phosphorylated hormonesensitive lipase (p-HSL) and total HSL in SKOV3ip1 cells cocultured with (+) and without (–)adipocytes and pretreated with 10 µM propranolol, a β-adrenergic receptor antagonist. (c)SKOV3ip1 cells were cocultured (1 h) with adipocytes, treated with isoproterenol (positivecontrol) or 14-22 amide (negative control) and protein kinase A (PKA) activity was assessed.(d) β-oxidation in MONTY-1 OvCa cells cocultured with primary human adipocytes (L-carnitine,positive control; etomoxir, negative control). The graph reports the average oxidation rate atthe indicated times ± s.e.m.
14-22 amide
PK
A a
ctiv
ity/m
g pr
otei
n(f
old
chan
ge)
Adi Iso-proterenol
0
1
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*
** P < 0.05
Alone
Adi +SKOV3ip1
p-HSL
Total HSL
β-tubulin
81,83 kDa
55 kDa
− − + +SKOV3ip1
Adipocytes
− + − +propanolol
*
*
*
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e15 kDa
P
45 kDa
M P M P M P M
FABP4
β-actin
a
c
Primary tumor (P)
Corresponding metastasis (M)
P < 0.001
FAB
P4
prot
ein
expr
essi
on
(log
expr
essi
on)
-2
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8d
p-ACC(Ser79)
β-actin
265 kDa
257 kDa
Total ACC
42 kDa
P M P M P M
p-A
CC
pro
tein
exp
ress
ion
(log
expr
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on)
P = 0.001
-2
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-1
-0.5
0
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P = 0.001
AC
C p
rote
in e
xpre
ssio
n
(lo
g ex
pres
sion
)
-2.2
-1.7
-1.2
-0.7
0.2
0.3
b
Supplementary Figure 7: Comparison of protein expression in human primary ovariantumors and corresponding omental metastases. (a) Reverse phase protein array heat map.Primary ovarian tumor and the corresponding omental metastatic tissues were collected from auniform cohort of 22 postmenopausal patients with advanced high-grade serous-papillary ovariancarcinoma (FIGO stage IIIC-IV). (b, d) Graphic representation of protein expression for individualpatients included in the array for phosphorylated acetyl CoA carboxylase (p-ACC), total ACC (b),and fatty acid binding protein 4 (FABP4) (d). (c, e) Confirmation of the protein array data usingimmunoblots for p-ACC, total ACC (c), and FABP4 (e) using fresh tumor samples from primaryovarian tumor (P) and corresponding omental metastatic tissues (M).
Supplementary Figure 8: Characterization of fatty acid binding protein 4 expression incocultures and human tissue. (a) Quantitative RT-PCR for fatty acid binding protein 4(FABP4) in ovarian (SKOV3ip1 and MONTY-1), colon (RKO), and breast cancer cells (MDA-MB-231 and T47D) cocultured with primary human omental adipocytes. Bars report mean foldchange relative to glyceraldehyde 3-phosphate dehydrogenase expression (GAPDH) ± s.e.m.(b,c) Immunohistochemical staining for FABP4 in human tissues from different organs (b) andin human adipose tissues from different anatomic locations (FABP4, top panel; hematoxylin andeosin (H & E), bottom panel; 100-400x) (c). Adipocytes and endothelial cells stain positive forFABP4. (d) FABP4 expression in adipocytes from distinct adipose tissue sites byimmunoblotting.
a
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mR
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ress
ion
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MDA-231breast
MONTY-1ovarian
RKOcolon
SKOV3ip1ovarian
T47Dbreast
*P < 0.001
*
* *
*
*Cancer cells Cancer cells + adipocytes
c
b
d
Thyroid Cervix
Fallopian tube
Parathyroid
Large bowel
Liver
Small bowel
Uterus
Placenta
Testis
200 µm200 µm
H &
EFA
BP
4
OmentalMesentericSubcutaneous
100 µm
15 kDaFABP4
GAPDH
Adipocytes
37 kDa
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Nature Medicine doi:10.1038/nm.2492
Supplementary Figure 9: Characterization of fatty acid binding protein 4 and tumor tissuesfrom knockout and wild-type mice. (a,b) Fatty acid binding protein 4 (FABP4), aP2, expression invisceral adipocytes from FABP4 knockout (FABP4-/-) and wild-type (WT) mice by quantitative RT-PCR(a) and immunoblotting (b). Bars report mean fold change relative to glyceraldehyde 3-phosphatedehydrogenase expression ± s.e.m. (c) Immunohistochemical staining for FABP4 in omentum fromFABP4-/- and WT mice (100x). (d) ID8 mouse ovarian cancer cells were injected intraperitoneally intoFABP4-/- or WT mice. Immunohistochemical staining of intraomental tumor sections from WT andFABP4-/- mice for markers of proliferation (Ki-67), microvessel density (CD31), and apoptosis(cleaved-caspase 3). Staining quantification is on the left and representative images are on the right(hematoxylin and eosin (H & E), 20x and 200x insets; CD31, 200x; Ki-67, 100x; and cleaved-caspase3, 400x). Bars report means (n = 5-9 mice/group) ± s.e.m.
d
b WT FABP4-/-
H &
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BP
4
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WT FABP4-/-
(aP2)
FAB
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50
100
150
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250
300 P < 0.001
FABP4 (15 kDa)
β-tubulin (55 kDa)
WT FABP4-/-a
WT FABP4-/-
Ki-67
H & E
CD31
Cleaved-caspase 3
100 µm100 µm
100 µm100 µm
1 mm1 mm
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0
10
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30
Mic
rove
ssel
de
nsity
(C
D31
)
P = 0.001
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Ki-6
7 po
sitiv
e ce
lls (
%)
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WT FABP4-/-0
2
4
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10
12P = 0.635
Cle
aved
-cas
pase
3po
sitiv
e ce
lls
c
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Nature Medicine doi:10.1038/nm.2492
Supplementary Methods
Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth
Kristin M. Nieman1, Hilary A. Kenny1, Carla V. Penicka1, Andras Ladanyi1, Rebecca Buell-Gutbrod2, Marion R. Zillhardt1, Iris L. Romero1, Mark S. Carey3, Gordon B. Mills3, Gökhan S. Hotamisligil4, S. Diane Yamada1, Marcus E. Peter5, Katja Gwin2 & Ernst Lengyel1 1Departments of Obstetrics and Gynecology/Section of Gynecologic Oncology – Center for Integrative Science, and 2Pathology, University of Chicago, Chicago, Illinois, USA.
3Department of Systems Biology, MD Anderson Cancer Center, Houston, Texas, USA. 4Department of Genetics and Complex Diseases and the Broad Institute of Harvard and MIT, Harvard School of Public Health, Boston, MA, USA.
5Department of Medicine/Division of Hematology and Oncology and the Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois, USA.
with anti-rabbit polymer labeled HRP-bound secondary reagent (DAKO Envision+ System-HRP,
Code K4002). Scoring of FABP4 protein expression in tissue sections was performed by two
pathologists (KG, RBG) as follows: 0=negative; 1=weak; 2=strong. Microvessel density was
performed by counting five random fields (400×, n=8-9). Ki-67 was counted in 250 tumor cells and
scored as percent positive (n=8-9). Cleaved-caspase 3 positive cells were counted in 5 random
fields (400×, n=5).
5
Nature Medicine doi:10.1038/nm.2492
Immunofluorescence. SKOV3ip1 cells were plated onto glass coverslips and cocultured with
primary human omental adipocytes using a modified ceiling culture as described18. Briefly, 50 µl
adipocytes (PCV) were plated in 2 ml DMEM/F12 containing 20% FBS. Coverslips with and without
SKOV3ip1 cells were set on the surface in contact with adipocytes for 3 d to allow the adipocytes to
attach. Coverslips were dried for 1 h and fixed in ice-cold acetone prior to incubation with p-AMPK
(1:100) and p-HSL (1:400) antibodies. The secondary antibody, Alexa Fluor 488 goat anti-rabbit
IgG, was used at 1:300. Coverslips were counterstained with Hoescht 33342 (1:2000). Images were
acquired on a Zeiss LSM 510 laser scanning confocal microscope (630× oil).
Statistical Analysis. The mean and the standard error of the mean (s.e.m) are reported. Data was
compared using two-tailed and paired Student’s t-tests. Differences were considered significant if P
< 0.05.
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Nature Medicine doi:10.1038/nm.2492
Supplementary References
Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth
Kristin M. Nieman1, Hilary A. Kenny1, Carla V. Penicka1, Andras Ladanyi1, Rebecca Buell-Gutbrod2, Marion R. Zillhardt1, Iris L. Romero1, Mark S. Carey3, Gordon B. Mills3, Gökhan S. Hotamisligil4, S. Diane Yamada1, Marcus E. Peter5, Katja Gwin2 & Ernst Lengyel1 1Departments of Obstetrics and Gynecology/Section of Gynecologic Oncology – Center for Integrative Science, and 2Pathology, University of Chicago, Chicago, Illinois, USA.
3Department of Systems Biology, MD Anderson Cancer Center, Houston, Texas, USA. 4Department of Genetics and Complex Diseases and the Broad Institute of Harvard and MIT, Harvard School of Public Health, Boston, MA, USA.
5Department of Medicine/Division of Hematology and Oncology and the Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois, USA.
1. Kenny,H.A., Krausz,T., Yamada,S.D., & Lengyel,E. Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells. Int J Cancer 121, 1463-1472 (2007).
2. Roby,K.F. et al. Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogenesis 21, 585-591 (2000).
3. Hertzel,A.V. et al. Identification and characterization of a small molecule inhibitor of fatty acid binding proteins. J Med Chem 52, 6024-6031 (2009).
4. Zou,H. et al. An orally available small-molecule inhibitor of c-Met PF-2341066, exhibits cytoreductive antitumor efficacy through antiporliferative and antiangiogenic mechanisms. Cancer Res 67, 4408-4417 (2007).
5. Hotamisligil,G.S. et al. Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein. Science 274, 1377-1379 (1996).
6. Robinson-Smith,T.M. et al. Macrophages mediate inflammation-enhanced metastasis of ovarian tumors in mice. Cancer Res 67, 5708-5716 (2007).
7. Drew,A. et al. Correlation of tumor-and stromal-derived MT1-MTP expression with progression of human ovarian tumors in SCID mice. Gynecol Oncol 95, 437-448 (2006).
8. Greenaway,J., Moorehead,R., Shaw,P., & Petrik,J. Epithelial-stromal interaction increases cell proliferation, survival and tumorigenicity in a mouse model of human epithelial ovarian cancer. Gynecol Oncol 108, 385-394 (2008).
9. Kaur,S. et al. b3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer. Am J Pathol 175, 2184-2196 (2009).
10. Sawada,K. et al. C-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion. Cancer Res 67, 1670-1680 (2007).
11. Sawada,K. et al. Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target. Cancer Res 68, 2329-2339 (2008).
12. Shell,S. et al. Let-7 expression defines two differentiation stages of cancer. Proc Natl Acad Sci USA 104, 11400-11405 (2007).
13. Tada,M., Diserens,A.C., Desbaillets,I., & De Tribolet,N. Analysis of cytokine receptor messenger RNA expression in human glioblastoma cells and normal astrocytes by reverse-transcription polymerase chain reaction. Journal of Neurosurg 80, 1063-1074 (1994).
14. Päth,G. et al. Human breast adipocytes express interleukin-6 (IL-6) and its receptor system: Increased IL-6 production by b-adrenergic activation and effects of IL-6 on adipocyte function. J Clin Endocrinol Metab 86, 2281-2288 (2001).
15. Naif,H.M. et al. CCR5 expression correlates with susceptibility of maturing monocytes to human immunodeficiency virus type 1 infection. J Virology 72, 830-836 (1998).
16. Kenny,H.A., Kaur,S., Coussens,L., & Lengyel,E. The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin. J Clin Invest 118, 1367-1379 (2008).
17. Zillhardt,M., Christensen,J., & Lengyel,E. An orally available small molecule inhibitor of c-Met, PF-2341066, reduces tumor burden in a pre-clinical model of ovarian cancer metastasis. Neoplasia 12, 1-10 (2010).
18. Zhang,H.H., Kumar,S., Barnett,A.H., & Eggo,M.C. Ceiling culture of mature human adipocytes: use in studies of adipocyte functions. J Endocrinol 164, 119-128 (2000).