Adipose derived stem cells inAdipose derived stem cells in endometrial cancers
Ann Klopp, MD PhDAdvances in Endometrial Cancer
Epidemiology and BiologyMarch 18th, 2014
Bone marrow origin of tumor Bone marrow origin of tumor stromastroma
/
XRTSCID
β-gal Tg RAG-1-/-, H-2bBMT Pancreas
Ca S.C.H-2d
4 wks
40% of the myofibroblastsin the cancer-induced stroma
f
2-4 wks
were of BM origin at 4 weeks.
X-gal
Ishii et al, BBRC, 309:232, 2003
MSC/ASCMSC/ASCMarrow stromal cells/Adipose stromal cells
TherapeuticImmune modulation for hematopoetic transplantionTissue engineeringMesenchymal stem cells Tissue engineering
Definitionl dhResident and circulating
Bone marrow Fat
Plastic adherentMesenchymal markersMultipotent
Endogenous functionsCharacteristicsCells can can be readily, g
Support hematopoesis Wound repair
s c c b d y,expanded and restransplanted.Migrate to sites of injury andtumors
Bone-marrow derived MSC engraft in MDA231 Breast Metastasis
Murine Lungs with MDA231 metastasis Normal Murine Lungsg g
Studeny, Marini, et al., Vol. 96, No. 21, November 3, 2004
Adipose is also a source of MSC/ASC Adipose is also a source of MSC/ASC hi h f thi h f t ttwhich form tumor which form tumor stromastroma
Zhang, Kolonin Cancer Research 2009
Does obesity increase ASC Does obesity increase ASC f ?f ?engraftment?engraftment?
Zhang, Kolonin Cancer Research 2012
Tumors in obese mice contain more ASC marker positive cellsTumors in obese mice contain more ASC marker positive cells
Hypothesis: visceral Hypothesis: visceral adipose tissue adipose tissue idid ASCASC i i li i lprovides provides ASC to ASC to intraperitonealintraperitoneal
tumorstumorstumorstumors
Visceral adiposity and endometrialVisceral adiposity and endometrial cancer risk in post-menopausal
womenWaist to hip ratio RRWaist to hip ratio RR
< 0.74 1.0
0 74 0 78 1 090.74-0.78 1.09
>0.78-0.83 1.22
>0.83 1.55
P-trend 0.005
Friedenreich et al Cancer Causes Control
Visceral adipose Peripheral adipose
Common site of metastasis of intra-abdominal cancers
Rare metastasis
Increases hypergycemia, Less metabolic effects on insulin hyperinsulinemia, hyperTG, impaired
glucose toleranceresistance
Drains through the liver Venous drainage through systemic veinsg g g y
Larger adipocytes (more insulin resistance)
Smaller adipocytes (higher uptake of FFA and TG)
Well vascularized and innervated Less well vascularized and innervated
More inflammatory cell infiltrating Less inflammatory cell infiltratey g y
Higher expression of pro-inflammatory cytokines (TNF-alpha, CRP, IL-6)
Less inflammatory cytokines
Accumulation supported by lower estrogen
Accumulation promoted by estrogen
Specimen Diagnosis Omentum BMI StageSpecimen Diagnosis Omentuminvolved
BMI Stage
OSC1 AdenoCA of endometrium and
ovary
- 25 Recurrent
OSC2 Granulosa cell tumor of the ovary
+ 31.4 Recurrent
sc ASC lipoaspirate n/a unknown n/asc-ASC lipoaspirate n/a unknown n/a
BM-MSC Normal bone marrow donors
n/a unknown n/a
Wi38 Fetal lung n/a n/a n/a
Unanswered questions• Do visceral adipose derived stromal cells
engraft and support endometrial cancers?engraft and support endometrial cancers?• Does the tissue source of ASC (visceral vs.
b ) h h h ?subcutaneous) change the ASC phenotype?• Does obesity support cancer progression
through ASC?– by altering the phenotype of ASC?– or just increasing the availability of ASC?
• Is ASC population abnormal in patients withIs ASC population abnormal in patients with metastatic cancer?
Mince omentum
Enzymatic digestion (collagenase and dispase) for 1 hour on shaker at 37 degreesfor 1 hour on shaker at 37 degrees
Centrifuge and resuspend in DMEM/10%FBSCentrifuge and resuspend in DMEM/10%FBS
Filter through 100μm and 40μm Nylon cell strainer
Centrifuge and resuspend pellet in red cell lysis buffer I b t i 37 d t b thIncubate in 37 degree water bath
Count and plate cellsCount and plate cells
In-vivo effects of OSC onIn vivo effects of OSC on endometrial xenograft formation
1.6 x 6
104 cells (sc)/mouse 3x/week
Klopp Clinical Cancer Research 2012
O-ASC engraft in endometrial
Periphery
xenograftsPeriphery Center
Klopp Clinical Cancer Research 2012
Conclusions• Omental ASC promote the growth of
endometrial xenografts by supporting tumor
Q i
endometrial xenografts by supporting tumor vasculature.
Questions
• Could differences in O-ASC and sc-ASC be attributed to isolation techniques?
Omental vs. subcutaneous derived ASC: P i d li i l lPaired clinical samples
Flou
rese
nce
Flou
rese
nce
F F
Days Days after 300uM carboplatinDays y p
Conclusion• O-ASC have similar effects as SC-ASC on
endometrial cancer cell proliferation,endometrial cancer cell proliferation, migration and chemosensitivity in-vitro.
Questions• Are differences in O-ASC and sc-ASC seen in-
vivo?• Does obesity alter the phenotype of ASC or
just increase the availability of ASC?
C57 mice fed high-fat diet developC57 mice fed high fat diet develop obesity and metabolic syndrome
50 Lean MiceObese Mice
Injection
30
40
Obese Mice
ht (g
)
10
20Wei
g
0 50 100 1501320
Days
ID8 ovarian cancers grow more rapidlyID8 ovarian cancers grow more rapidly in mice with diet-induced obesity
t] lean
[tota
l cou
n
1500000
2000000 leanobese
nesc
ence
500000
1000000
days
lum
i
0 2 4 6 8 10 120
ASC proliferationASC proliferation
2000000
Le-SC-ASC
umbe
r
1000000
1500000Le SC ASC
Ob-SC-ASC
Le-V-ASC
cell
nu
500000
1000000
Ob-V-ASC
0 5 10 150
days
In-vitro adipocyte differentiationp ySubQ Visceral
Lean
Obese
adipogenesis( total OD)100000
**
Opt
ical
Den
sity
20000
40000
60000
80000**
**Lea
n-SC-A
SC
Ob-SC-A
SCLea
n-V-A
SC
Ob-V-A
SC
0
In-vitro osteocyte differentiationVisceralSubQ
y
Lean
Obese
osteogenesis(total OD)
ptic
al D
ensi
ty
200000
400000
600000** **
Op
Lean-S
C-ASC
Ob-SC-A
SCLea
n-V-A
SC
Ob-V-A
SC
0
VisceralSubQ
Lean
Obese
Ki67 Vessel Nuclear
Ki67 in tumor
8
10 * 30
4+ p
ixel
s
****
perc
ent K
i67+
pixe
ls
0
2
4
6
10
20en
t GSL
I is
olec
tin B
4
groups
p
Le-SC-A
SC O
b-SC-A
SC
Le-V-A
SC
Ob-V-A
SC
0
Le-SC Ob-SC Le-V Ob-V0
perc
e
VisceralSubQ
Lean
Obese
Perilipin (pixel)
els
4 **Perilipin Vessel Nuclear
erce
nt p
erili
pin+
pix
e
1
2
3
groups
pe
Le-SC-A
SCOb-S
C-ASC
Le-V-A
SC
Ob-V-A
SC
0
Analysis of malignant ascities
ascites Macrophage in ascites
me
(ml)
3
4
5
cent
20
30
40 *
volu
m
C C C C
0
1
2
perc
C C C C
0
10
20
Le-SC-A
SC O
b-SC-A
SC
Le-V-A
SC
Ob-V-A
SC
Le-SC-A
SC O
b-SC-A
SC
Le-V-A
SC
Ob-V-A
SC
F4/80 Vessel Nuclear VisceralSubQ
Lean
Lean
Obese
Ob-V-ASC
6
8
10
4/80
+ pi
xels
* *****
Le-SC Ob-SC Le-V Ob-V0
2
4
perc
ent F
4
MSC increase MCF-7 mammosphere formationMSC increase MCF-7 mammosphere formation
MCF-7 alone 20% MSC
Day 5
E-Cadherin
N-Cadherin
Fibronectin
RhoC
Tenascin
Vimentin
Actin
Ann H.KloppPLoS One 2010
ASC spheroid formation in ID8-CM
200
250** **
VisceralSubQ
num
bers
50
100
150
Lean
200 **
Le-SC-C
M
Ob-SC-C
M
Le-V-C
M
Ob-V-C
M
0
size
(um
)
50
100
150
Obese
Le-SC-ct
rlLe-S
C-CM
Le-V-ct
rlLe-V
-CM
Ob-SC-ct
rlOb-S
C-CM
Ob-V-ct
rlOb-V
-CM
0
ID8 spheroid formation in ASC-CM
250*
VisceralSubQ
num
bers
50
100
150
200
Lean
ctrl
Le-SC
Ob-SC
Le-V Ob-V
0
250
size
(um
)
50
100
150
200
Obese
ctrl
Le-SC
Ob-SC
Le-V Ob-V
0Le-V-ASC
Ob-V-ASC
Characterizing ASC samplesCharacterizing ASC samples
Specimen Diagnosis Oment BMISpecimen Diagnosis Omentum
involved
BMI
OSC1 AdenoCA of endometrium and
ovary
- 25
OSC4 Serous ovarian + 32.5cancer
OSC5 Serous ovarian cancer
+ 34
sc-ASC lipoaspirate n/a unknown
BM-MSC Normal bone marrow donors
n/a unknownmarrow donors
Nowicka PLOS 2013
Hec1A spheroid formationnu
mbe
r
re v
olum
e
Hec
1a sp
here
Hec
1a sp
her
H
Control O-ASC1 O-ASC4Control O-ASC1 O-ASC4
ASC secretome: Mass spectrometry
Present in O-ASC Present in O-ASC4
Inflammatory cytokines TGF-B, IL-6
Extracellular matrix modeling
Fibronectin, TIMP1, thrombospondin1, Plasminogen activatorinhibitor
Periostin, vitronectin, thrombospondin2, lysyloxidase, and collagens(1 2 3 5 6)inhibitor (1,2,3,5,6)
Mesenchymal markers Vimentin, fibronectin
t k l t l t licytoskeletal transgelin
Migration/metastasis Profilin-1 Petraxin
ConclusionsConclusions• Omental ASC promote the growth and vascularization of endometrial
xenografts.• Individual heterogeneity in ASC isolates
– Candidate secreted factors include periostin, MMP2, LOXL2• Obesity and tissue source of murine ASC does not alter ASC morphologyObesity and tissue source of murine ASC does not alter ASC morphology,
MSC cell surface marker expression or alter in-vitro effects of ASC on ID8 proliferation or migration
• Obesity impairs differentiation potential of ASC from both subcutaneousObesity impairs differentiation potential of ASC from both subcutaneous and visceral adipose
• Obesity had most consistent effects of ASC from subcutaneous tissues – Increasing– Increasing
• In-vivo ID8 tumor growth• ASC sphere formation and tumorisphere formation• Macrophage infiltration• Macrophage infiltration
– Decreasing• Intra-tumoral adipocytes
Future directionsFuture directions
• Identify critical ASC <-> endometrial cancerIdentify critical ASC < > endometrial cancer signals– Characterizing ASC from patients with and without– Characterizing ASC from patients with and without
cancer
• Determine if effects of tissue source and• Determine if effects of tissue source and obesity are seen in clinical ASC samples.I ti t i t f i ht l d i• Investigate impact of weight loss and exercise on ASC.
AcknowledgmentsAcknowledgments
Lab Travis Solley Ola Nowicka, PhD Z Yh PhD Zan Yhang, PhD. Hadley Sharp, M.D.
Radiation Oncology Wendy Woodward, MD PhDy ,
Gynecologic Oncology Karen Lu, MD Russell Broaddus M.D., Ph.D. Sam Mok Ph D Sam Mok, Ph.D. Rosie Schmandt PhD
Leukemia Michael Andreeff, MD, PhD
Institute for Molecular Medicine Michael Kolonin, Ph.D.
Funding from OCRF, ACS-IRG and endometrial and ovarian spore
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