Adipose derived stem cells inAdipose derived stem cells in endometrial cancers

Ann Klopp, MD PhDAdvances in Endometrial Cancer

Epidemiology and BiologyMarch 18th, 2014

Tumor Stroma

Where does it come from?

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

Kidd Plos 2012

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 vs. BMIp y

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

In-vivo effects of O-ASC on endometrial xenograft formation

Klopp Clinical Cancer Research 2012

O-ASC engraft in endometrial

Periphery

xenograftsPeriphery Center

Klopp Clinical Cancer Research 2012

O-ASC increase tumor vascularity

Klopp Clinical Cancer Research 2012

O-ASC increase tumor vascularity

Klopp Clinical Cancer Research 2012

O-ASC increase tumor vessel maturity

O-ASC increase tumor vessel maturityO ASC increase tumor vessel maturity

Klopp Clinical Cancer Research 2012

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

Adipose densityy

10 ***

6

8

10

cm^3

) ***

2

4

Volu

me

(c

Le-SC Le-V Ob-SC Ob-V0

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 isolation

Lean Obese

SubQL-V-ASC

Visceral

Le-V-ASCLe-SC-ASC Ob-V-ASCOb-SC-ASC

CD29

CD34

CD31

32

CD11b

Le-V-ASCLe-SC-ASC Ob-V-ASCOb-SC-ASC

CD73

CD90

CD105

33

CD45

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

SubQ Visceral

Obese LeanObese

Lean Obese

Lean

P=0.05 P=0.321

Lean

Obese

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

O-ASC and cancer cell migration

HEC1A

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